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Sample records for a-1-acid glycoprotein concentration

  1. Effects of Mycoplasma gallisepticum vaccination on serum a1-acid glycoprotein concentrations in commercial layer chickens

    USDA-ARS?s Scientific Manuscript database

    Increases in circulating acute phase protein (APP) levels, as an integral component of the acute phase response, occur in reaction to systemic infections in animals. However, no previous research has been conducted to monitor possible changes in APP levels of birds in response to pre-lay vaccinatio...

  2. The immune system modulator a1-acid glycoprotein inhibits insulin and IGF1 induced protein synthesis in C2C12 myotubes

    USDA-ARS?s Scientific Manuscript database

    Alpha-1 acid glycoprotein (AGP) has previously been demonstrated by our laboratory to be negatively correlated with growth rate in newborn piglets. However, a mechanism of action for AGP in growth has not been identified. Previous research has demonstrated that AGP can modify adipose tissue metabo...

  3. Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method.

    PubMed

    Kamoda, Satoru; Nakanishi, Yasuharu; Kinoshita, Mitsuhiro; Ishikawa, Rika; Kakehi, Kazuaki

    2006-02-17

    Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

  4. Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study.

    PubMed

    Albani, J R; Sillen, A; Plancke, Y D; Coddeville, B; Engelborghs, Y

    2000-07-24

    White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.

  5. Determination of the concentrations of oligosaccharides, complex type carbohydrates, and glycoproteins using the phenol-sulfuric acid method.

    PubMed

    Saha, S K; Brewer, C F

    1994-02-17

    The concentrations of methyl glycosides, oligosaccharides, glycopeptides, and glycoproteins can be accurately determined by using calibration curves composed of the appropriate monosaccharide(s) obtained with a modified version of the colorimetric phenol-sulfuric acid method. Calibration curves of micrograms sugar vs. 490 nm for Man, Glc, or Gal are shown to provide reliable determinations (typically +/- 3-4%) of corresponding methyl glycosides and linear and branched-chain oligosaccharides containing the corresponding reactive hexose residue. For complex oligosaccharides containing a known mixture of reactive hexose units, the appropriate mixture of monosaccharides are shown to provide equally accurate calibration curves for concentration determinations. In the case of the soybean agglutinin, which is a tetramer possessing one Man9 oligomannose-type chain per subunit, the protein concentration was determined from the Man calibration curve which agreed with that obtained from the molar extinction coefficient of the protein.

  6. Pregnancy-associated glycoprotein (PAG) concentration in plasma and milk samples for early pregnancy diagnosis in Lacaune dairy sheep.

    PubMed

    El Amiri, B; Sousa, N M; Alvarez Oxiley, A; Hadarbach, D; Beckers, J F

    2015-04-01

    In the present study, four RIA systems (RIA-1 to -4) based on two antisera raised against ovine pregnancy-associated glycoproteins (ovPAGs), combined with an ovine or a bovine PAG tracer were used to measure PAG concentrations in plasma and milk samples of dairy ewes. Blood and milk samples were collected on different days of gestation: 0, 18, 20, 22, 25, 28, 32, 42, and 49. From day 20 onward, the PAG in plasma could be detected in all pregnant ewes using the four RIA systems. By using milk, except for RIA-1, the other systems showed a sensitivity of 100% from day 28 of gestation onward. In plasma, PAG concentrations were higher in multiple than in single pregnancies, while no clear relationship was observed in milk. In conclusion, milk is a good alternative to plasma for early pregnancy diagnosis in sheep from day 28 to day 42. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. The third-generation P-glycoprotein inhibitor tariquidar may overcome bacterial multidrug resistance by increasing intracellular drug concentration.

    PubMed

    Leitner, I; Nemeth, J; Feurstein, T; Abrahim, A; Matzneller, P; Lagler, H; Erker, T; Langer, O; Zeitlinger, M

    2011-04-01

    The use of efflux pump inhibitors may be a powerful strategy to overcome transporter-mediated bacterial multidrug resistance. In the present study, we set out to investigate the potency of tariquidar, a third-generation P-glycoprotein inhibitor in clinical development, for overcoming bacterial resistance towards ciprofloxacin. Staphylococcus aureus 29213 (SA29213) and S. aureus 1199B (SA1199B), which overexpresses the multidrug transporter NorA, as well as Pseudomonas aeruginosa 27853 and Stenotrophomonas maltophilia BAA-85, which expresses SmeDEF, were exposed to ciprofloxacin in the presence and absence of tariquidar or, for comparative reasons, elacridar. Activity of both P-glycoprotein inhibitors was evaluated by determination of MICs and time-kill curves, and by quantification of uptake of ciprofloxacin into bacterial cells. Activity of tariquidar and elacridar was comparable for S. aureus strains, and both dose-dependently increased susceptibility towards ciprofloxacin. Highest effects were observed for SA1199B, where the addition of tariquidar resulted in a 10-fold reduction of the ciprofloxacin MIC, while no effect was observed for P. aeruginosa. For S. maltophilia, elacridar but not tariquidar improved susceptibility. Uptake of [14C]ciprofloxacin and modification of susceptibility showed significant correlations (r=0.89, P<0.0001). Tariquidar had no intrinsic activity against any strain tested. We conclude that tariquidar has potent inhibitory effect against certain bacterial efflux pumps in vitro. Their high activity at clinically achievable concentrations might yield this class of drugs promising for future applications in infectious diseases.

  8. Altered brain concentrations of citalopram and escitalopram in P-glycoprotein deficient mice after acute and chronic treatment.

    PubMed

    Karlsson, Louise; Carlsson, Björn; Hiemke, Christoph; Ahlner, Johan; Bengtsson, Finn; Schmitt, Ulrich; Kugelberg, Fredrik C

    2013-11-01

    According to both in vitro and in vivo data P-glycoprotein (P-gp) may restrict the uptake of several antidepressants into the brain, thus contributing to the poor success rate of current antidepressant therapies. The therapeutic activity of citalopram resides in the S-enantiomer, whereas the R-enantiomer is practically devoid of serotonin reuptake potency. To date, no in vivo data are available that address whether the enantiomers of citalopram and its metabolites are substrates of P-gp. P-gp knockout (abcb1ab (-/-)) and wild-type (abcb1ab (+/+)) mice underwent acute (single-dose) and chronic (two daily doses for 10 days) treatment with citalopram (10mg/kg) or escitalopram (5mg/kg) Serum and brain samples were collected 1-6h after the first or last i.p. injection for subsequent drug analysis by an enantioselective HPLC method. In brain, 3-fold higher concentrations of S- and R-citalopram, and its metabolites, were found in abcb1ab (-/-) mice than in abcb1ab (+/+) mice after both acute and chronic citalopram treatments. After escitalopram treatment, the S-citalopram brain concentration was 3-5 times higher in the knockout mice than in controls. The results provide novel evidence that the enantiomers of citalopram are substrates of P-gp. Possible clinical and toxicological implications of this finding need to be further elucidated. Copyright © 2013 Elsevier B.V. and ECNP. All rights reserved.

  9. Influence of ionic strength and beta2-glycoprotein I concentration on agglutination of like-charged phospholipid membranes.

    PubMed

    Perutková, Šárka; Frank-Bertoncelj, Mojca; Rozman, Blaž; Kralj-Iglič, Veronika; Iglič, Aleš

    2013-11-01

    The effect of ionic strength on adhesion between negatively charged giant unilamellar vesicles induced by beta2-glycoprotein I (β2-GPI) was studied experimentally and theoretically. Measuring the effective angle of contact between adhering vesicles indicated that the strength of adhesion between vesicles decreases with increasing ionic strength, and increases with concentration of β2-GPI. In the theoretical part we focused on the study of the average orientation of β2-GPI near the charged membrane and its role in mediating the attractive interactions between the vesicles. β2-GPI proteins were modelled as rods with internal distribution of electric charge. The predictions of Monte Carlo simulations show orthogonal orientation of some of the membrane attached β2-GPI in narrow gap between two vesicles. On the contrary, at larger distances between vesicles the proteins are parallelly attached to the membrane surface. A local minimum of the free energy corresponding to β2-GPI-mediated adhesion of two neighbouring vesicles was predicted. The strength of adhesion was confirmed to decrease at high ionic strength. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Glycoprotein synthesis

    DOEpatents

    Shultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

    2007-04-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  11. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA; Zhang, Zhiwen [San Diego, CA

    2007-05-15

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  12. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-02-27

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  13. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-16

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  14. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-02

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  15. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2005-08-09

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  16. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-07-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  17. Glycoprotein synthesis

    DOEpatents

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

    2009-07-14

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  18. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2006-10-31

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  19. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-08-28

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  20. Glycoproteins: Properties

    NASA Astrophysics Data System (ADS)

    Wittmann, Valentin

    This chapter focuses on the biological roles of the glycans contained in glycoproteins. Today we know there is no unifying function for the carbohydrates present in glycoproteins. They rather span the complete spectrum from being obviously unimportant to being crucial for the survival of an organism. In a crude scheme, their biological functions can be classified into two groups. On one hand, the carbohydrates can modify intrinsic properties of a protein by altering its size, charge, solubility, accessibility, structure, or dynamic properties. On the other hand, the glycans themselves may be specifically recognized by carbohydrate-binding proteins and thus participate in adhesion processes and signal transduction. A selection of some of these processes, that are well characterized, will be highlighted.

  1. Effects of Eimeria acervulina infection severity on growth performance, apparent ileal amino acid digestibility, and plasma concentrations of amino acids, carotenoids, and α1-acid glycoprotein in broilers.

    PubMed

    Rochell, S J; Parsons, C M; Dilger, R N

    2016-07-01

    An experiment was conducted to evaluate growth performance, apparent ileal digestibility (AID) of amino acids, and plasma concentrations of amino acids, carotenoids, and α1-acid glycoprotein, an acute-phase protein, in broilers inoculated with graded doses of E. acervulina oocysts. Ross 308 male broilers (400 total) were housed in battery cages from 1 to 21 d post-hatch and received common corn-soybean meal-based diets throughout the experiment. At 9 d post-hatch, birds were individually weighed and allotted to 4 treatment groups with 10 replicate cages of 10 birds per cage. At 15 d post-hatch, all birds were inoculated with 1 mL of distilled water that contained 0, 2.5 × 10(5), 5.0 × 10(5), or 1.0 × 10(6) sporulated E. acervulina oocysts. At 21 d, birds were euthanized for collection of blood and ileal digesta. Body weight gain and feed efficiency decreased linearly (P < 0.05) with increasing E. acervulina dose. With the exception of Trp and Gly, AID values decreased (P < 0.05) linearly or quadratically for all amino acids by an average of 2.6 percentage units for birds inoculated with 1.0 × 10(6) oocysts compared with uninfected birds. Infection with E. acervulina caused a quadratic decrease (P < 0.05) in plasma carotenoid concentrations. Plasma concentrations of Arg and Tyr decreased linearly (P < 0.05) with increasing E. acervulina inoculation dose and plasma Gln and Asn decreased quadratically (P < 0.01). Linear increases (P < 0.05) were observed for plasma Lys, Leu, Ile, Val, Pro, and Orn as E. acervulina inoculation dose increased. Plasma α1-acid glycoprotein of broilers was not influenced (P > 0.05) by E. acervulina infection. In conclusion, E. acervulina challenge adversely impacted growth performance, plasma carotenoids, and AID of amino acids in a dose-dependent manner. However, plasma amino acid responses to graded E. acervulina inoculation doses varied considerably among amino acids. Thus, these results indicated that alterations

  2. Mechanism of inhibition of P-glycoprotein mediated efflux by Pluronic P123/F127 block copolymers: relationship between copolymer concentration and inhibitory activity.

    PubMed

    Wei, Zhang; Yuan, Shi; Hao, Junguo; Fang, Xiaoling

    2013-02-01

    The aim of this study was to clarify the relationship between the concentration of Pluronic P123/F127 block copolymers and P-glycoprotein (P-gp) inhibitory potency. Modulation of multidrug resistance (MDR) by Pluronic P123/F127 was evaluated in P-gp over-expressing human breast cancer cell line MCF-7/ADR and its non-P-gp over-expressing counterpart MCF-7 cells. Four different probes (known as P-gp substrates) including rhodamine 123 (R-123), rhodamine 6G (R-6G), doxorubicin (DOX), and paclitaxel (PTX) were applied to investigate the impact of Pluronic P123/F127 copolymers with different concentrations on the intracellular accumulation of these probes. Additionally, the intracellular ATP and mitochondrial transmembrane potential in MCF-7/ADR cells were determined over a wide concentration range of Pluronic P123/F127. Furthermore, the endocytic mechanisms of Pluronic micelles were performed. It was suggested that P-gp substrate hydrophobicity and the concentration of P123/F127 copolymers had little impact on P-gp inhibitory activity of Pluronic P123/F127 itself. Intracellular ATP depletion was the main mechanism of Pluronic P123/F127 for P-gp inhibition. In vitro cytotoxicity study was also conducted in order to compare cytotoxic effect among different PTX formulations. It indicated that the IC50 of PTX-loaded Pluronic P123/F127 mixed micelles was 6.3-fold lower than free PTX and 2.3-fold lower than Taxol, respectively. Therefore, Pluronic P123/F127 polymeric micelles could be considered a promising drug delivery system to overcome MDR in cancer therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Circulating concentrations of pregnancy associated glycoproteins (PAGs) are associated with embryo/fetal survival but not ovulatory follicle size in suckled beef cows

    USDA-ARS?s Scientific Manuscript database

    GnRH-induced ovulation of small dominant follicles resulted in increased late embryonic/fetal mortality around the time of embryo-uterine attachment. Pregnancy associated glycoproteins (PAGs) are secreted by binucleated trophoblast cells into the maternal circulation and have been used to monitor t...

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

    PubMed

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

    1992-08-01

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

  5. An endoglycosidase-assisted LC-MS/MS-based strategy for the analysis of site-specific core-fucosylation of low-concentrated glycoproteins in human serum using prostate-specific antigen (PSA) as example.

    PubMed

    Lang, Robert; Leinenbach, Andreas; Karl, Johann; Swiatek-de Lange, Magdalena; Kobold, Uwe; Vogeser, Michael

    2018-05-01

    Recently, site-specific fucosylation of glycoproteins has attracted attention as it can be associated with several types of cancers including prostate cancer. However, individual glycoproteins, which might serve as potential cancer markers, often are very low-concentrated in complex serum matrices and distinct glycan structures are hard to detect by immunoassays. Here, we present a mass spectrometry-based strategy for the simultaneous analysis of core-fucosylated and total prostate-specific antigen (PSA) in human serum in the low ng/ml concentration range. Sample preparation comprised an immunoaffinity capture step to enrich total PSA from human serum using anti-PSA antibody coated magnetic beads followed by consecutive two-step on-bead partial deglycosylation with endoglycosidase F3 and tryptic digestion prior to LC-MS/MS analysis. The method was shown to be linear from 0.5 to 60 ng/ml total PSA concentrations and allows the simultaneous quantification of core-fucosylated PSA down to 1 ng/ml and total PSA lower than 0.5 ng/ml. The imprecision of the method over two days ranged from 9.7-23.2% for core-fucosylated PSA and 10.3-18.3% for total PSA depending on the PSA level. The feasibility of the method in native sera was shown using three human specimens. To our knowledge, this is the first MS-based method for quantification of core-fucosylated PSA in the low ng/ml concentration range in human serum. This method could be used in large patient cohorts as core-fucosylated PSA may be a diagnostic biomarker for the differentiation of prostate cancer and other prostatic diseases, such as benign prostatic hyperplasia (BPH). Furthermore, the described strategy could be used to monitor potential changes in site-specific core-fucosylation of other low-concentrated glycoproteins, which could serve as more specific markers ("marker refinement") in cancer research. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy-Associated Glycoproteins 1 and 2 in Foetal Fluids.

    PubMed

    Mur-Novales, R; López-Gatius, F; Serrano-Pérez, B; García-Ispierto, I; Darwich, L; Cabezón, O; de Sousa, N M; Beckers, J F; Almería, S

    2016-04-01

    Plasma concentrations of PAG-1 are used for pregnancy diagnosis and as a marker of placental/foetal well-being, while those of PAG-2 may be an indicator of abortion risk in Neospora caninum-infected cows. Studies have shown that N. caninum infection modifies PAG-1 and PAG-2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG-1, PAG-2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG-2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non-infected foetuses. In infected foetuses, significantly higher concentrations of PAG-1 (p < 0.001) and PAG-2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non-infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG-1 and PAG-2 levels and pH values than fluids from non-infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH. © 2016 Blackwell Verlag GmbH.

  7. HIV-1 envelope glycoprotein

    SciTech Connect

    Caulfield, Michael; Cupo, Albert; Dean, Hansi

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  8. N-Glycoprotein SRMAtlas

    PubMed Central

    Hüttenhain, Ruth; Surinova, Silvia; Ossola, Reto; Sun, Zhi; Campbell, David; Cerciello, Ferdinando; Schiess, Ralph; Bausch-Fluck, Damaris; Rosenberger, George; Chen, Jingchung; Rinner, Oliver; Kusebauch, Ulrike; Hajdúch, Marián; Moritz, Robert L.; Wollscheid, Bernd; Aebersold, Ruedi

    2013-01-01

    Protein biomarkers have the potential to transform medicine as they are clinically used to diagnose diseases, stratify patients, and follow disease states. Even though a large number of potential biomarkers have been proposed over the past few years, almost none of them have been implemented so far in the clinic. One of the reasons for this limited success is the lack of technologies to validate proposed biomarker candidates in larger patient cohorts. This limitation could be alleviated by the use of antibody-independent validation methods such as selected reaction monitoring (SRM). Similar to measurements based on affinity reagents, SRM-based targeted mass spectrometry also requires the generation of definitive assays for each targeted analyte. Here, we present a library of SRM assays for 5568 N-glycosites enabling the multiplexed evaluation of clinically relevant N-glycoproteins as biomarker candidates. We demonstrate that this resource can be utilized to select SRM assay sets for cancer-associated N-glycoproteins for their subsequent multiplexed and consistent quantification in 120 human plasma samples. We show that N-glycoproteins spanning 5 orders of magnitude in abundance can be quantified and that previously reported abundance differences in various cancer types can be recapitulated. Together, the established N-glycoprotein SRMAtlas resource facilitates parallel, efficient, consistent, and sensitive evaluation of proposed biomarker candidates in large clinical sample cohorts. PMID:23408683

  9. Enhancement of human monocyte phagocytic function by alpha 2HS glycoprotein.

    PubMed Central

    Lewis, J G; André, C M

    1981-01-01

    Human alpha 2HS glycoprotein was isolated from normal adult serum and its effect on monocyte phagocytic function (in vitro) was investigated. The results demonstrate that alpha 2HS glycoprotein enhances the ability of peripheral blood monocytes to take up radiolabelled latex particles compared to control cells. Enhanced uptake required the simultaneous presence of cells, substrate and alpha 2HS glycoprotein. This uptake was dependent on the concentration of alpha 2HS glycoprotein offering the possibility that reduced levels of this protein, as in malignant and inflammatory disease, may result in decreased phagocytic function. PMID:7203533

  10. Envelope Glycoprotein of Arenaviruses

    PubMed Central

    Burri, Dominique J.; da Palma, Joel Ramos; Kunz, Stefan; Pasquato, Antonella

    2012-01-01

    Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses. PMID:23202458

  11. Salivary Mucin 19 Glycoproteins

    PubMed Central

    Culp, David J.; Robinson, Bently; Cash, Melanie N.; Bhattacharyya, Indraneel; Stewart, Carol; Cuadra-Saenz, Giancarlo

    2015-01-01

    Saliva functions in innate immunity of the oral cavity, protecting against demineralization of teeth (i.e. dental caries), a highly prevalent infectious disease associated with Streptococcus mutans, a pathogen also linked to endocarditis and atheromatous plaques. Gel-forming mucins are a major constituent of saliva. Because Muc19 is the dominant salivary gel-forming mucin in mice, we studied Muc19−/− mice for changes in innate immune functions of saliva in interactions with S. mutans. When challenged with S. mutans and a cariogenic diet, total smooth and sulcal surface lesions are more than 2- and 1.6-fold higher in Muc19−/− mice compared with wild type, whereas the severity of lesions are up to 6- and 10-fold higher, respectively. Furthermore, the oral microbiota of Muc19−/− mice display higher levels of indigenous streptococci. Results emphasize the importance of a single salivary constituent in the innate immune functions of saliva. In vitro studies of S. mutans and Muc19 interactions (i.e. adherence, aggregation, and biofilm formation) demonstrate Muc19 poorly aggregates S. mutans. Nonetheless, aggregation is enhanced upon adding Muc19 to saliva from Muc19−/− mice, indicating Muc19 assists in bacterial clearance through formation of heterotypic complexes with salivary constituents that bind S. mutans, thus representing a novel innate immune function for salivary gel-forming mucins. In humans, expression of salivary MUC19 is unclear. We find MUC19 transcripts in salivary glands of seven subjects and demonstrate MUC19 glycoproteins in glandular mucous cells and saliva. Similarities and differences between mice and humans in the expression and functions of salivary gel-forming mucins are discussed. PMID:25512380

  12. [Characterization of stability of polypeptides and glycoproteins by capillary electrophoresis].

    PubMed

    Wang, Yong; Gao, Peifang; Zhao, Xinying; Qu, Feng

    2013-06-01

    A capillary electrophoresis (CE) method was developed for the stability characterization of polypeptides and glycoproteins. Angiotensin II (Ang II), phytagglutinin (PHA), bovine thrombin (B-Thr), human thrombin (H-Thr) and horseradish peroxidase (HRP) were used as polypeptide and glycoprotein mode molecules. The parameters affecting the analysis efficiency for Ang II, such as sample concentration, running buffer, pH and ionic strength of sample solution were optimized, as for the glycoprotein, capillary conditions, charge state of sample, running buffer and applied voltage were optimized. It showed that the Ang II was stable when kept in borate buffer (0.02 mol/L pH 7.4) at 4 degrees C for 48 h. The four glycoproteins were quite stable in borate buffer (0.2 mol/L pH 7.4) at 20, 4, -20 degrees C for 48 h, and also kept stable at - 20 degrees C when deposited over one week and less than four weeks. HRP was the only one that kept stable when deposited over two weeks and less than four weeks. This method is effective, rapid, simple and low-cost and can be widely used for the stability characterization of polypeptides and glycoproteins.

  13. Proteomic Screening of Glycoproteins in Human Plasma for Frailty Biomarkers

    PubMed Central

    Shamsi, Kinza S.; Pierce, Anson; Ashton, Aaron S.; Halade, Dipti G.; Richardson, Arlan

    2012-01-01

    The application of proteomics methodology for analyzing human blood samples is of increasing importance as a noninvasive method for understanding, detecting, and monitoring disease. In particular, glycoproteomic analysis may be useful in the study of age-related diseases and syndromes, such as frailty. This study demonstrates the use of methodology for isolating plasma glycoproteins using lectins, comparing the glycoproteome by frailty status using two-dimensional polyacrylamide gel electrophoresis and identifying glycoproteins using mass spectrometry. In a pilot study, we found seven glycoproteins to differ by at least twofold in prefrail compared with nonfrail older adults, including haptoglobin, transferrin, and fibrinogen, consistent with known inflammatory and hematologic changes associated with frailty. Enzyme-linked immunosorbent assay analysis found that plasma transferrin concentration was increased in frail and prefrail older adults compared with nonfrail, confirming our proteomic findings. This work provides evidence for using a reproducible methodology for conducting clinical proteomic comparative studies of age-related diseases. PMID:22219522

  14. Interaction of human erythrocyte MN glycoprotein with rabbit IgG immunoglobulins.

    PubMed

    Białkowska, H; Morawiecki, A

    1978-01-01

    The interaction of rabbit non-specific IgG and human erythrocyte glycoprotein was investigated using the solvent perturbation difference spectroscopy method. This interaction manifested itself by decreasing accessibility of chromophores to perturbants. Masking of the chromophores was abolished by low detergent concentrations and by changes of native IgG structure by 3 M urea. The sialic acid residues of the glycoprotein were necessary for this effect but probably not due to simple electrostatic interactions. It seems that the IgG-glycoprotein interaction requires intact both--the IgG molecule structure and the structure of the glycoprotein micelle. Interaction of this kind was not observed between glycoprotein and some other proteins as bovine serum albumin, alpha-chymotrypsynogen and human IgA.

  15. Elution of tightly bound solutes from concanavalin A Sepharose Factors affecting the desorption of cottonmouth venom glycoproteins

    PubMed Central

    Soper, Anastasiya S.; Aird, Steven D.

    2007-01-01

    Some glycoproteins bind so tightly to concanavalin A Sepharose that common desorption techniques are ineffective, so a systematic exploration of factors affecting desorption of cottonmouth venom glycoproteins was undertaken. Glycoprotein desorption is greatly improved by introducing up to four pauses of 5-10 min duration into the elution step. Eluent concentrations above 250 mM methylglucoside or 500 mM methyl-mannoside reduced glycoprotein desorption. Eluent NaCl diminished glycoprotein desorption. Most venom glycoproteins desorb more readily as pH diminishes from 6.0 to 4.0, but phosphodiesterase shows the opposite pattern. Eluents recommended by the supplier for desorbing solutes or for column cleaning were ineffectual. PMID:17449042

  16. Purification of a recombinant human respiratory syncytial virus chimeric glycoprotein using reversed-phase chromatography and protein refolding in guanidine hydrochloride.

    PubMed

    Wells, P A; Garlick, R L; Lyle, S B; Tuls, J L; Poorman, R A; Brideau, R J; Wathen, M W

    1994-08-01

    FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu(2+)-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.

  17. P-Glycoprotein-ATPase Modulation: The Molecular Mechanisms

    PubMed Central

    Li-Blatter, Xiaochun; Beck, Andreas; Seelig, Anna

    2012-01-01

    P-glycoprotein-ATPase is an efflux transporter of broad specificity that counteracts passive allocrit influx. Understanding the rate of allocrit transport therefore matters. Generally, the rates of allocrit transport and ATP hydrolysis decrease exponentially with increasing allocrit affinity to the transporter. Here we report unexpectedly strong down-modulation of the P-glycoprotein-ATPase by certain detergents. To elucidate the underlying mechanism, we chose 34 electrically neutral and cationic detergents with different hydrophobic and hydrophilic characteristics. Measurement of the P-glycoprotein-ATPase activity as a function of concentration showed that seven detergents activated the ATPase as expected, whereas 27 closely related detergents reduced it significantly. Assessment of the free energy of detergent partitioning into the lipid membrane and the free energy of detergent binding from the membrane to the transporter revealed that the ratio, q, of the two free energies of binding determined the rate of ATP hydrolysis. Neutral (cationic) detergents with a ratio of q = 2.7 ± 0.2 (q > 3) followed the aforementioned exponential dependence. Small deviations from the optimal ratio strongly reduced the rates of ATP hydrolysis and flopping, respectively, whereas larger deviations led to an absence of interaction with the transporter. P-glycoprotein-ATPase inhibition due to membrane disordering by detergents could be fully excluded using 2H-NMR-spectroscopy. Similar principles apply to modulating drugs. PMID:22455921

  18. Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts.

    PubMed

    Gitz, Eelo; Koekman, Cornelis A; van den Heuvel, Dave J; Deckmyn, Hans; Akkerman, Jan W; Gerritsen, Hans C; Urbanus, Rolf T

    2012-12-01

    Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-D-glucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future.

  19. Assembly of a functional HCV glycoprotein heterodimer.

    PubMed

    Lavie, Muriel; Goffard, Anne; Dubuisson, Jean

    2007-07-01

    The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have remained limited because of the lack of a robust cell culture system to amplify this virus. A few years ago, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins. This model allowed the characterization of functional E1E2 envelope glycoproteins. The data obtained can now be confirmed with the help of a newly developed cell-culture system that allows efficient amplification of HCV (HCVcc). Here, we present the recent data that have been accumulated on the assembly of the functional HCV glycoprotein heterodimer.

  20. Dynamics of the Hydration Water of Antifreeze Glycoproteins.

    PubMed

    Groot, Carien C M; Meister, Konrad; DeVries, Arthur L; Bakker, Huib J

    2016-12-01

    Antifreeze glycoproteins (AFGPs) are unique proteins that inhibit the growth of ice by a mechanism that is still unclear. We study the dynamics of water in aqueous solutions of small and large isoforms of AFGPs using polarization-resolved femtosecond infrared spectroscopy. We find that a fraction of the water molecules is strongly slowed down by the interaction with the antifreeze glycoprotein surface. The fraction of slow water molecules scales with the size and concentration of AFGP, and is similar to the fraction of slow water observed for nonantifreeze proteins, both at room temperature and close to biologically relevant working temperatures. We observe that inhibiting AFGP antifreeze activity using borate buffer induces no changes in the dynamics of water hydrating the AFGP. Our findings support a mechanism in which the sugar unit of AFGP forms the active ice-binding site.

  1. Histidine-rich glycoprotein and idiopathic pulmonary fibrosis.

    PubMed

    Ernst, G; Dantas, E; Sabatté, J; Caro, F; Salvado, A; Grynblat, P; Geffner, J

    2015-12-01

    Histidine-rich glycoprotein (HRG) is an enigmatic glycoprotein able to interact with a variety of ligands such as IgG, complement components, heparan sulfate, thrombospondin, fibrinogen and plasminogen. HRG is present at high concentrations in plasma and there is evidence indicating that it is able to modulate the course of biological processes such as angiogenesis, fibroblast proliferation, complement activation, coagulation and fibrinolysis. Because these processes are involved in the pathogeneses of lung fibrosis we here analyzed a possible link between HRG and idiopathic pulmonary fibrosis (IPF). We found that plasma concentrations of HRG are significantly diminished in IPF patients compared to healthy subjects. Moreover, we found a positive correlation between HRG plasma levels and forced vital capacity (FVC) values, suggesting that plasma concentration of HRG would be a useful indicator of disease activity in IPF. HRG has been described as a negative acute phase reactant able to accumulate at sites of tissue injury. Hence, we also measured the concentrations of HRG in BAL samples from IPF patients. We found that the concentrations of HRG in samples from IPF patients were significantly higher compared to controls, suggesting that the reduced concentration of HRG in plasma from IPF patients could be due, at least in part, to an enhanced uptake of this protein in the lung. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. A double responsive smart upconversion fluorescence sensing material for glycoprotein.

    PubMed

    Guo, Ting; Deng, Qiliang; Fang, Guozhen; Yun, Yaguang; Hu, Yongjin; Wang, Shuo

    2016-11-15

    A novel strategy was developed to prepare double responsive smart upconversion fluorescence material for highly specific enrichment and sensing of glycoprotein. The novel double responsive smart sensing material was synthesized by choosing Horse radish peroxidase (HRP) as modal protein, the grapheme oxide (GO) as support material, upconversion nanoparticles (UCNPs) as fluorescence signal reporter, N-isopropyl acrylamide (NIPAAM) and 4-vinylphenylboronic acid (VPBA) as functional monomers. The structure and component of smart sensing material was investigated by transmission electron microscopy (TEM), Scanning electron microscopy (SEM), X-ray photoelectron spectroscopic (XPS) and Fourier transform infrared (FTIR), respectively. These results illustrated the smart sensing material was prepared successfully. The recognition characterizations of smart sensing material were evaluated, and results showed that the fluorescence intensity of smart sensing material was reduced gradually, as the concentration of protein increased, and the smart sensing material showed selective recognition for HRP among other proteins. Furthermore, the recognition ability of the smart sensing material for glycoprotein was regulated by controlling the pH value and temperature. Therefore, this strategy opens up new way to construct smart material for detection of glycoprotein. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Human cytomegalovirus: glycoproteins associated with virions and dense bodies.

    PubMed Central

    Stinski, M F

    1976-01-01

    The glycoproteins associated with the membranes of cytomegalovirions and dense bodies were characterized by their relative mobility, percentage of glucosamine incorporation, and molecular weight. Eight glycopolypeptides were repeatedly detectable. Three glycopolypeptides of higher molecular weight with low levels of glucosamine incorporation were occasionally detectable. These latter glycopolypeptides may be precursors or aggregates of the glycopolypeptides with lower molecular weights. The glycoproteins associated with the membranes were on the surface, as determined by iodination with 125I of virions and dense bodies partially purified in gradients of D-sorbitol. Velocity centrifugation in linear gradients of D-sorbitol was used to obtain concentrated and partially purified preparations of infectious cytomegalovirus. Viral infectivity and the membranes of cytomegalovirions and dense bodies were stable in gradients of sorbitol, but cellular contaminants were not completely removed. Additional centrifugation in CsCl separated both cellular contaminants and viral nucleocapsids from virions and dense bodies. Many dense bodies, which are considered to be aberrant forms of cytomegalovirus, had the same size, sedimentation properties, and density as virions. Consequently, they were not separable from virions by various centrifugation techniques. Electron microscopy demonstrated that purified virions and dense bodies were qualitatively free of extraneous material and that each dense body was bounded by a membrane, as evidenced by its double-tract appearance. Antisera to a preparation of purified virions and dense bodies, or to their glycoproteins, contained antibodies that neutralized viral infectivity and reacted with antigens in cells infected with cytomegalovirus. However, these same antisera did not contain antibodies that reacted with uninfected cells. The glycoproteins associated with the membranes of cytomegalovirions and dense bodies are considered to be

  4. Intestinal P-glycoprotein inhibitors, benzoxanthone analogues.

    PubMed

    Chae, Song Wha; Lee, Jaeok; Park, Jung Hyun; Kwon, Youngjoo; Na, Younghwa; Lee, Hwa Jeong

    2018-02-01

    The inhibitors of P-glycoprotein (P-gp) which limits an access of exogenous compounds in the luminal membrane of the intestine have been studied to enhance the intestinal P-gp-mediated absorption of anticancer drugs. Inhibition of the efflux pump by synthesized benzoxanthone derivatives was investigated in vitro and in vivo. MCF-7/ADR cell line was used for cytotoxicity assay and [ 3 H]-daunomycin (DNM) accumulation/efflux study. Eight benzoxanthone analogues were tested for their effects on DNM cytotoxicity. Among them, three analogues were selected for the accumulation/efflux and P-gp ATPase studies. Paclitaxel (PTX), a P-gp substrate anticancer drug, was orally administered to rats with/without compound 1 (8,10-bis(thiiran-2-ylmethoxy)-7H-benzo[c]xanthen-7-one). The pharmacokinetic parameters of PTX in the presence/absence of compound 1 were evaluated from the plasma concentration-time profiles. Compound 1 increased the DNA accumulation to 6.5-fold and decreased the DNM efflux to approximately 1/2 in the overexpressing P-gp cell line. Relative bioavailability (RB) of PTX in rats was significantly increased up to 3.2-fold by compound 1 (0.5 or 2 mg/kg). Benzoxanthone analogue, compound 1 is strongly suggested to be a promising inhibitor of P-gp to improve an oral absorption of compounds for cancer therapy. © 2017 Royal Pharmaceutical Society.

  5. Genetics Home Reference: glycoprotein VI deficiency

    MedlinePlus

    ... protein called glycoprotein VI (GPVI). This protein is embedded in the outer membrane of blood cell fragments ... erythematosus (SLE). Autoimmune disorders occur when the immune system malfunctions and attacks the body's own cells and ...

  6. Platelet Glycoprotein Ib-IX and Malignancy

    DTIC Science & Technology

    2010-09-01

    whether adjunct anti-GP Ib-IX therapy could benefit the breast cancer patient with malignant disease. Body Below we list the 3 Specific Aims from our...Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D...AND SUBTITLE Platelet Glycoprotein Ib-IX and Malignancy 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-08-1-0576 5c

  7. Bioactivity of proteins isolated from Lactobacillus plantarum L67 treated with Zanthoxylum piperitum DC glycoprotein.

    PubMed

    Song, S; Oh, S; Lim, K-T

    2015-06-01

    Lactobacilli in the human gastrointestinal tract have beneficial effects on the health of their host. To enhance these effects, the bioactivity of lactobacilli can be fortified through exogenous dietary or pharmacological agents, such as glycoproteins. To elucidate the inductive effect of Zanthoxylum piperitum DC (ZPDC) glycoprotein on Lactobacillus plantarum L67, we evaluated the radical-scavenging activity, anti-oxidative enzymes (SOD, GPx and CAT), growth rate, ATPase activity and β-galactosidase activity of this strain. When Lact. plantarum L67 was treated with ZPDC glycoprotein at different concentrations, the intensities of a few SDS-PAGE bands were slightly changed. The amount of a 23 kDa protein was increased upon treatment with increasing concentrations of ZPDC glycoprotein. The results of this study indicate that the radical-scavenging activity for O2(-) and OH¯, but not for the DPPH radical, increased in a concentration-dependent manner after treatment with ZPDC glycoprotein. The activation of anti-oxidative enzymes (SOD, GPx and CAT), growth rate and β-galactosidase activity also increased in a concentration-dependent manner in response to ZPDC glycoprotein treatment, whereas ATPase activity was decreased. In summary, ZPDC glycoprotein stimulated an increase in the bioactivity of Lact. plantarum L67. Significance and impact of the study: This study demonstrated that Lactobacillus plantarum L67 possesses anti-oxidative activity. This strain of lactic bacteria has been known to have various probiotic uses, such as yogurt starters and dietary additional supplements. We found, through this experiment, that the protein has a strong anti-oxidative character, and the activity can be enhanced by treatment with Zanthoxylum piperitum DC (ZPDC) glycoprotein. This study may be application of Lact. plantarum L67 treated by ZPDC glycoprotein in yogurt fermentation. It could be one of the avenues of minimizing yogurt postacidification during storage. In addition

  8. Elevated alpha-1-acid glycoprotein reduces the volume of distribution and systemic clearance of saquinavir.

    PubMed

    Holladay, J W; Dewey, M J; Michniak, B B; Wiltshire, H; Halberg, D L; Weigl, P; Liang, Z; Halifax, K; Lindup, W E; Back, D J

    2001-03-01

    The purpose of this study was to characterize the relationship between plasma protein binding and the pharmacokinetic disposition of saquinavir during a normal and elevated alpha-1-acid glycoprotein condition. The extent of plasma binding of [14C]saquinavir to human plasma, human albumin, and human alpha-1-acid glycoprotein was also assessed. Transgenic mice, which overexpress plasma alpha-1-acid glycoprotein, and control mice were given a single intravenous injection of saquinavir (10 mg/kg) and plasma samples were harvested as a function of time. The extent of [14C]saquinavir (0.5-30 microg/ml) plasma protein binding in each group of mice was determined by ultrafiltration. Plasma saquinavir concentrations from in vivo administration were determined by high performance liquid chromatography with tandem mass spectrometry. Saquinavir binding in human plasma and control mouse plasma was similar (approximately 3% unbound). In contrast, the extent of binding was significantly increased in transgenic mice (1.5% unbound). Furthermore, saquinavir was more extensively bound to alpha-1-acid glycoprotein than to albumin (2.1 versus 11.5% unbound). The systemic clearance and volume of distribution of saquinavir were significantly reduced in transgenic mice compared with control mice. The results of this study show that alpha-1-acid glycoprotein is the predominant plasma protein to which saquinavir binds. In addition, elevations in plasma alpha-1-acid glycoprotein considerably alter the pharmacokinetic disposition of saquinavir. This is consistent with the observations that systemic exposure to saquinavir in human immunodeficiency virus patients is greater than that in healthy volunteers and that alpha-1-acid glycoprotein levels increase with the degree of HIV infection.

  9. Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein.

    PubMed

    Albani, J R

    2006-11-06

    Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels.

  10. Improved platelet survival after cold storage by prevention of glycoprotein Ibα clustering in lipid rafts

    PubMed Central

    Gitz, Eelo; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan W.; Gerritsen, Hans C.; Urbanus, Rolf T.

    2012-01-01

    Background Storing platelets for transfusion at room temperature increases the risk of microbial infection and decreases platelet functionality, leading to out-date discard rates of up to 20%. Cold storage may be a better alternative, but this treatment leads to rapid platelet clearance after transfusion, initiated by changes in glycoprotein Ibα, the receptor for von Willebrand factor. Design and Methods: We examined the change in glycoprotein Ibα distribution using Förster resonance energy transfer by time-gated fluorescence lifetime imaging microscopy. Results Cold storage induced deglycosylation of glycoprotein Ibα ectodomain, exposing N-acetyl-Dglucosamine residues, which sequestered with GM1 gangliosides in lipid rafts. Raft-associated glycoprotein Ibα formed clusters upon binding of 14-3-3ζ adaptor proteins to its cytoplasmic tail, a process accompanied by mitochondrial injury and phosphatidyl serine exposure. Cold storage left glycoprotein Ibα surface expression unchanged and although glycoprotein V decreased, the fall did not affect glycoprotein Ibα clustering. Prevention of glycoprotein Ibα clustering by blockade of deglycosylation and 14-3-3ζ translocation increased the survival of cold-stored platelets to above the levels of platelets stored at room temperature without compromising hemostatic functions. Conclusions We conclude that glycoprotein Ibα translocates to lipid rafts upon cold-induced deglycosylation and forms clusters by associating with 14-3-3ζ. Interference with these steps provides a means to enable cold storage of platelet concentrates in the near future. PMID:22733027

  11. Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: limitations for integral membrane glycoproteins

    PubMed Central

    Lajko, Michelle; Haddad, Alexander F.; Robinson, Carolyn A.; Connolly, Sarah A.

    2015-01-01

    Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane. PMID:25958131

  12. Schisandrin B--a novel inhibitor of P-glycoprotein.

    PubMed

    Qiangrong, Pan; Wang, Tao; Lu, Qinghua; Hu, Xun

    2005-09-23

    P-glycoprotein-mediated drug efflux is one of the major causes of the cancer multidrug resistance (MDR). Inhibition of P-glycoprotein could reverse cancer MDR. Here, we show that schisandrin B, a naturally occurring compound from Schisandra chinensis (Turcz.) Baill, bears strong potency to inhibit P-glycoprotein. Schisandrin B reversed the drug resistance of four MDR cell lines characterized with overexpression of P-glycoprotein and fully restored the intracellular drug accumulation by interacting with P-glycoprotein. Schisandrin B has a core structure of dibenzocyclooctadiene, representing a novel P-glycoprotein inhibitor. To our best knowledge, the role of schisandrin B to inhibit P-glycoprotein has not been reported.

  13. Intracellular lectins are involved in quality control of glycoproteins

    PubMed Central

    YAMAMOTO, Kazuo

    2014-01-01

    Glycoprotein quality control is categorized into three kinds of reactions; the folding of nascent glycoproteins, ER-associated degradation of misfolded or unassembled glycoproteins, and transport and sorting of correctly folded glycoproteins. In all three processes, N-glycans on the glycoproteins are used as tags that are recognized by intracellular lectins. We analyzed the functions of these intracellular lectins and their sugar-binding specificities. The results clearly showed that the A, B, and C-arms of high mannose-type glycans participate in the folding, transport and sorting, and degradation, respectively, of newly synthesized peptides. After correctly folded glycoproteins are transported to the Golgi apparatus, N-glycans are trimmed into Man3GlcNAc2 and then rebuilt into various complex-type glycans in the Golgi, resulting in the addition of diverse sugar structures that allow glycoproteins to play various roles outside of the cells. PMID:24522156

  14. The complexity of glycoprotein-derived glycans

    PubMed Central

    VLIEGENTHART, Johannes F. G.

    2017-01-01

    A brief review is presented of our studies on the structure of glycoprotein-derived glycans. The emphasis is on the introduction of high-resolution 1H-NMR spectroscopy for the unambiguous determination of primary structures. For this purpose, we developed the structural reporter group concept. Structural reporters are defined as unique markers of structural elements in the NMR spectra. Application of this concept led to the discovery of numerous new structures. Furthermore, a number of structures presented in the literature could be corrected. The results are relevant for insight in the various steps in glycan metabolism in health and disease, for the function and mode of action of glycans in vivo and for the interpretation of structural information obtained through other techniques. The strength of the approach is further shown for several highly complex glycoproteins, carrying very heterogeneous and complicated glycans. PMID:28190870

  15. Sav1866 from Staphylococcus aureus and P-glycoprotein: similarities and differences in ATPase activity assessed with detergents as allocrites.

    PubMed

    Beck, Andreas; Aänismaa, Päivi; Li-Blatter, Xiaochun; Dawson, Roger; Locher, Kaspar; Seelig, Anna

    2013-05-14

    The ATP-binding cassette exporters Sav1866 from Staphylococcus aureus and P-glycoprotein are known to share a certain sequence similarity and disposition for cationic allocrites. Conversely, the two ATPases react very differently to neutral detergents that have previously been shown to be inhibitory allocrites for P-glycoprotein. To gain insight into the functional differences of the two proteins, we compared their basal and detergent-stimulated ATPase activity. P-Glycoprotein was investigated in NIH-MDR1-G185 plasma membrane vesicles and Sav1866 in lipid vesicles exhibiting a membrane packing density and a surface potential similar to those of the plasma membrane vesicles. Under basal conditions, Sav1866 revealed a lower catalytic efficiency and concomitantly a more pronounced sodium chloride and pH dependence than P-glycoprotein. As expected, the cationic allocrites (alkyltrimethylammonium chlorides) induced similar bell-shaped activity curves as a function of concentration for both exporters, suggesting stimulation upon binding of the first and inhibition upon binding of the second allocrite molecule. However, the neutral allocrites (n-alkyl-β-d-maltosides and n-ethylene glycol monododecyl ethers) reduced P-glycoprotein's ATPase activity at concentrations well below their critical micelle concentration (CMC) but strongly enhanced Sav1866's ATPase activity even at concentrations above their CMC. The lack of ATPase inhibition at high concentrations of neutral of detergents could be explained by their comparatively low binding affinity for the transmembrane domains of Sav1866, which seems to prevent binding of a second inhibitory molecule. The high ATPase activity in the presence of hydrophobic, long chain detergents moreover revealed that Sav1866, despite its lower basal catalytic efficiency, is a more efficient floppase for lipidlike amphiphiles than P-glycoprotein.

  16. Platelet Glycoprotein lb-1X and Malignancy

    DTIC Science & Technology

    2010-09-01

    therapy could benefit the breast cancer patient with malignant disease. Body Below we list the 3 Specific Aims from our original submission (blue font...Muller WJ and Pollard JW. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human...08-1-0576 TITLE: Platelet Glycoprotein lb-1X and Malignancy PRINCIPAL INVESTIGATOR: Dr. Jerry Ware

  17. Characterization of Glycoproteins in Equine Herpesvirus-1

    PubMed Central

    MAHMOUD, Hassan Y. A. H.; ANDOH, Kiyohiko; HATTORI, Shiho; TERADA, Yutaka; NOGUCHI, Keita; SHIMODA, Hiroshi; MAEDA, Ken

    2013-01-01

    ABSTRACT In this study, we attempted to express twelve glycoproteins of equine herpesvirus-1 (EHV-1) in 293T cells and to characterize these using monoclonal antibodies (MAbs) and horse sera against EHV-1. Expression of glycoprotein B (gB), gC, gD, gG, gI and gp2 was recognized by immunoblot analysis using horse sera, but that of gE, gH, gK, gL, gM and gN was not. Four MAbs recognized gB, four recognized gC and one recognized gp2. Two MAbs against gB cross-reacted with EHV-4. Interestingly, coexpression of gE and gI and gM and gN enhanced their antigenicity. Furthermore, immunoblot analysis of gp2 showed that different molecular masses of gp2 were recognized by the MAb against gp2 and horse sera against EHV-1. In this study, it was demonstrated that at least six glycoproteins were immunogenic to horses, and coexpression of gE and gI and gM and gN was important for enhancement of antigenicity. PMID:23748975

  18. Effect of P-glycoprotein modulators on the human extraneuronal monoamine transporter.

    PubMed

    Martel, F; Keating, E; Azevedo, I

    2001-06-22

    The aim of this work was to investigate the effect of P-glycoprotein modulators on human extraneuronal monoamine transporter (EMT)-mediated transport. The experiments were performed using a cell line from human embryonic kidney (HEK293 cells) stably transfected with pcDNA3hEMT (293(hEMT)), or with pcDNA3 alone (293(control)). Of the P-glycoprotein modulators tested, rhodamine123, verapamil and daunomycin concentration-dependently inhibited EMT-mediated uptake of [3H]1-methyl-4-phenylpyridinium ([3H]MPP(+)). The corresponding IC(50)'s were found to be 3.6, 37 and 130 microM, respectively. By contrast, vinblastine, digitoxin and cyclosporine A were devoid of effect. The endogenous organic cation tyramine, but not choline, inhibited EMT-mediated transport (IC(50) of 468 microM). Moreover, L-arginine and L-histidine (up to 1 mM) did not affect [3H]MPP(+) uptake. Finally, MPP(+) and tyramine trans-stimulated [3H]MPP(+) uptake, but rhodamine123 had no effect, and verapamil and daunomycin trans-inhibited [3H]MPP(+) uptake. In conclusion, this study shows that several cationic modulators of P-glycoprotein inhibit EMT-mediated transport. As a consequence, the interaction of P-glycoprotein modulators with EMT must be taken into account, and the consequences of this interaction must not be forgotten when using such drugs in vivo.

  19. Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

    PubMed Central

    Wang, Lai-Xi; Amin, Mohammed N.

    2014-01-01

    Summary Glycoproteins are an important class of biomolecules involved in a number of biological recognition processes. However, natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ in the structures of the pendent glycans, which are difficult to separate in pure glycoforms. As a result, synthetic homogeneous glycopeptides and glycoproteins have become indispensable probes for detailed structural and functional studies. A number of elegant chemical and biological strategies have been developed for synthetic construction of tailor-made, full-size glycoproteins to address specific biological problems. In this review, we highlight recent advances in chemical and chemoenzymatic synthesis of homogeneous glycoproteins. Selected examples are given to demonstrate the applications of tailor-made, glycan-defined glycoproteins for deciphering glycosylation functions. PMID:24439206

  20. Solubilization of human erythrocyte membrane glycoproteins by triton X-100.

    PubMed Central

    Pratt, R S; Cook, G M

    1979-01-01

    1. The enzymic removal of sialic acid residues from the glycoproteins of the human erythrocyte decreases the solubilization of membrane glycoprotein by Triton X-100. 2. The solubilization of asialoglycoprotein by Triton X-100 may be restored by the addition of borate. 3. Use of this non-ionic detergent in the presence of borate, as a general procedure for the mild solubilization of membrane glycoproteins deficient in sialic acid residues, is discussed. PMID:39541

  1. Native functionality and therapeutic targeting of arenaviral glycoproteins.

    PubMed

    Crispin, Max; Zeltina, Antra; Zitzmann, Nicole; Bowden, Thomas A

    2016-06-01

    Surface glycoproteins direct cellular targeting, attachment, and membrane fusion of arenaviruses and are the primary target for neutralizing antibodies. Despite significant conservation of the glycoprotein architecture across the arenavirus family, there is considerable variation in the molecular recognition mechanisms used during host cell entry. We review recent progress in dissecting these infection events and describe how arenaviral glycoproteins can be targeted by small-molecule antivirals, the natural immune response, and immunoglobulin-based therapeutics. Arenaviral glycoprotein-mediated assembly and infection pathways present numerous opportunities and challenges for therapeutic intervention. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Native functionality and therapeutic targeting of arenaviral glycoproteins

    PubMed Central

    Crispin, Max; Zeltina, Antra; Zitzmann, Nicole; Bowden, Thomas A.

    2016-01-01

    Surface glycoproteins direct cellular targeting, attachment, and membrane fusion of arenaviruses and are the primary target for neutralising antibodies. Despite significant conservation of the glycoprotein architecture across the arenavirus family, there is considerable variation in the molecular recognition mechanisms used during host cell entry. We review recent progress in dissecting these infection events and describe how arenaviral glycoproteins can be targeted by small-molecule antivirals, the natural immune response and immunoglobulin-based therapeutics. Arenaviral glycoprotein-mediated assembly and infection pathways present numerous opportunities and challenges for therapeutic intervention. PMID:27104809

  3. Subunit structure of the glycoprotein complex of avian tumor virus.

    PubMed Central

    Leamnson, R N; Halpern, M S

    1976-01-01

    Envelope glycoprotein of avian tumor virus is linked by disulfide bonds in a structure that we have designated VGP to stand for viral glycoprotein. VGP appears to contain one molecule of gp85 and one of gp37. Under nonreducing conditions, VGP is the only glycoprotein component that is stable in the presence of ionic detergent, although in the presence of nonionic detergent two or more VGPs are associated in discrete complexes. The disulfide bonds linking viral glycoprotein are formed before release of virus from infected cells. PMID:178931

  4. Blocking Antibody Access to Neutralizing Domains on Glycoproteins Involved in Entry as a Novel Mechanism of Immune Evasion by Herpes Simplex Virus Type 1 Glycoproteins C and E▿

    PubMed Central

    Hook, Lauren M.; Huang, Jialing; Jiang, Ming; Hodinka, Richard; Friedman, Harvey M.

    2008-01-01

    Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins. PMID:18480440

  5. Oligosaccharides of the glycoprotein of rabies virus.

    PubMed Central

    Dietzschold, B

    1977-01-01

    The number of oligosaccharide side chains on rabies virus glycoprotein (G-protein) was investigated. Analysis of glycopeptides obtained by protease digestion of desialated G-protein revealed three discrete glycopeptides. Comparison of the protease digestion products from desialated and from untreated G-protein indicated a heterogeneity among the glycopeptides in the sialic acid content. Two major tryptic glycopeptides were isolated from desialated rabies virus G-protein and analyzed after protease digestion; one contained two oligosaccharide side chains and the other contained a single oligosaccharide side chain. PMID:886651

  6. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  7. Annotating Human P-Glycoprotein Bioassay Data.

    PubMed

    Zdrazil, Barbara; Pinto, Marta; Vasanthanathan, Poongavanam; Williams, Antony J; Balderud, Linda Zander; Engkvist, Ola; Chichester, Christine; Hersey, Anne; Overington, John P; Ecker, Gerhard F

    2012-08-01

    Huge amounts of small compound bioactivity data have been entering the public domain as a consequence of open innovation initiatives. It is now the time to carefully analyse existing bioassay data and give it a systematic structure. Our study aims to annotate prominent in vitro assays used for the determination of bioactivities of human P-glycoprotein inhibitors and substrates as they are represented in the ChEMBL and TP-search open source databases. Furthermore, the ability of data, determined in different assays, to be combined with each other is explored. As a result of this study, it is suggested that for inhibitors of human P-glycoprotein it is possible to combine data coming from the same assay type, if the cell lines used are also identical and the fluorescent or radiolabeled substrate have overlapping binding sites. In addition, it demonstrates that there is a need for larger chemical diverse datasets that have been measured in a panel of different assays. This would certainly alleviate the search for other inter-correlations between bioactivity data yielded by different assay setups.

  8. HIV-1 Envelope Glycoprotein Biosynthesis, Trafficking, and Incorporation

    PubMed Central

    Checkley, Mary Ann; Luttge, Benjamin G.; Freed, Eric O.

    2011-01-01

    The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor, gp160, that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and co-receptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation, and the role of specific membrane microdomains in this process. Here we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions. PMID:21762802

  9. Ammonia transport in the kidney by Rhesus glycoproteins

    PubMed Central

    Verlander, Jill W.

    2014-01-01

    Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport. PMID:24647713

  10. Varicella-Zoster Virus Glycoproteins: Entry, Replication, and Pathogenesis

    PubMed Central

    Yang, Edward; Arvin, Ann M.

    2016-01-01

    Varicella-zoster virus (VZV), an alphaherpesvirus that causes chicken pox (varicella) and shingles (herpes zoster), is a medically important pathogen that causes considerable morbidity and, on occasion, mortality in immunocompromised patients. Herpes zoster can afflict the elderly with a debilitating condition, postherpetic neuralgia, triggering severe, untreatable pain for months or years. The lipid envelope of VZV, similar to all herpesviruses, contains numerous glycoproteins required for replication and pathogenesis. Purpose of Review To summarize the current knowledge about VZV glycoproteins and their roles in cell entry, replication and pathogenesis. Recent Findings The functions for some VZV glycoproteins are known, such as gB, gH and gL in membrane fusion, cell-cell fusion regulation, and receptor binding properties. However, the molecular mechanisms that trigger or mediate VZV glycoproteins remains poorly understood. Summary VZV glycoproteins are central to successful replication but their modus operandi during replication and pathogenesis remain elusive requiring further mechanistic based studies. PMID:28367398

  11. Varicella-Zoster Virus Glycoproteins: Entry, Replication, and Pathogenesis.

    PubMed

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2016-12-01

    Varicella-zoster virus (VZV), an alphaherpesvirus that causes chicken pox (varicella) and shingles (herpes zoster), is a medically important pathogen that causes considerable morbidity and, on occasion, mortality in immunocompromised patients. Herpes zoster can afflict the elderly with a debilitating condition, postherpetic neuralgia, triggering severe, untreatable pain for months or years. The lipid envelope of VZV, similar to all herpesviruses, contains numerous glycoproteins required for replication and pathogenesis. To summarize the current knowledge about VZV glycoproteins and their roles in cell entry, replication and pathogenesis. The functions for some VZV glycoproteins are known, such as gB, gH and gL in membrane fusion, cell-cell fusion regulation, and receptor binding properties. However, the molecular mechanisms that trigger or mediate VZV glycoproteins remains poorly understood. VZV glycoproteins are central to successful replication but their modus operandi during replication and pathogenesis remain elusive requiring further mechanistic based studies.

  12. Solubilization of glycoproteins of envelope viruses by detergents

    SciTech Connect

    Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.

    1986-11-20

    The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-..beta..-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis.more » Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines.« less

  13. Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria.

    PubMed

    Karav, Sercan; Le Parc, Annabelle; Leite Nobrega de Moura Bell, Juliana Maria; Frese, Steven A; Kirmiz, Nina; Block, David E; Barile, Daniela; Mills, David A

    2016-06-15

    Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel

  14. Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

    PubMed Central

    Karav, Sercan; Le Parc, Annabelle; Leite Nobrega de Moura Bell, Juliana Maria; Frese, Steven A.; Kirmiz, Nina; Block, David E.; Barile, Daniela

    2016-01-01

    ABSTRACT Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as

  15. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes

    PubMed Central

    2013-01-01

    Background Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Methods Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Results Optimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays. DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. Conclusions This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for

  16. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes.

    PubMed

    Falconar, Andrew K I; Romero-Vivas, Claudia M E

    2013-04-22

    Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Optimal quenching of endogenous human serum peroxidases was attained using 3% H(2)O(2) in H(2)0 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays.DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for dot-blot assays can be performed

  17. The Metabolic Origins of Mannose in Glycoproteins*♦

    PubMed Central

    Ichikawa, Mie; Scott, David A.; Losfeld, Marie-Estelle; Freeze, Hudson H.

    2014-01-01

    Mannose in N-glycans is derived from glucose through phosphomannose isomerase (MPI, Fru-6-P ↔ Man-6-P) whose deficiency causes a congenital disorder of glycosylation (CDG)-Ib (MPI-CDG). Mannose supplements improve patients' symptoms because exogenous mannose can also directly contribute to N-glycan synthesis through Man-6-P. However, the quantitative contributions of these and other potential pathways to glycosylation are still unknown. We developed a sensitive GC-MS-based method using [1,2-13C]glucose and [4-13C]mannose to measure their contribution to N-glycans synthesized under physiological conditions (5 mm glucose and 50 μm mannose). Mannose directly provides ∼10–45% of the mannose found in N-glycans, showing up to a 100-fold preference for mannose over exogenous glucose based on their exogenous concentrations. Normal human fibroblasts normally derive 25–30% of their mannose directly from exogenous mannose, whereas MPI-deficient CDG fibroblasts with reduced glucose flux secure 80% of their mannose directly. Thus, both MPI activity and exogenous mannose concentration determine the metabolic flux into the N-glycosylation pathway. Using various stable isotopes, we found that gluconeogenesis, glycogen, and mannose salvaged from glycoprotein degradation do not contribute mannose to N-glycans in fibroblasts under physiological conditions. This quantitative assessment of mannose contribution and its metabolic fate provides information that can help bolster therapeutic strategies for treating glycosylation disorders with exogenous mannose. PMID:24407290

  18. Glycoprotein Expression in Human Milk During Lactation

    PubMed Central

    Froehlich, John W.; Dodds, Eric D.; Barboza, Mariana; McJimpsey, Erica L.; Seipert, Richard R.; Francis, Jimi; An, Hyun Joo; Freeman, Samara; German, J. Bruce; Lebrilla, Carlito B.

    2010-01-01

    While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first ten days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor. PMID:20415418

  19. Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.

    PubMed

    Safa, A R

    1988-10-01

    Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido[3,5-3H]benzoyl)aminomethyl verapamil and N-(p-azido[3-125I]salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistant DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein. Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil. Photoaffinity labeling was also inhibited by 50 microM concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Bay K8644, a calcium channel agonist, also inhibited P-glycoprotein photolabeling. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 microM compared to that by verapamil at 8 microM. Photolabeling was also partially inhibited by two of the drugs to which these cells are cross-resistant, doxorubicin and actinomycin D, at 100 microM, but not by colchicine. These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.

  20. Effect of rifampin, an inducer of CYP3A and P-glycoprotein, on the pharmacokinetics of risperidone.

    PubMed

    Kim, Kyoung-Ah; Park, Pil-Whan; Liu, Kwang-Hyeon; Kim, Kwon-Bok; Lee, Heon-Jeong; Shin, Jae-Gook; Park, Ji-Young

    2008-01-01

    The authors studied the effect of rifampin, a dual inducer of CYP3A and P-glycoprotein, on the pharmacokinetics and pharmacodynamics of risperidone in humans. Ten healthy male subjects were treated daily for 7 days with 600 mg rifampin or with placebo. On day 6, a single dose of 1 mg risperidone was administered. Plasma risperidone and 9-hydroxyrisperidone concentrations were measured. Rifampin significantly decreased the mean area under the plasma concentration-time curve by 51% for risperidone, by 43% for 9-hydroxyrisperidone, and by 45% for the active moieties (risperidone + 9-hydroxyrisperidone). Rifampin also decreased the peak plasma concentration of risperidone by 38%, 9-hydroxyrisperidone by 46%, and the active moieties by 41%. The apparent oral clearance of risperidone approximately doubled after rifampin treatment. Thus, rifampin reduced the exposure to risperidone, probably because of a decrease in its bioavailability through the induction of CYP3A and probably P-glycoprotein.

  1. Effect of P-glycoprotein on flavopiridol sensitivity

    PubMed Central

    Boerner, S A; Tourne, M E; Kaufmann, S H; Bible, K C

    2001-01-01

    Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC 50 s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CHRC5 (higher Pgp) cells were 90.2 ± 6.6 nM and 117 ± 2.3 nM, respectively (P< 0.01), suggesting that Pgp might have a modest effect on flavopiridol action. Consistent with this hypothesis, pretreatment with either quinidine or verapamil (inhibitors of Pgp-mediated transport) sensitized CHRC5 cells to the antiproliferative effects of flavopiridol. Because of concern that colony forming assays might not accurately reflect cytotoxicity, we also examined flavopiridol-treated cells by trypan blue staining and flow cytometry. These assays confirmed that flavopiridol was less toxic to cells expressing higher levels of Pgp. Further experiments revealed that flavopiridol inhibited the binding of [3H]-azidopine to Pgp in isolated membrane vesicles, but only at high concentrations. Collectively, these results identify flavopiridol as a weak substrate for Pgp. © 2001 Cancer Research Campaign www.bjcancer.com PMID:11355953

  2. Oxygen radicals stimulate guinea pig gallbladder glycoprotein secretion in vitro

    SciTech Connect

    Hale, W.B.; Turner, B.; LaMont, J.T.

    1987-11-01

    In several animal models of cholelithiasis, and in humans with gallstones, hypersecretion of gallbladder mucin is observed. This study was undertaken to determine the effect of oxygen radicals on guinea pig gallbladder glycoprotein secretion in organ culture. Mucosal explants were incubated with ({sup 3}H)glucosamine hydrochloride to label glycoproteins, then exposed to oxygen radicals generated by chelated ferric iron and ascorbic acid. Marked stimulation of glycoprotein release was observed after a 30-min exposure to the oxygen radical-generating system, and the effect was inhibited by mannitol. The stimulatory effect of hydroxyl radical was not accompanied by leakage of intracellular lactate dehydrogenase. Parallelmore » experiments with human granulocytes activated with f-Met-Leu-Phe and coincubated with gallbladder explants revealed similar results. These results indicate that oxygen radicals, especially the hydroxyl radical (OH), are capable of stimulating rapid release of mucous-type glycoproteins from gallbladder epithelium.« less

  3. Using Single Lectins to Enrich Glycoproteins in Conditioned Media.

    PubMed

    Sethi, Manveen K; Fanayan, Susan

    2015-08-03

    Lectins are sugar-binding proteins that can recognize and bind to carbohydrates conjugated to proteins and lipids. Coupled with mass spectrometry technologies, lectin affinity chromatography is becoming a popular approach for identification and quantification of glycoproteins in complex samples such as blood, tumor tissues, and cell lines. Given the commercial availability of a large number of lectins that recognize diverse sugar structures, it is now possible to isolate and study glycoproteins for biological and medical research. This unit provides a general guide to single-lectin-based enrichment of glycoproteins from serum-free conditioned media. Due to the unique carbohydrate specificity of most lectins and the complexity of the samples, optimization steps may be required to evaluate different elution buffers and methods as well as binding conditions, for each lectin, for optimal recovery of bound glycoproteins. Copyright © 2015 John Wiley & Sons, Inc.

  4. A model for mucus glycoprotein structure.

    PubMed

    Silberberg, A

    1987-01-01

    An approximately 500,000Da subunit characterizes the structural glycoprotein of mucus. This unit is composed of a rod-like very heavily glycosylated protein chain with an unglycosylated cysteine rich region at its end. It is proposed that the 'bare' peptide portion of the subunit forms itself into a lectin by undergoing a disulfide bond stabilised conformational fold. The lectin binding site, R, is assumed to be selective for a rare sugar sequence, which, when present, creates a binding site R. The site R has to be relatively rare. The average number less than s greater than of sidechains which contain R per subunit will only be of order 1. When less than s greater than less than or equal to 1 reasonably long, chain-like aggregates are formed which behave like coiling polymer chains with the subunit the Kuhn statistical element. When less than s greater than much greater than 1 the entire system forms one structure. Data obtained from the literature are analysed. They favor finite size, separate chains. The Kuhn statistical element length, derived from the data, is shown to agree well with the lectin model hypothesis.

  5. nES GEMMA Analysis of Lectins and Their Interactions with Glycoproteins - Separation, Detection, and Sampling of Noncovalent Biospecific Complexes

    NASA Astrophysics Data System (ADS)

    Engel, Nicole Y.; Weiss, Victor U.; Marchetti-Deschmann, Martina; Allmaier, Günter

    2017-01-01

    In order to better understand biological events, lectin-glycoprotein interactions are of interest. The possibility to gather more information than the mere positive or negative response for interactions brought mass spectrometry into the center of many research fields. The presented work shows the potential of a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) to detect weak, noncovalent, biospecific interactions besides still unbound glycoproteins and unreacted lectins without prior liquid phase separation. First results for Sambucus nigra agglutinin, concanavalin A, and wheat germ agglutinin and their retained noncovalent interactions with glycoproteins in the gas phase are presented. Electrophoretic mobility diameters (EMDs) were obtained by nES GEMMA for all interaction partners correlating very well with molecular masses determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the individual molecules. Moreover, EMDs measured for the lectin-glycoprotein complexes were in good accordance with theoretically calculated mass values. Special focus was laid on complex formation for different lectin concentrations and binding specificities to evaluate the method with respect to results obtained in the liquid phase. The latter was addressed by capillary electrophoresis on-a-chip (CE-on-a-chip). Of exceptional interest was the fact that the formed complexes could be sampled according to their size onto nitrocellulose membranes after gas-phase separation. Subsequent immunological investigation further proved that the collected complex actually retained its native structure throughout nES GEMMA analysis and sampling.

  6. Clinical relevance of β₂-glycoprotein-I plasma levels in antiphospholipid syndrome (APS).

    PubMed

    Banzato, Alessandra; Pengo, Vittorio

    2014-06-01

    Antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid (aPL) antibodies associated with thrombosis or pregnancy morbidity. The antibodies mainly involved in this disorder are directed against β2-glycoprotein I (β2-GPI). β2-GPI plasma level is usually not reported in studies on APS, because it is not regarded as relevant to the diagnosis and prognosis of APS. Nevertheless its measurement may be important for understanding the pathophysiology of the syndrome. This review summarizes available data from the literature on plasma concentrations of β2-GPI in patients with different antibody profiles.

  7. Properties of Replication-Competent Vesicular Stomatitis Virus Vectors Expressing Glycoproteins of Filoviruses and Arenaviruses

    PubMed Central

    Garbutt, Michael; Liebscher, Ryan; Wahl-Jensen, Victoria; Jones, Steven; Möller, Peggy; Wagner, Ralf; Volchkov, Viktor; Klenk, Hans-Dieter; Feldmann, Heinz; Ströher, Ute

    2004-01-01

    Replication-competent recombinant vesicular stomatitis viruses (rVSVs) expressing the type I transmembrane glycoproteins and selected soluble glycoproteins of several viral hemorrhagic fever agents (Marburg virus, Ebola virus, and Lassa virus) were generated and characterized. All recombinant viruses exhibited rhabdovirus morphology and replicated cytolytically in tissue culture. Unlike the rVSVs with an additional transcription unit expressing the soluble glycoproteins, the viruses carrying the foreign transmembrane glycoproteins in replacement of the VSV glycoprotein were slightly attenuated in growth. Biosynthesis and processing of the foreign glycoproteins were authentic, and the cell tropism was defined by the transmembrane glycoprotein. None of the rVSVs displayed pathogenic potential in animals. The rVSV expressing the Zaire Ebola virus transmembrane glycoprotein mediated protection in mice against a lethal Zaire Ebola virus challenge. Our data suggest that the recombinant VSV can be used to study the role of the viral glycoproteins in virus replication, immune response, and pathogenesis. PMID:15113924

  8. A glycosulphatase that removes sulphate from mucus glycoprotein.

    PubMed Central

    Roberton, A M; McKenzie, C G; Sharfe, N; Stubbs, L B

    1993-01-01

    A novel glycosulphatase has been purified from a mucus glycopeptide-degrading Prevotella from the colon. The purified enzyme removed inorganic [35S]sulphate from 35S-labelled native rat gastric mucus glycoprotein. Desulphation of mucus glycoprotein was initially rapid (19% complete after 10 min) but then plateaued, reaching only 33% after 3 h. Crude periplasmic extracts could remove 79% of the radioactivity as inorganic sulphate. These results suggest that steric hindrance may limit the access of the purified glycosulphatase to the mucus glycoprotein oligosaccharide chains in the absence of glycosidases, and/or that the enzyme may have the wrong specificity for some of the remaining sulphated sugars in the chains. The apparent molecular mass of the enzyme was 111 kDa as judged from gel exclusion chromatography, and it appeared to be composed of two identical subunits. The enzyme was localized in the periplasm of the bacterium, and using pig gastric mucus glycopeptide as a growth substrate markedly increased enzyme levels. Enzymic activity increased at the end of the growth phase. The substrate specificity of the enzyme was tested against low-molecular-mass sulphated molecules. The monosaccharides glucose 6-sulphate and N-acetylglucosamine 6-sulphate were rapidly desulphated, the latter being the major sulphated sugar in some mucus glycoproteins. Lactose 6-sulphate, galactose 6-sulphate, sulphated steroids and unsaturated disaccharide sulphate breakdown products from chondroitin sulphate were not desulphated. Glycosulphatases which can remove sulphate from mucus glycoproteins may play an important role in the degradation of highly sulphated mucus glycoproteins in the digestive tract, and could modify the effectiveness of mucus glycoproteins in mucosal protection. Images Figure 2 PMID:8352735

  9. Physical Properties of the Glycoprotein Mucin

    NASA Astrophysics Data System (ADS)

    Matthews, Garrett; Davis, William; Superfine, Richard; Boucher, Richard

    2003-03-01

    Epithelial cell surfaces are covered by a protective gel known as mucus. The physiological function of this gel depends on its rheological properties, and these properties are largely derived from the secreted glycoprotein mucin. The genetic disease Cystic Fibrosis (CF) is characterized by the adhesion of thick, viscous mucus on these tissues. In the lungs, this results in the interruption of mucus transport thus compromising the first line of defense against pathogens in these tissues. In order to restore the flow of tracheobronchial mucus out of the body, knowledge of the molecular and physical properties of mucin and mucin solutions would be greatly beneficial. The present model for these molecules is that of a long linear strand consisting of highly glycosylated regions linked by cystein-rich globular regions. It is thought that the globular regions may interact either through intermolecular disulfide bonds or through hydrophobic interactions. It has also been speculated that the glycosylated regions may have lectin-like interactions. In the present work, single mucin molecules were imaged at high resolution using atomic force microscopy (AFM). Phase mode imaging was used to map the interactions between functionalized AFM tips and the molecular topography. Additionally, using force-distance curves with the AFM, the adhesion between mucin bound tips and cell surface glycocalyx and glycocalyx-like model surfaces, was measured. And, finally, the viscoelastic properties of mucin solutions were measured using the recently developed technique, single particle tracking microrheology. A model is being developed that will incorporate the properties of mucins beginning at the single molecule and ending with the bulk viscoelastic properties.

  10. Neutralization epitope of varicella zoster virus on native viral glycoprotein gp118 (VZV glycoprotein gpIII).

    PubMed

    Montalvo, E A; Grose, C

    1986-03-01

    Varicella-zoster virus (VZV) specifies the formation of several glycoproteins, including a 118,000-Da mature structural product (gp118). The biologic and biochemical properties of gp118 were studied after production of murine monoclonal antibodies to both a lowpassage laboratory strain (VZV-32) and an attenuated vaccine strain (VZV-Oka). Structural analyses performed with the three glycosidases endo-beta-N-acetylglucosaminidase H (endoglycosidase H), endo-beta-N-acetylglucosaminidase F (endoglycosidase F), and endo-alpha-N-acetylgalactosaminidase demonstrated that gp118 was predominantly an N-linked complex type glycoprotein built upon a polypeptide backbone of approximately 79,000 Da. Sialic acid residues were present on the mature glycoprotein, but these terminal sugars were absent from the partially glycosylated intermediate forms recovered from monensin-treated infected cultures. Unlike another VZV-specified glycoprotein gp98, no new oligosaccharide moieties were observed on gp118 after addition of tunicamycin to VZV-infected cultures. By plaque reduction assays with a panel of monoclonal antibodies, we defined an epitope on this glycoprotein which elicited a complement-independent neutralizing antibody response of high magnitude. The epitope was highly conserved, since it was present on a laboratory VZV strain, wild type isolates, as well as the attenuated vaccine strain (VZV-Oka). Competitive blocking experiments with the same anti-gp118 monoclonal antibodies indicated that four neutralizing antibodies were directed against similar or identical epitopes whereas one nonneutralizing antibody reacted with a different antigenic site. Thus, this study demonstrates the presence of an immunodominant neutralization epitope on native viral glycoprotein gp118. Under a new consensus nomenclature, this glycoprotein will be designated VZV gpIII.

  11. Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters.

    PubMed

    Wang, Fun-In; Deng, Ming-Chung; Huang, Yu-Liang; Chang, Chia-Yi

    2015-06-29

    Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field.

  12. Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters

    PubMed Central

    Wang, Fun-In; Deng, Ming-Chung; Huang, Yu-Liang; Chang, Chia-Yi

    2015-01-01

    Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field. PMID:26131960

  13. Biotin- and glycoprotein-coated microspheres: potential surrogates for studying filtration of cryptosporidium parvum in porous media.

    PubMed

    Pang, Liping; Nowostawska, Urszula; Weaver, Louise; Hoffman, Gabrielle; Karmacharya, Anjuman; Skinner, Alexandra; Karki, Naveena

    2012-11-06

    Cryptosporidium parvum is a waterborne pathogen, yet no suitable surrogate has been established for quantifying its filtration removal in porous media. Carboxyl polystyrene microspheres with size, density, and shape similar to C. parvum were coated with biotin (free and containing amine, NH(2)) and glycoprotein. These biomolecules have isoelectric points similar to C. parvum (pH ≈ 2), and glycoprotein is a major type of surface protein that oocysts possess. Zeta potential (ζ) and filtration removal of particles in sand of two different grain sizes were examined. Compared to unmodified microspheres, modified microspheres achieved a superior match to the oocysts in ζ, concentration, mass recovery, and collision coefficient. They showed the same log reduction in concentration as oocysts, whereas results from unmodified microspheres deviated by 1 order of magnitude. Of the three types of modified microspheres, glycoprotein-coated microspheres best resembled oocyst concentration, despite having ζ similar to NH(2)-biotin-coated microspheres, suggesting that surface protein also played an important role in particle attachment on solid surfaces. With further validation in environmental conditions, the surrogates developed here could be a cost-effective new tool for assessing oocyst filtration in porous media, for example, to evaluate the performance of sand filters in water and wastewater treatment, water recycling through riverbank filtration, and aquifer recharge.

  14. Multiple Drug Transport Pathways through human P-Glycoprotein(†)

    PubMed Central

    McCormick, James W.; Vogel, Pia D.; Wise, John G.

    2015-01-01

    P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

  15. Alteration in P-glycoprotein functionality affects intrabrain distribution of quinidine more than brain entry-a study in rats subjected to status epilepticus by kainate.

    PubMed

    Syvänen, Stina; Schenke, Maarten; van den Berg, Dirk-Jan; Voskuyl, Rob A; de Lange, Elizabeth C

    2012-03-01

    This study aimed to investigate the use of quinidine microdialysis to study potential changes in brain P-glycoprotein functionality after induction of status epilepticus (SE) by kainate. Rats were infused with 10 or 20 mg/kg quinidine over 30 min or 4 h. Plasma, brain extracellular fluid (brain ECF), and end-of-experiment total brain concentrations of quinidine were determined during 7 h after the start of the infusion. Effect of pretreatment with tariquidar (15 mg/kg, administered 30 min before the start of the quinidine infusion) on the brain distribution of quinidine was assessed. This approach was repeated in kainate-treated rats. Quinidine kinetics were analyzed with population modeling (NONMEM). The quinidine microdialysis assay clearly revealed differences in brain distribution upon changes in P-glycoprotein functionality by pre-administration of tariquidar, which resulted in a 7.2-fold increase in brain ECF and a 40-fold increase in total brain quinidine concentration. After kainate treatment alone, however, no difference in quinidine transport across the blood-brain barrier was found, but kainate-treated rats tended to have a lower total brain concentration but a higher brain ECF concentration of quinidine than saline-treated rats. This study did not provide evidence for the hypothesis that P-glycoprotein function at the blood-brain barrier is altered at 1 week after SE induction, but rather suggests that P-glycoprotein function might be altered at the brain parenchymal level.

  16. P-Glycoprotein Transport of Neurotoxic Pesticides.

    PubMed

    Lacher, Sarah E; Skagen, Kasse; Veit, Joachim; Dalton, Rachel; Woodahl, Erica L

    2015-10-01

    P-glycoprotein (P-gp) has been associated with a number of neurodegenerative diseases, including Parkinson's disease, although the mechanisms remain unclear. Altered transport of neurotoxic pesticides has been proposed in Parkinson's disease, but it is unknown whether these pesticides are P-gp substrates. We used three in vitro transport models, stimulation of ATPase activity, xenobiotic-induced cytotoxicity, and inhibition of rhodamine-123 efflux, to evaluate P-gp transport of diazinon, dieldrin, endosulfan, ivermectin, maneb, 1-methyl-4-phenyl-4-phenylpyridinium ion (MPP(+)), and rotenone. Diazinon and rotenone stimulated ATPase activity in P-gp-expressing membranes, with Vmax values of 22.4 ± 2.1 and 16.8 ± 1.0 nmol inorganic phosphate/min per mg protein, respectively, and Km values of 9.72 ± 3.91 and 1.62 ± 0.51 µM, respectively, compared with the P-gp substrate verapamil, with a Vmax of 20.8 ± 0.7 nmol inorganic phosphate/min per mg protein and Km of 0.871 ± 0.172 μM. None of the other pesticides stimulated ATPase activity. We observed an increased resistance to MPP(+) and rotenone in LLC-MDR1-WT cells compared with LLC-vector cells, with 15.4- and 2.2-fold increases in EC50 values, respectively. The resistance was reversed in the presence of the P-gp inhibitor verapamil. None of the other pesticides displayed differential cytotoxicity. Ivermectin was the only pesticide to inhibit P-gp transport of rhodamine-123, with an IC50 of 0.249 ± 0.048 μM. Our data demonstrate that dieldrin, endosulfan, and maneb are not P-gp substrates or inhibitors. We identified diazinon, MPP(+), and rotenone as P-gp substrates, although further investigation is needed to understand the role of P-gp transport in their disposition in vivo and associations with Parkinson's disease. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  17. Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non-parietal cells.

    PubMed Central

    Heim, H. K.; Oestmann, A.; Sewing, K. F.

    1991-01-01

    1. Glycoprotein and protein synthesis in and release from pig isolated, enriched gastric mucous cells were measured by the incorporation of N-acetyl-[14C]-D-glucosamine and [3H]-L-leucine, respectively, into cellular and released acid precipitable material. 2. Histamine and activators of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) system maximally stimulated total protein and glycoprotein synthesis in and release from the cells at concentrations of histamine (10 microM), forskolin (10-100 microM), 3-isobutyl-1-methylxanthine (100 microM), and dibutyryl cyclic AMP (1-3 mM), respectively. In the presence of 3-isobutyl-1-methylxanthine (30 microM) histamine stimulation was enhanced. 3. As shown by gel chromatography, stimulation by histamine (100 microM), forskolin (10 microM), 3-isobutyl-1-methylxanthine (100 microM) and dibutyryl cyclic AMP (1 mM) resulted in a release of high molecular weight (approximately 2 x 10(6) daltons) glycoproteins from the cells. The histamine H2-receptor antagonist, ranitidine (100 microM), blocked the effect of histamine. 4. We conclude that cyclic AMP-dependent processes are involved in the regulation of protein and glycoprotein synthesis in and the release of high molecular weight (mucous) glycoproteins from pig gastric non-parietal cells and that histamine may be a physiological activator of this system. PMID:1724626

  18. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein III aids in the diagnosis of an inherited deficiency of this serum protein and a variety of other...

  19. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein I aids in the diagnosis of an inherited deficiency of this serum protein. (b) Classification...

  20. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein III aids in the diagnosis of an inherited deficiency of this serum protein and a variety of other...

  1. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein I aids in the diagnosis of an inherited deficiency of this serum protein. (b) Classification...

  2. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein I aids in the diagnosis of an inherited deficiency of this serum protein. (b) Classification...

  3. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein I aids in the diagnosis of an inherited deficiency of this serum protein. (b) Classification...

  4. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein III aids in the diagnosis of an inherited deficiency of this serum protein and a variety of other...

  5. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein I aids in the diagnosis of an inherited deficiency of this serum protein. (b) Classification...

  6. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein III aids in the diagnosis of an inherited deficiency of this serum protein and a variety of other...

  7. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein III aids in the diagnosis of an inherited deficiency of this serum protein and a variety of other...

  8. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... alpha-1-glycoproteins (a group of plasma proteins found in the alpha-1 group when subjected to electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in the...

  9. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... alpha-1-glycoproteins (a group of plasma proteins found in the alpha-1 group when subjected to electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in the...

  10. Screening of extraction methods for glycoproteins from jellyfish ( Rhopilema esculentum) oral-arms by high performance liquid chromatography

    NASA Astrophysics Data System (ADS)

    Ren, Guoyan; Li, Bafang; Zhao, Xue; Zhuang, Yongliang; Yan, Mingyan; Hou, Hu; Zhang, Xiukun; Chen, Li

    2009-03-01

    In order to select an optimum extraction method for the target glycoprotein (TGP) from jellyfish ( Rhopilema esculentum) oral-arms, a high performance liquid chromatography (HPLC)-assay for the determination of the TGP was developed. Purified target glycoprotein was taken as a standard glycoprotein. The results showed that the calibration curves for peak area plotted against concentration for TGP were linear ( r = 0.9984, y = 4.5895 x+47.601) over concentrations ranging from 50 to 400 mgL-1. The mean extraction recovery was 97.84% (CV2.60%). The fractions containing TGP were isolated from jellyfish ( R. esculentum) oral-arms by four extraction methods: 1) water extraction (WE), 2) phosphate buffer solution (PBS) extraction (PE), 3) ultrasound-assisted water extraction (UA-WE), 4) ultrasound-assisted PBS extraction (UA-PE). The lyophilized extract was dissolved in Milli-Q water and analyzed directly on a short TSK-GEL G4000PWXL (7.8 mm×300 mm) column. Our results indicated that the UA-PE method was the optimum extraction method selected by HPLC.

  11. Beta2-glycoprotein I dependent anticardiolipin antibodies and lupus anticoagulant in patients with recurrent pregnancy loss.

    PubMed

    Kumar, K S D; Jyothy, A; Prakash, M S; Rani, H S; Reddy, P P

    2002-01-01

    The present study was aimed to define the incidence of antiphospholipid antibodies of different types lupus anticoagulant (LAC), venereal disease research laboratory test (VDRL) and Beta2-glycoprotein I dependent anticardiolipin antibodies Beta2 I aCL) in our cohort of population experiencing recurrent pregnancy loss (RPL) from Andhra Pradesh, South India. A referral case-control study at a tertiary centre over a period of 5 years. 150 couples experiencing 3 or more recurrent pregnancy losses with similar number of matched controls. LAC activity was measured by the activated partial thromboplastin time (aPTT) according to the method of Proctor and Rapaport with relevant modifications. VDRL analysis was performed by the kit method supplied by Ranbaxy Diagnostics Limited and Beta2 Glycoprotein I dependent anticardiolipin antibodies were estimated by ELISA kit (ORGen Tech, GmbH, Germany) with human Beta2 Glycoprotein I as co-factor. Statistical analysis was performed using Student's t test. LAC activity was found positive in 11 women (10.28%). The mean +/- SE Beta2 I aCL concentration in the study group was 14.53 (micro/ml) +/- 1.79 (range 0 to 90.4 micro/ml) which was higher than the control group with a mean +/- SE of 7.26 (micro/ml) +/- 0.40 (range 0 to 18 u/ml). The binding of the antibodies to the antigen was observed in 40.24% (n=33) of the cases compared to 6.09% (n=5) in controls. VDRL test was positive in 7(2.34%) individuals (3 couples and 1 male partner) and none among controls. The present study indicates the importance of antiphospholipid antibodies in women experiencing RPL and suggests the usefulness of screening for these antibodies as a mandatory routine for instituting efficient therapeutic regimens for a successful outcome of pregnancy.

  12. Detection of glycoproteins in the Acanthamoeba plasma membrane

    SciTech Connect

    Paatero, G.I.L.; Gahmberg, C.G.

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presencemore » of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.« less

  13. QUANTITATIVE MASS SPECTROMETRIC ANALYSIS OF GLYCOPROTEINS COMBINED WITH ENRICHMENT METHODS

    PubMed Central

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:148–165, 2015. PMID:24889823

  14. Glycoprotein expression by adenomatous polyps of the colon

    NASA Astrophysics Data System (ADS)

    Roney, Celeste A.; Xie, Jianwu; Xu, Biying; Jabour, Paul; Griffiths, Gary; Summers, Ronald M.

    2008-03-01

    Colon cancer is the second leading cause of cancer related deaths in the United States. Specificity in diagnostic imaging for detecting colorectal adenomas, which have a propensity towards malignancy, is desired. Adenomatous polyp specimens of the colon were obtained from the mouse model of colorectal cancer called adenomatous polyposis coli-multiple intestinal neoplasia (APC Min). Histological evaluation, by the legume protein Ulex europaeus agglutinin I (UEA-1), determined expression of the glycoprotein α-L-fucose. FITC-labelled UEA-1 confirmed overexpression of the glycoprotein by the polyps on fluorescence microscopy in 17/17 cases, of which 13/17 included paraffin-fixed mouse polyp specimens. In addition, FITC-UEA-1 ex vivo multispectral optical imaging of 4/17 colonic specimens displayed over-expression of the glycoprotein by the polyps, as compared to non-neoplastic mucosa. Here, we report the surface expression of α-L-fucosyl terminal residues by neoplastic mucosal cells of APC specimens of the mouse. Glycoprotein expression was validated by the carbohydrate binding protein UEA-1. Future applications of this method are the development of agents used to diagnose cancers by biomedical imaging modalities, including computed tomographic colonography (CTC). UEA-1 targeting to colonic adenomas may provide a new avenue for the diagnosis of colorectal carcinoma by CT imaging.

  15. Magnetic enzyme reactors for isolation and study of heterogeneous glycoproteins

    NASA Astrophysics Data System (ADS)

    Korecká, Lucie; Ježová, Jana; Bílková, Zuzana; Beneš, Milan; Horák, Daniel; Hradcová, Olga; Slováková, Marcela; Viovy, Jean-Louis

    2005-05-01

    The newly developed magnetic micro- and nanoparticles with defined hydrophobicity and porosity were used for the preparation of magnetic enzyme reactors. Magnetic particles with immobilized proteolytic enzymes trypsin, chymotrypsin and papain and with enzyme neuraminidase were used to study the structure of heterogeneous glycoproteins. Factors such as the type of carrier, immobilization procedure, operational and storage stability, and experimental conditions were optimized.

  16. Hemostatic Function, Survival, and Membrane Glycoprotein Changes in Young versus Old Rabbit Platelets

    PubMed Central

    Blajchman, Morris A.; Senyi, Andrew F.; Hirsh, Jack; Genton, Edward; George, James N.

    1981-01-01

    relationship among all the glycoprotein peaks was equal and the only changes observed were quantitative, with young platelets having significantly more membrane glycoprotein per cell than old platelets and normal platelets. Normal platelets had intermediate concentrations of each glycoprotein. These results demonstrate that young platelets are hemostatically more effective in vivo than old platelets. The data are compatible with the hypothesis that platelets age in the circulation by losing membrane fragments and then after becoming senescent, are removed from the circulation by a random process. PMID:7298853

  17. [Purification and cytotoxicity of glycoprotein isolated from Dendrobium huoshanense].

    PubMed

    Deng, Hui; Chen, Nai-Dong; Dai, Jun; Chen, Nai-Fu

    2017-01-01

    Dendrobium huoshanense is a rare traditional Chinese medicinal herb, and the anti-tumor activity of its polysaccharides is a research hotspot in traditional Chinese medicine resources domain. This study aims to explore the material basis for the anti-tumor activity of polysaccharide. D. huoshanense was used as raw material in the experiment, and the different protein components were obtained through low salt solution extraction and ammonium sulfate fractional precipitation. Then glycoprotein components were determined by SDS-PAGE electrophoresis staining, and were further isolated and purified by DEAE ion column and Sephadex gel column. At the same time, MTT assay was used in detecting the cytotoxicity of different products on HepG2 cells in vitro. As a result, three kinds of glycoprotein components RG1, RG2, RG3 with relative molecular mass of 22.5, 19.8, 15.6 kDa were gained, and the IC₅₀ of three compounds on human liver cancer cell HepG2 was 534.23 mg•L⁻¹, meanwhile IC₅₀ of single glycoprotein component RG1, RG2 was 432.96, 413.91 mg•L⁻¹ respectively, and glycoprotein component RG3 had no cytotoxicity on HepG2 cells. All in all, the experiment results suggested that two kinds of glycoproteins components with relative molecular mass of 22.5, 19.8 kDa may be one of the material basis for anti-tumor activity of D. huoshanense polysaccharide, and they had a synergistic effect. Copyright© by the Chinese Pharmaceutical Association.

  18. Urinary excretion of beta 2-glycoprotein-1 (apolipoprotein H) and other markers of tubular malfunction in "non-tubular" renal disease.

    PubMed Central

    Flynn, F. V.; Lapsley, M.; Sansom, P. A.; Cohen, S. L.

    1992-01-01

    AIM: To determine whether urinary beta 2-glycoprotein-1 assays can provide improved discrimination between chronic renal diseases which are primarily of tubular or glomerular origin. METHODS: Urinary beta 2-glycoprotein-1, retinol-binding protein, alpha 1-microglobulin, beta 2-microglobulin, N-acetyl-beta-D-glucosa-minidase and albumin were measured in 51 patients with primary glomerular disease, 23 with obstructive nephropathy, and 15 with polycystic kidney disease, and expressed per mmol of creatinine. Plasma beta 2-glycoprotein-1 was assayed in 52 patients and plasma creatinine in all 89. The findings were compared between the diagnostic groups and with previously published data relating to primary tubular disorders. RESULTS: All 31 patients with plasma creatinine greater than 200 mumol/l excreted increased amounts of beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin, and 29 had increased N-acetyl-beta-D-glucosaminidase; the quantities were generally similar to those found in comparable patients with primary tubular pathology. Among 58 with plasma creatinine concentrations under 200 mumol/l, increases in beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin excretion were less common and much smaller, especially in those with obstructive nephropathy and polycystic disease. The ratios of the excretion of albumin to the other proteins provided the clearest discrimination between the patients with glomerular or tubular malfunction, but an area of overlap was present which embraced those with obstructive nephropathy and polycystic disease. CONCLUSIONS: Increased excretion of beta 2-glycoprotein-1 due to a raised plasma concentration or diminution of tubular reabsorption, or both, is common in all the forms of renal disease investigated, and both plasma creatinine and urinary albumin must be taken into account when interpreting results. Ratios of urinary albumin: beta 2-glycoprotein-1 greater than 1000 are highly suggestive

  19. Urinary excretion of beta 2-glycoprotein-1 (apolipoprotein H) and other markers of tubular malfunction in "non-tubular" renal disease.

    PubMed

    Flynn, F V; Lapsley, M; Sansom, P A; Cohen, S L

    1992-07-01

    To determine whether urinary beta 2-glycoprotein-1 assays can provide improved discrimination between chronic renal diseases which are primarily of tubular or glomerular origin. Urinary beta 2-glycoprotein-1, retinol-binding protein, alpha 1-microglobulin, beta 2-microglobulin, N-acetyl-beta-D-glucosa-minidase and albumin were measured in 51 patients with primary glomerular disease, 23 with obstructive nephropathy, and 15 with polycystic kidney disease, and expressed per mmol of creatinine. Plasma beta 2-glycoprotein-1 was assayed in 52 patients and plasma creatinine in all 89. The findings were compared between the diagnostic groups and with previously published data relating to primary tubular disorders. All 31 patients with plasma creatinine greater than 200 mumol/l excreted increased amounts of beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin, and 29 had increased N-acetyl-beta-D-glucosaminidase; the quantities were generally similar to those found in comparable patients with primary tubular pathology. Among 58 with plasma creatinine concentrations under 200 mumol/l, increases in beta 2-glycoprotein-1, retinol-binding protein, and alpha 1-microglobulin excretion were less common and much smaller, especially in those with obstructive nephropathy and polycystic disease. The ratios of the excretion of albumin to the other proteins provided the clearest discrimination between the patients with glomerular or tubular malfunction, but an area of overlap was present which embraced those with obstructive nephropathy and polycystic disease. Increased excretion of beta 2-glycoprotein-1 due to a raised plasma concentration or diminution of tubular reabsorption, or both, is common in all the forms of renal disease investigated, and both plasma creatinine and urinary albumin must be taken into account when interpreting results. Ratios of urinary albumin: beta 2-glycoprotein-1 greater than 1000 are highly suggestive of primary glomerular disease and

  20. Interactions between cortisol and Rhesus glycoprotein expression in ureogenic toadfish, Opsanus beta.

    PubMed

    Rodela, Tamara M; McDonald, M Danielle; Walsh, Patrick J; Gilmour, Kathleen M

    2012-01-15

    In their native environment, gulf toadfish excrete equal quantities of ammonia and urea. However, upon exposure to stressful conditions in the laboratory (i.e. crowding, confinement or air exposure), toadfish decrease branchial ammonia excretion and become ureotelic. The objective of this study was to determine the influences of cortisol and ammonia on ammonia excretion relative to expression of Rhesus (Rh) glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). In vivo infusions and/or injections were used to manipulate corticosteroid activity and plasma ammonia concentrations in ureotelic toadfish. Metyrapone treatment to lower circulating cortisol levels resulted in a 3.5-fold elevation of ammonia excretion rates, enhanced mRNA expression of two of the toadfish Rh isoforms (Rhcg1 and Rhcg2), and decreased branchial and hepatic GS activity. Correspondingly, cortisol infusion decreased ammonia excretion 2.5-fold, a change that was accompanied by reduced branchial expression of all toadfish Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) and a twofold increase in hepatic GS activity. In contrast, maintenance of high circulating ammonia levels by ammonia infusion enhanced ammonia excretion and Rh expression (Rhag, Rhbg and Rhcg2). Toadfish treated with cortisol showed an attenuated response to ammonia infusion with no change in Rh mRNA expression or GS activity. In summary, the evidence suggests that ammonia excretion in toadfish is modulated by cortisol-induced changes in both Rh glycoprotein expression and GS activity.

  1. Surface (glyco-)proteins: primary structure and crystallization under microgravity conditions

    NASA Astrophysics Data System (ADS)

    Claus, H.; Akca, E.; Schultz, N.; Karbach, G.; Schlott, B.; Debaerdemaeker, T.; De Clercq, J.-P.; König, H.

    2001-08-01

    The Archaea comprise microorganisms that live under environmental extremes, like high temperature, low pH value or high salt concentration. Their cells are often covered by a single layer of (glyco)protein subunits (S-layer) in hexagonal arrangement. In order to get further hints about the molecular mechanisms of protein stabilization we compared the primary and secondary structures of archaeal S-layer (glyco)proteins. We found an increase of charged amino acids in the S-layer proteins of the extreme thermophilic species compared to their mesophilic counterparts. Our data and those of other authors suggest that ionic interactions, e.g., salt bridges seem to be played a major role in protein stabilization at high temperatures. Despite the differences in the growth optima and the predominance of some amino acids the primary structures of S-layers revealed also a significant degree of identity between phylogenetically related archaea. These obervations indicate that protein sequences of S-layers have been conserved during the evolution from extremely thermophilic to mesophilic life. To support these findings the three-dimensional structure of the S-layer proteins has to be elucidated. Recently, we described the first successful crystallization of an extreme thermophilic surface(glyco)protein under microgravity conditions.

  2. In vitro and in vivo investigations on fluoroquinolones; effects of the P-glycoprotein efflux transporter on brain distribution of sparfloxacin.

    PubMed

    de Lange, E C; Marchand, S; van den Berg, D; van der Sandt, I C; de Boer, A G; Delon, A; Bouquet, S; Couet, W

    2000-12-01

    The role of mdr1a-encoded P-glycoprotein on transport of several fluoroquinolones across the blood-brain barrier was investigated. In vitro, P-glycoprotein substrates were selected by using a confluent monolayer of MDR1-LLC-PK1 cells. The inhibition of fluoroquinolones (100 microM) on transport of rhodamine-123 (1 microM) was compared with P-glycoprotein inhibitors verapamil (20 microM) and SDZ PSC 833 (2 microM). Subsequently, transport polarity of fluoroquinolones was studied. Sparfloxacin showed the strongest inhibition (26%) and a large polarity in transport, by P-glycoprotein activity. In vivo, using mdr1a (-/-) and wild-type mice, brain distribution of pefloxacin, norfloxacin, ciprofloxacin, fleroxacin and sparfloxacin was determined at 2, 4, and 6 h following intra-arterial infusion (50 nmol/min). Brain distribution of sparfloxacin was clearly higher in mdr1a (-/-) mice compared with wild-type mice. Sparfloxacin was infused (50 nmol/min) for 1, 2, 3 and 4 h in which intracerebral microdialysis was performed. At 4 h, in vivo recovery (dynamic-no-net-flux method) was 6.5+/-2.2 and 1.5+/-0.5%; brain(ECF) concentrations were 5.1+/-0.2 and 26+/-21 microM; and total brain concentrations were 7.2+/-0.3 and 23+/-0.3 microM in wild-type and mdr1a (-/-) mice, respectively. Plasma concentrations were similar (18.4+/-0.7 and 17.9+/-0.5 microM, respectively). In conclusion, sparfloxacin enters the brain poorly mainly because of P-glycoprotein activity at the blood-brain barrier.

  3. Marine Natural Products with P-Glycoprotein Inhibitor Properties

    PubMed Central

    Lopez, Dioxelis; Martinez-Luis, Sergio

    2014-01-01

    P-glycoprotein (P-gp) is a protein belonging to the ATP-binding cassette (ABC) transporters superfamily that has clinical relevance due to its role in drug metabolism and multi-drug resistance (MDR) in several human pathogens and diseases. P-gp is a major cause of drug resistance in cancer, parasitic diseases, epilepsy and other disorders. This review article aims to summarize the research findings on the marine natural products with P-glycoprotein inhibitor properties. Natural compounds that modulate P-gp offer great possibilities for semi-synthetic modification to create new drugs and are valuable research tools to understand the function of complex ABC transporters. PMID:24451193

  4. Markers of Ovarian Cancer Using a Glycoprotein/Antibody Array

    DTIC Science & Technology

    2011-05-01

    DATE: May 2011 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick...alternative method to accomplish the same task. This i nvolves ex traction o f the fucosylated glycoproteins us ing a l ectin c olumn and i sotopic ...proteins and performed quantitative i sotopic proteomics for the 22 c ancer and 12 b enign s amples. The da ta i s currently being analyzed to search

  5. Ice growth in supercooled solutions of antifreeze glycoproteins

    NASA Technical Reports Server (NTRS)

    Harrison, K.; Hallett, J.; Burcham, T. S.; Feeney, R. E.; Kerr, W. L.

    1987-01-01

    The effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of antifreeze glycoproteins are reported. To isolate the influence of different solutions and supercooling alone, a system was devised that nucleated crystals in the middle of a uniformly supercooled sample. Alternatively, single crystals of selected orientation were inserted into free liquid surface. A crystallization rate up to five times greater than that in pure water was found. A mechanism explaining these results is suggested.

  6. Markers of Ovarian Cancer Using a Glycoprotein/Antibody Array

    DTIC Science & Technology

    2014-05-01

    detection: unique lectin binding patterns as a tool for classifying normal, chronic pancreatitis and pancreatic cancer sera. J Proteome Res, 2007...6, 1864-1874. 5. Li C, Simeone DM, Brenner DE, Anderson MA, Shedden KA, Ruffin MT, et al. Pancreatic Cancer Serum Detection Using a Lectin/Glyco...Glycoproteomics Using Lectin Selected Sialic Acid Glycoproteins with Mass Spectrometric Analysis: Application to Pancreatic Cancer Serum, J

  7. Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

    PubMed

    Rasheed, Muhammad Asif; Ansari, Abdur Rahman; Ihsan, Awais; Navid, Muhammad Tariq; Ur-Rehman, Shahid; Raza, Sohail

    2018-03-01

    Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Glycoprotein analysis using enzymatic digestion and MALDI-TOF MS

    NASA Astrophysics Data System (ADS)

    Kornfeld, Rich; Kenny, James W.; Weinberger, Scot R.; Yang, Yi; Orlando, Ron

    1996-04-01

    A sensitive and facile method is described to identify the glycosylation sites and site-specific heterogeneity in the carbohydrate portion of glycoproteins. In this procedure, the peptide backbone of the glycoprotein is cleaved enzymatically. The peptide/glycopeptide mixture is divided into three fractions. The first fraction is analyzed directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), while the other two aliquots are analyzed by MALDI-TOF MS after enzymatic release of the N-linked and N- and O-linked chains. Comparison of these mass spectra provides the molecular weight of each carbohydrate side chain and of the peptide to which it is attached. This information combined with the protein's amino acid sequence identifies the glycosylation sites and provides information concerning site-specific oligosaccharide heterogeneity. This approach is faster and simpler than procedures currently used for glycosylation site mapping and can be performed on as little as 10 picomoles of glycoprotein.

  9. Identification of glycoproteins from mouse skin tumors and plasma

    PubMed Central

    Tian, Yuan; Kelly-Spratt, Karen S.; Kemp, Christopher J.; Zhang, Hui

    2010-01-01

    Plasma has been the focus of testing different proteomic technologies for the identification of biomarkers due to its ready accessibility. However, it is not clear if direct proteomic analysis of plasma can be used to discover new marker proteins from tumor that are associated with tumor progression. Here, we reported that such proteins can be detected in plasma in a chemical induced skin cancer mouse model. We analyzed glycoproteins from both benign papillomas and malignant carcinomas from mice using our recently developed platform, solid-phase extraction of glycopeptides (SPEG) and mass spectrometry, and identified 463 unique N-linked glycosites from 318 unique glycoproteins. These include most known extracellular proteins that have been reported to play roles in skin cancer development such as thrombospondin, cathepsins, epidermal growth factor receptor, cell adhesion molecules, cadherins, integrins, tuberin, fibulin, TGFβ receptor, etc. We further investigated whether these tumor proteins could be detected in plasma from tumor bearing mice using isotope labeling and 2D-LC-MALDI-MS/MS. Two tumor glycoproteins, Tenascin-C and Arylsulfatase B, were identified and quantified successfully in plasma from tumor bearing mice. This result indicates that analysis of tumor associated proteins in tumors and plasma by method using glycopeptide capture, isotopic labeling, and mass spectrometry can be used as a discovery tool to identify candidate tumor proteins that may be detected in plasma. PMID:21072318

  10. Processing of virus-specific glycoproteins of varicella zoster virus

    SciTech Connect

    Namazue, J.; Campo-Vera, H.; Kitamura, K.

    1985-05-01

    Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with (/sup 3/H)glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gpmore » 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.« less

  11. Bladder Cancer Associated Glycoprotein Signatures Revealed by Urinary Proteomic Profiling

    PubMed Central

    Kreunin, Paweena; Zhao, Jia; Rosser, Charles; Urquidi, Virginia; Lubman, David M.; Goodison, Steve

    2009-01-01

    Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including α-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from non-tumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine. PMID:17518487

  12. Comparison of glycoprotein expression between ovarian and colon adenocarcinomas.

    PubMed

    Multhaupt, H A; Arenas-Elliott, C P; Warhol, M J

    1999-10-01

    Tumor-associated antigens may be expressed as surface glycoproteins. These molecules undergo qualitative and quantitative modifications during cell differentiation and malignant transformation. During malignant transformation, incomplete glycosylation is common, and certain glycosylation pathways are preferred. These antigens might help distinguish between ovarian and colonic adenocarcinomas in the primary and metastatic lesions. Different cytokeratins have been proposed as relatively organ-specific antigens. We used monoclonal antibodies against T1, Tn, sialosyl-Tn, B72.3, CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use in distinguishing between these 2 entities. A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot be distinguished from colonic adenocarcinomas using immunohistochemistry.

  13. Stimulation of glycoprotein and protein synthesis in isolated pig gastric mucosal cells by prostaglandins.

    PubMed Central

    Heim, H K; Oestmann, A; Sewing, K F

    1990-01-01

    The purpose of this study is to evaluate the effects of different prostaglandin derivatives on protein and glycoprotein synthesis and secretion in isolated and enriched pig gastric mucous cells, as measured by the incorporation of [3H]L-leucine and N-acetyl-[14C]D-glucosamine respectively into acid insoluble macromolecules (AIM). PGE2 and 16,16-dimethyl-PGE2 enhanced the incorporation of the amino sugar into cellular (EC50 8 and 75 nmol/l) and secreted (EC50 30 and 270 nmol/l) AIM in a concentration dependent manner during a 20 hours incubation. After incubation for eight hours or more they also stimulated the incorporation of [3H]L-leucine into cellular AIM. PGF2 alpha was considerably less potent (EC50 greater than 1 mumol/l) than the E-type prostaglandins. Iloprost, a stable prostacyclin analogue, was ineffective. PMID:2338265

  14. Urinary epitectin (MUC-1 glycoprotein) in the menstrual cycle and interstitial cystitis.

    PubMed

    Erickson, D R; Mast, S; Ordille, S; Bhavanandan, V P

    1996-09-01

    We compared interstitial cystitis and control urine specimens for epitectin (MUC-1 glycoprotein), an epithelial mucin. Urinary epitectin was measured in 28 patients with interstitial cystitis and 26 healthy controls. Ten controls provided multiple urine samples to determine whether urinary epitectin changes with the menstrual cycle. Epitectin levels were stable throughout the menstrual cycle. Interstitial cystitis cases had decreased urinary epitectin-to-creatinine ratios (mean 3.89 versus 6.38 micrograms./mg. creatinine for controls, p = 0.0035) and epitectin concentrations (mean 1.96 versus 4.30 micrograms./ml., respectively, p = 0.0005). Decreased mean urinary epitectin levels may reflect a cause (epithelial mucin deficiency) or a consequence of interstitial cystitis.

  15. The antigenic domain 1 of human cytomegalovirus glycoprotein B contains an intramolecular disulphide bond.

    PubMed

    Speckner, A; Kropff, B; Knör, S; Mach, M

    2000-11-01

    Glycoprotein B (gB, gpUL55) is the major antigen recognized by the neutralizing humoral immune response against human cytomegalovirus (HCMV). The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids (aa 552-635) for antibody binding. In this study, the structural requirements for antibody binding to AD-1 have been determined. The domain was expressed in prokaryotic and eukaryotic systems and analysed in immunoblots under reducing and non-reducing conditions. In addition, AD-1 was purified in an immunologically active form and the concentration of sulphydryl groups was determined. The data clearly show that the only form that is recognized by antibodies is a disulphide-linked monomer of AD-1. The disulphide bond is formed between cysteines at amino acid positions 573 and 610 of gB.

  16. The human fibroblast receptor for gp86 of human cytomegalovirus is a phosphorylated glycoprotein.

    PubMed Central

    Keay, S; Baldwin, B

    1992-01-01

    A human embryonic lung (HEL) cell receptor for gp86 of human cytomegalovirus that functions in virus-cell fusion was further characterized. Anti-idiotype antibodies that mimic gp86 were used to immunoprecipitate the 92.5-kDa fibroblast membrane receptor for gp86, which was preincubated with various endoglycosidases. The receptor, which has a pI ranging from 5.3 to 5.6, appears to be a glycoprotein with primarily N-linked sugar residues, some of which have high concentrations of mannose and some of which are complex oligosaccharides. Western blots (immunoblots) of electrophoretically transferred receptor incubated with various biotinylated lectins confirmed the presence of sugar moieties, including N-acetylglucosamine, glucose or mannose, and galactose, but not fucose or N-acetylgalactosamine. This gp86 receptor from uninfected HEL cells also incorporated radiolabeled phosphate from orthophosphoric acid, indicating that it is a constitutively phosphorylated receptor. Images PMID:1321272

  17. Role of beta 2-glycoprotein I and anti-phospholipid antibodies in activation of protein C in vitro.

    PubMed Central

    Keeling, D M; Wilson, A J; Mackie, I J; Isenberg, D A; Machin, S J

    1993-01-01

    AIMS--To investigate the effect of beta 2-glycoprotein I (beta 2 GPI) on the thrombin/thrombomodulin dependent activation of protein C; and to determine whether beta 2 GPI dependent anticardiolipin antibodies have any effect. METHODS--Protein C was activated by thrombin in the presence of thrombomodulin and phospholipid vesicles in an in vitro system. The effect of adding purified beta 2 GPI to this system was observed. Affinity purified anticardiolipin antibodies and total IgG from patients with anticardiolipin antibodies and the lupus anticoagulant were studied for their effects on protein C activation in the presence and absence of beta 2 GPI. RESULTS--beta 2-Glycoprotein I had no effect on the activity of preformed activated protein C. When the phospholipid vesicles were incubated with beta 2 GPI before the addition of protein C, the activation of protein C was inhibited in a dose dependent manner. With phosphatidylserine:phosphatidylcholine vesicles at a concentration of 1 microM:2 microM, beta 2 GPI began to inhibit the reaction at a concentration of 15 nM, and at 4 microM (the normal plasma concentration) the activation of protein C was reduced to 40%. Anticardiolipin antibodies had no demonstrable effect. CONCLUSIONS--beta 2-Glycoprotein I inhibits protein C activation in an in vitro system. Its physiological role is unknown but it has potential procoagulant as well as anticoagulant properties. An effect of antiphospholipid antibodies on protein C activation, which might explain their association with thrombosis, could not be shown. PMID:8227406

  18. Effect of binding of Calcofluor White on the carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) on the structure and dynamics of the protein moiety. A fluorescence study.

    PubMed

    Albani, J R

    2001-08-23

    Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously studied at low and high concentrations of Calcofluor compared to that of the protein. alpha1-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. At equimolar concentrations of Calcofluor and alpha1-acid glycoprotein, the fluorophore displays free motions [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94], while at high concentration of Calcofluor, its surrounding microenvironment is rigid, inducing the rigidity of the fluorophore itself [Albani, J. R.; Sillen, A.; Plancke, Y. D.; Coddeville, B.; Engelborghs, Y. Carbohydr. Res. 2000, 327, 333-340]. In the present work, red-edge excitation spectra and steady-state anisotropy studies performed on Trp residues in the presence of Calcofluor, showed that the apparent dynamics of Trp residues are not modified. However, deconvoluting the emission spectra with two different methods into different components, reveals that the structure of the protein matrix has been disrupted in the presence of high Calcofluor concentrations.

  19. Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein.

    PubMed

    Ledwitch, Kaitlyn V; Gibbs, Morgan E; Barnes, Robert W; Roberts, Arthur G

    2016-10-15

    The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Effect of human alpha 2HS glycoprotein on mouse macrophage function.

    PubMed Central

    Lewis, J G; André, C M

    1980-01-01

    alpha 2HS glycoprotein was isolated from normal adult serum. The ability of alpha 2HS glycoprotein to promote the endocytosis of radiolabelled DNA and radiolabelled latex particles by mouse macrophages was investigated. The results using both radiolabelled latex particles and radiolabelled DNA show that alpha 2HS glycoprotein enhances the ability of mouse macrophages to take up these radiolabelled substrates as compared to control cells. Images Figure 1 Figure 2 PMID:7439929

  1. The anti-Parkinson drug budipine is exported actively out of the brain by P-glycoprotein in mice.

    PubMed

    Uhr, Manfred; Ebinger, Martin; Rosenhagen, Marcus C; Grauer, Markus T

    P-glycoprotein, a product of the ABCB1 gene, is a plasma membrane transporter that exports certain drugs as well as endogenous substances against a concentration gradient in the intestines, kidney and testes. It also constitutes an important part of the blood-brain barrier, where it exports its substrates out of the brain back into the circulation. To investigate whether the uptake of the anti-Parkinson drug budipine into the brain is mediated by P-glycoprotein, abcb1ab(-/-) double knock-out mice and wild-type control mice received budipine continuously over 11 days via implanted osmotic infusion pumps at the rate of 30ug over 24h. Concentrations of the drug in plasma, brain, and organs were measured with HPLC. Budipine concentrations in the abcb1ab knock-out animals were 3.1 times higher than in control mice. This study confirms the important role P-gp plays at the blood-brain barrier and shows that budipine is a substrate of P-gp.

  2. Involvement of Endoplasmic Reticulum Chaperones in the Folding of Hepatitis C Virus Glycoproteins

    PubMed Central

    Choukhi, Amélie; Ung, Sophana; Wychowski, Czeslaw; Dubuisson, Jean

    1998-01-01

    The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding. PMID:9557669

  3. PREPARATION AND FURTHER CHARACTERIZATION OF THE MN GLYCOPROTEIN OF HUMAN ERYTHROCYTE MEMBRANES

    PubMed Central

    Cleve, Hartwig; Hamaguchi, Hideo; Hütteroth, Thomas

    1972-01-01

    Human erythrocyte membrane glycoproteins were solubilized and recovered in the aqueous phase after extraction of red cell ghosts with a mixture of chloroform and methanol. The major glycoprotein, the so-called MN glycoprotein, was prepared from this phase by gel filtration on Sepharose 4B columns in 6 M guanidine hydrochloride. By SDS-acrylamide gel electrophoresis the MN glycoproteins from the three major genetic types, MM, MN, and NN, were found to be relatively free from minor glycoprotein contaminants. The carbohydrate and amino acid composition did not reveal significant differences between the three MN genotypes. The specific activities of the purified glycoproteins were determined for inhibition of agglutinating anti-M and anti-N rabbit antisera and for inhibition of myxovirus hemagglutination. It was, furthermore, established that the purified MN glycoproteins contain a receptor for Phaseolus vulgaris phytohemagglutination. The red cell MN glycoprotein inhibits partially lymphocyte stimulation induced by unfractionated Phaseolus vulgaris phytohemagglutinin; the red cell MN glycoprotein does not influence the lymphocyte stimulation induced by purified Phaseolus vulgaris mitogen. PMID:4673323

  4. Isomeric N,N-bis(cyclohexanol)amine aryl esters: the discovery of a new class of highly potent P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors.

    PubMed

    Teodori, Elisabetta; Martelli, Cecilia; Salerno, Milena; Darghal, Nacira; Dei, Silvia; Garnier-Suillerot, Arlette; Gualtieri, Fulvio; Manetti, Dina; Scapecchi, Serena; Romanelli, Maria Novella

    2007-02-22

    A new series of P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors having a N,N-bis(cyclohexanol)amine scaffold have been designed, following the frozen analog approach. With respect to the parent flexible molecules, the new compounds show improved potency and efficacy. Among them, compound 1d, on anthracycline-resistant erythroleukemia K562 cells, is able to completely reverse Pgp-dependent MDR at low nanomolar concentration.

  5. Effect of P-glycoprotein modulation on the clinical pharmacokinetics and adverse effects of morphine

    PubMed Central

    Drewe, Jürgen; Ball, Howard A; Beglinger, Christoph; Peng, Bin; Kemmler, Andreas; Schächinger, Hartmut; Haefeli, Walter E

    2000-01-01

    Aims To investigate the effect of acute P-glycoprotein inhibition by the multidrug-resistance (MDR) modulator valspodar (SDZ PSC 833; PSC) on the pharmacokinetics, and potentially adverse pharmacodynamic effects of morphine, and its principal pharmacologically active metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Methods In a double-blind, three-way crossover study, the pharmacokinetic and potentially adverse pharmacodynamic effects (reaction time, transcutaneous PCO2, blood pressure) of morphine were compared with and without acute inhibition ofP-glycoprotein by PSC. The effects of PSC alone were also evaluated. The study was performed in 18 healthy male volunteers and pharmacodynamic effects analysed by measuring the area under the effect (AUE) curve. 150 mg PSC (or its placebo) was given as an i.v. infusion over 2 h. With the expected inhibition of Pgp 1 h after starting PSC infusion, 7.5 morphine HCl (or its placebo) was infused over 2 h. Results The infusion of PSC resulted in blood concentrations expected to inhibit Pgp mediated transport. While the pharmacokinetics of plasma morphine and M6G. were unaffected there was a small but statistically significant increase in the AUC and Cmax of M3G (11.8 and 8.3%, respectively). The t½ and tmax were unaffected. The pharmacokinetic parameters of PSC were not affected by coadministration with morphine. PSC did not significantly affect the adverse events of morphine, as assessed by spontaneous reporting. Compared with PSC alone, morphine elicited an increase in reaction time (Emax 48 ms, compared with the predose absolute reaction time of 644 ms), which was not detected by the alertness-drowsiness score, indicating only slight sedation. There was a significant decrease in systolic blood pressure (Emin−9 mmHg), and a trend for a fall in diastolic blood pressure (Emin−14.5 mmHg) and respiratory rate (Emin−1.8 breath·min−1). For all these parameters, the effects of PSC/morphine were

  6. Comparison of In Vitro Assays in Selecting Radiotracers for In Vivo P-Glycoprotein PET Imaging

    PubMed Central

    Savolainen, Heli; Cantore, Mariangela; van de Steeg, Evita; Colabufo, Nicola A.; Elsinga, Philip H.; Windhorst, Albert D.

    2017-01-01

    Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer´s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R)-N-[18F]fluoroethylverapamil, (R)-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM) and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM), P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands. PMID:29036881

  7. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates.

    PubMed

    Kwong, P D; Wyatt, R; Majeed, S; Robinson, J; Sweet, R W; Sodroski, J; Hendrickson, W A

    2000-12-15

    The gp120 exterior envelope glycoprotein of HIV-1 binds sequentially to CD4 and chemokine receptors on cells to initiate virus entry. During natural infection, gp120 is a primary target of the humoral immune response, and it has evolved to resist antibody-mediated neutralization. We previously reported the structure at 2.5 A of a gp120 core from the HXBc2 laboratory-adapted isolate in complex with a 2 domain fragment of CD4 and the antigen binding fragment of a human antibody. This revealed atomic details of gp120-receptor interactions and suggested multiple mechanisms of immune evasion. We have now extended the HXBc2 structure in P222, crystals to 2.2 A. The enhanced resolution enabled a more accurate modeling of less-well-ordered regions and provided conclusive identification of the density in the central cavity at the crux of the gp120-CD4 interaction as isopropanol from the crystallization medium. We have also determined the structure of a gp120 core from the primary clinical HIV-1 isolate, YU2, in the same ternary complex but in a C2 crystal lattice. Comparisons of HXBc2 and YU2 showed that while CD4 binding was rigid, portions of the gp120 core were conformationally flexible; overall differences were minor, with sequence changes concentrated on a surface expected to be exposed on the envelope oligomer. Despite dramatic antigenic differences between primary and laboratory-adapted HIV-1, the gp120 cores from these isolates are remarkably similar. Taken together with chimeric substitution and sequence analysis, this indicates that neutralization resistance is specified by quaternary interactions involving the major variable loops and thus affords a mechanism for viral adaptation. Conservation of the central cavity suggests the possibility of therapeutic inhibitors. The structures reported here extend in detail and generality our understanding of the biology of the gp120 envelope glycoprotein.

  8. P-Glycoprotein in skin contributes to transdermal absorption of topical corticosteroids.

    PubMed

    Hashimoto, Naoto; Nakamichi, Noritaka; Yamazaki, Erina; Oikawa, Masashi; Masuo, Yusuke; Schinkel, Alfred H; Kato, Yukio

    2017-04-15

    ATP binding cassette transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are expressed in skin, but their involvement in transdermal absorption of clinically used drugs remains unknown. Here, we examined their role in transdermal absorption of corticosteroids. Skin and plasma concentrations of dexamethasone after dermal application were reduced in P-gp and BCRP triple-knockout (Mdr1a/1b/Bcrp -/- ) mice. The skin concentration in Mdr1a/1b/Bcrp -/- mice was reduced in the dermis, but not in the epidermis, indicating that functional expression of these transporters in skin is compartmentalized. Involvement of these transporters in dermal transport of dexamethasone was also supported by the observation of a higher epidermal concentration in Mdr1a/1b/Bcrp -/- than wild-type mice during intravenous infusion. Transdermal absorption after dermal application of prednisolone, but not methylprednisolone or ethinyl estradiol, was also lower in Mdr1a/1b/Bcrp -/- than in wild-type mice. Transport studies in epithelial cell lines transfected with P-gp or BCRP showed that dexamethasone and prednisolone are substrates of P-gp, but are minimally transported by BCRP. Thus, our findings suggest that P-gp is involved in transdermal absorption of at least some corticosteroids in vivo. P-gp might be available as a target for inhibition in order to deliver topically applied drugs and cosmetics in a manner that minimizes systemic exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Disulfide bonds in hepatitis C virus glycoprotein E1 control the assembly and entry functions of E2 glycoprotein.

    PubMed

    Wahid, Ahmed; Helle, François; Descamps, Véronique; Duverlie, Gilles; Penin, François; Dubuisson, Jean

    2013-02-01

    Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein.

  10. Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

    PubMed Central

    Wahid, Ahmed; Helle, François; Descamps, Véronique; Duverlie, Gilles; Penin, François

    2013-01-01

    Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein. PMID:23175356

  11. QSAR studies of macrocyclic diterpenes with P-glycoprotein inhibitory activity.

    PubMed

    Sousa, Inês J; Ferreira, Maria-José U; Molnár, Joseph; Fernandes, Miguel X

    2013-02-14

    Multidrug resistance (MDR) represents a major limitation for cancer chemotherapy. There are several mechanisms of MDR but the most important is associated with P-glycoprotein (P-gp) overexpression. The development of modulators of P-gp that are able to re-establish drug sensitivity of resistant cells has been considered a promising approach for overcoming MDR. Macrocyclic lathyrane and jatrophane-type diterpenes from Euphorbia species were found to be strong MDR reversing agents. In this study we applied quantitative structure-activity relationship (QSAR) methodology in order to identify the most relevant molecular features of macrocyclic diterpenes with P-gp inhibitory activity and to determine which structural modifications can be performed to improve their activity. Using experimental biological data at two concentrations (4 and 40 μg/ml), we developed a QSAR model for a set of 51 bioactive diterpenic compounds which includes lathyrane and jatrophane-type diterpenes and another model just for jatrophanes. The cross-validation correlation values for all diterpenes QSAR models developed for biological activities at compound concentrations of 4 and 40 μg/ml were 0.758 and 0.729, respectively. Regarding the prediction ability, we get R²(pred) values of 0.765 and 0.534 for biological activities at compound concentrations of 4 and 40 μg/ml, respectively. Applying the cross-validation test to jatrophanes QSAR models, we obtained 0.680 and 0.787 for biological activities at compound concentrations of 4 and 40 μg/ml concentrations, respectively. For the same concentrations, the obtained R²(pred) values for jatrophanes models were 0.541 and 0.534, respectively. The obtained models were statistically valid and showed high prediction ability. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. The Ameliorated Pharmacokinetics of VP-16 in Wistar Rats: A Possible Role of P-Glycoprotein Inhibition by Pharmaceutical Excipients.

    PubMed

    Akhtar, Naseem; Ahad, Abdul; Khan, Mohd Faiyaz; Allaham, Ayman; Talegaonkar, Sushama

    2017-04-01

    The selection of suitable functional excipients with low toxicity index and having P-glycoprotein inhibitory characteristics represents a major innovative step in designing a promising formulation for oral chemotherapy. This study was aimed at investigating the chemosensitizing effect of selected pharmaceutical excipients to improve the in vivo pharmacokinetic performance of VP-16. The pharmaceutical excipients having P-glycoprotein inhibitory activity were screened by shake flask method for their VP-16 solubilization capacity. The cumulative amount of VP-16 was determined with or without the selected pharmaceutical excipients at three different concentrations (0.1 % w/v, 0.5 % w/v and 1 % w/v) by an everted gut sac technique. Moreover, pharmacokinetic studies were also performed to determine the oral bioavailability assessment of VP-16 in albino male Wistar rats. The absorptive transport from mucosal-to-serosal (M → S) and secretory transport from serosal-to-mucosal (S → M) for VP-16 solution over 90 min were found to be (3.58 ± 0.32) × 10 -6 and (14.63 ± 3.11) × 10 -6  cm/s, respectively, with a net efflux of 4.08. Addition of verapamil (200 µM), a P-glycoprotein inhibitor, elevated the transport from M → S [Papp from (3.58 ± 0.32) to (9.66 ± 1.55) × 10 -6 cm/s, p < 0.05] and lowered the S → M [Papp from (14.63 ± 3.11) to (13.35 ± 2.01) × 10 -6 cm/s, p < 0.01], with a net efflux of 1.38. The relative bioavailability of VP-16 following oral administration (4.5 mg/kg) in rats was increased significantly (p < 0.01) in presence of Labrasol micellar solution at a concentration of 5 % (w/v) when compared with VP-16 solution alone. The findings suggest that pharmaceutical excipients may be employed in the development of drug delivery systems to improve the oral bioavailability of drugs having low solubility and/or less permeability as a result of substantial P-glycoprotein mediated efflux.

  13. [The preparation and evaluation of the quality control materials for detection of platelet membrane glycoproteins by flow cytometry].

    PubMed

    Liu, Y Q; Gong, Y; Qu, C X; You, R; Li, L P; Xing, L S; Wang, J Z

    2017-04-11

    Objective: To prepare the quality control material for detection of platelet membrane glycoproteins by flowcytometry and evaluate the appearance traits, homogeneity and stability of it. Methods: Fresh platelets from the blood group O donors were fixed by the certain concentration of aldehyde solution and then washed by the imidazole buffer. After that, adding certain concentration of lyophilized protection solution into the preparations. The preparations were dispensed to be lyophilized and then were kept refrigerated in 2-8 ℃.According to the protocol of control of lyophilized biological products, the quality indicator for monitoring the prepared process, containing the appearance traits, the residual water, the platelet recovery and the rehydration quality were evaluated. The homogeneity and stability of these preparations were evaluated according to the CNAS-GL03 Guidance on evaluating the homogeneity and stability of samples used for proficiency testing and the ISO Guide 35 Reference material - general and statistical principles for certification . Results: The appearance traits and the rehydration quality of the quality control materials meeted the requirements, with the residual water distributed between 3.96% to 4.04% and the platelet recovery rate ranged from 68% to 72%.The homogeneity evaluation showed that there was no significant difference among the groups( P >0.05). The stability test indicated that the positive rate of platelet membrane glycoproteins CD42b, CD41 and CD62P of the quality control material was -0.14%, -0.14% and 0.74%, respectively, at 16 weeks after storage. There was no linear trend between the percentage of positive platelets with membrane glycoproteins and time( P >0.05). Conclusions: The quality control material for detection of platelet membrane glycoproteins by flow cytometry prepared by us meets the needs of the appearance traits, the residual water, the rehydration quality, the homogeneity and the longtime stability.It is

  14. Characterization of Monomeric Intermediates during VSV Glycoprotein Structural Transition

    PubMed Central

    Albertini, Aurélie A.; Mérigoux, Cécile; Libersou, Sonia; Madiona, Karine; Bressanelli, Stéphane; Roche, Stéphane; Lepault, Jean; Melki, Ronald; Vachette, Patrice; Gaudin, Yves

    2012-01-01

    Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (Gth, aa residues 1–422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, Gth is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition. PMID:22383886

  15. Molecular modeling, simulation and docking study of ebola virus glycoprotein.

    PubMed

    Ahmad, Nasir; Farman, Aqsa; Badshah, Syed Lal; Ur Rahman, Ata; Ur Rashid, Haroon; Khan, Khalid

    2017-03-01

    Ebola virus (EBOV) is a filamentous, enveloped, non-segmented, negative-strand ribonucleic acid (RNA) virus which belongs to family Filoviridae. Ebola virus includes different glycoproteins each of which plays their roles in different aspects of viral life cycle. In this study secreted glycoprotein (Q7T9E0) of Ebola virus was acquired from Uniprot. The formation of alpha helix and beta sheets of secondary structures were predicted through online servers. Higher flexibility and disordered regions of proteins were determined through RONN, GLOBPLOT and DISSEMBLE. Three dimensional (3D) structure of the protein was built through homology modeling techniques and MOE software. The validation and evaluation of the refined models were determined with two stereochemical tests i-e RAMPAGE and ERRAT servers. Further docking studies of given protein was performed with different derivatives of two antiviral drugs dronedarone and amiodarone through MOE. Docking score and binding affinity of respective derivatives demonstrate that these might be used as protein receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Modeling of Oligosaccharides within Glycoproteins from Free-Energy Landscapes.

    PubMed

    Turupcu, Aysegül; Oostenbrink, Chris

    2017-09-25

    In spite of the abundance of glycoproteins in biological processes, relatively little three-dimensional structural data is available for glycan structures. Here, we study the structure and flexibility of the vast majority of mammalian oligosaccharides appearing in N- and O-glycosylated proteins using a bottom up approach. We report the conformational free-energy landscapes of all relevant glycosidic linkages as obtained from local elevation simulations and subsequent umbrella sampling. To the best of our knowledge, this represents the first complete conformational library for the construction of N- and O-glycan structures. Next, we systematically study the effect of neighboring residues, by extensively simulating all relevant trisaccharides and one tetrasaccharide. This allows for an unprecedented comparison of disaccharide linkages in large oligosaccharides. With a small number of exceptions, the conformational preferences in the larger structures are very similar as in the disaccharides. This, finally, allows us to suggest several efficient approaches to construct complete N- and O-glycans on glycoproteins, as exemplified on two relevant examples.

  17. F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein.

    PubMed

    Lim, S H; Salton, M R

    1981-12-14

    It has been claimed (Andreu, JM, Warth, R. and Muñoz, E. (1978) FEBS Letter, 86, 1-5) that the F1-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F1-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F1 preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F1-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F1-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (alpha, beta, gamma, delta and epsilon) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F1-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F1-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.

  18. Glycoprotein NMB: an Emerging Role in Neurodegenerative Disease.

    PubMed

    Budge, Kevin M; Neal, Matthew L; Richardson, Jason R; Safadi, Fayez F

    2017-08-30

    Neurodegeneration is characterized by severe neuronal loss leading to the cognitive and physical impairments that define various neurodegenerative diseases. Neuroinflammation is one hallmark of neurodegenerative diseases and can ultimately contribute to disease progression. Increased inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1 β), and tumor necrosis factor-α (TNF-α) are associated with Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Unfortunately, current therapeutic options lack ability to stop or effectively slow progression of these diseases and are primarily aimed at alleviating symptoms. Thus, it is crucial to discover novel treatment candidates for neurodegenerative diseases. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type-I transmembrane glycoprotein first identified in a melanoma cell line. GPNMB augments bone mineral deposition by stimulating osteoblast differentiation. Aside from its anabolic function in the bone, emerging evidence suggests that GPNMB has anti-inflammatory and reparative functions. GPNMB has also been demonstrated to be neuroprotective in an animal model of ALS, cerebral ischemia, and other disease models. Given these discoveries, GPNMB should be investigated as a potential therapeutic option for multiple neurodegenerative diseases.

  19. Reconstitution in vitro of neurotoxin-responsive ion efflux by using membrane glycoproteins of neuroblastoma cells.

    PubMed Central

    Giovanni, M Y; Glick, M C

    1983-01-01

    Glycoproteins were purified from a clonal cell line of mouse neuroblastoma, N-18, labeled metabolically with L-[3H]fucose. The purified radioactive glycoproteins were reconstituted into artificial phosphatidylcholine vesicles. When the vesicles were preloaded with cesium acetate and treated with neurotoxins to activate the Na+ channel, a shift in intravesicular density was observed to a less dense position after centrifugation on sucrose gradients. This shift was partially inhibited by tetrodotoxin, which prevents the activation of the Na+ channel. A similarly derived fraction of [14C]fucose-containing glycoproteins from a neuroblastoma cell line that does not possess excitable membranes, N1A-103, was reconstituted into phospholipid vesicles, and, after preloading with cesium ions, the fraction was combined with those of the 3H-labeled glycoproteins of the differentiated cells, N-18, which have excitable membranes. Only the 3H-labeled glycoprotein-containing vesicles were responsive to the neurotoxins, as shown by a shift in intravesicular density on sucrose gradients. These results are interpreted as a demonstration of the reconstitution of glycoproteins to form the activated Na+ channel. Comparison of the radioactive glycoprotein profiles after polyacrylamide gel electrophoresis showed that glycoproteins of Mr 200,000, Mr 165,000, and Mr 65,000 were common to the reconstituted fractions that were biologically active. PMID:6308628

  20. Semisynthetic lectin-4-dimethylaminopyridine conjugates for labeling and profiling glycoproteins on live cell surfaces.

    PubMed

    Hayashi, Takahiro; Sun, Yedi; Tamura, Tomonori; Kuwata, Keiko; Song, Zhining; Takaoka, Yousuke; Hamachi, Itaru

    2013-08-21

    Glycoproteins on cell surfaces play important roles in biological processes, including cell-cell interaction/signaling, immune response, and cell differentiation. Given the diversity of the structure of glycans, labeling and imaging of selected glycoproteins are challenging, although several promising strategies have been developed recently. Here, we design and construct semisynthetic reactive lectins (sugar-binding proteins) that are able to selectively label glycoproteins. Congerin II, an animal galectin, and wheat germ agglutinin are conjugated with 4-dimethylaminopyridine (DMAP), a well-known acyl transfer catalyst by our affinity-guided DMAP method and Cu(I)-assisted click chemistry. Selective labeling of glycoproteins is facilitated by the DMAP-tethered lectin catalysts both in vitro and on living cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis enabled us to isolate labeled glycoproteins that are uniquely exposed on distinct cell lines. Furthermore, the combination of immunoprecipitation with mass spectrometry (MS)-fingerprinting techniques allowed us to characterize 48 glycoproteins endogenously expressed on HeLa cells, and some low-abundant glycoproteins, such as epidermal growth factor receptor (EGFR) and neuropilin-1, were successfully identified. Our results demonstrate that semisynthetic DMAP-tethered lectins provide a new tool for labeling and profiling glycoproteins on living cells.

  1. Concanavalin A acceptor glycoproteins: a new type of marker for the classification of tumour cells.

    PubMed Central

    Koch, G. L.; Smith, M. J.

    1983-01-01

    The Con A acceptor glycoproteins of murine and human tumour cell lines revealed by two-dimensional fingerprinting on polyacrylamide gels fall into two main categories: constant glycoproteins expressed by all cell lines and variable glycoproteins which are only expressed by particular tumour cell lines. Since the number of variable glycoproteins on a typical fingerprint is 50, fingerprints from different cell lines are readily distinguishable. However the variable glycoproteins are not expressed idiosyncratically and cell lines derived from similar classes of tumours express similar patterns of the variable glycoproteins. For example, murine fibrosarcomas express patterns which are virtually identical with one another. Characteristic patterns are also expressed by murine macrophage tumour lines, human carcinomas and human B lymphoblastoid cells. Thus, the variable glycoproteins behave as a set of linked markers which are indicators of the type of normal pre-neoplastic precursor cell from which a tumour is derived and appear to be a new type of marker for tumour cell classification. Antibodies to these glycoproteins could prove useful in tumour localisation and diagnosis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:6849797

  2. Concanavalin A acceptor glycoproteins: a new type of marker for the classification of tumour cells.

    PubMed

    Koch, G L; Smith, M J

    1983-04-01

    The Con A acceptor glycoproteins of murine and human tumour cell lines revealed by two-dimensional fingerprinting on polyacrylamide gels fall into two main categories: constant glycoproteins expressed by all cell lines and variable glycoproteins which are only expressed by particular tumour cell lines. Since the number of variable glycoproteins on a typical fingerprint is 50, fingerprints from different cell lines are readily distinguishable. However the variable glycoproteins are not expressed idiosyncratically and cell lines derived from similar classes of tumours express similar patterns of the variable glycoproteins. For example, murine fibrosarcomas express patterns which are virtually identical with one another. Characteristic patterns are also expressed by murine macrophage tumour lines, human carcinomas and human B lymphoblastoid cells. Thus, the variable glycoproteins behave as a set of linked markers which are indicators of the type of normal pre-neoplastic precursor cell from which a tumour is derived and appear to be a new type of marker for tumour cell classification. Antibodies to these glycoproteins could prove useful in tumour localisation and diagnosis.

  3. Purification and characterization of a soluble glycoprotein from garlic (Allium sativum) and its in vitro bioactivity.

    PubMed

    Wang, Yan; Zou, Tingting; Xiang, Minghui; Jin, Chenzhong; Zhang, Xuejiao; Chen, Yong; Jiang, Qiuqing; Hu, Yihong

    2016-10-02

    A soluble glycoprotein was purified to homogeneity from ripe garlic (Allium sativum) bulbs using ammonium sulfate precipitation, Sephadex G-100 gel filtration, and diethylaminoethyl-52 cellulose anion-exchange chromatography. A native mass of 55.7 kDa estimated on gel permeation chromatography and a molecular weight of 13.2 kDa observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis supported that the glycoprotein is a homotetramer. β-Elimination reaction result suggested that the glycoprotein is an N-linked type. Fourier-transform infrared spectroscopy proved that it contains sugar. Gas chromatography-mass spectrometer analysis showed that its sugar component was galactose. The glycoprotein has 1,1-diphenyl-2-picrylhydrazil free radical scavenging activity and the peroxidation inhibition ability to polyunsaturated fatty acid. These results indicated that the glycoprotein has potential for food additives, functional foods, and even biotechnological and medical applications.

  4. Increased platelet expression of glycoprotein IIIa following aspirin treatment in aspirin-resistant but not aspirin-sensitive subjects

    PubMed Central

    Floyd, Christopher N; Goodman, Timothy; Becker, Silke; Chen, Nan; Mustafa, Agnesa; Schofield, Emma; Campbell, James; Ward, Malcolm; Sharma, Pankaj; Ferro, Albert

    2014-01-01

    Aims Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. Methods Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B2, and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. Results In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. Conclusions In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker. PMID:25099258

  5. Adipokine zinc-α2-glycoprotein regulated by growth hormone and linked to insulin sensitivity.

    PubMed

    Balaz, Miroslav; Ukropcova, Barbara; Kurdiova, Timea; Gajdosechova, Lucia; Vlcek, Miroslav; Janakova, Zuzana; Fedeles, Jozef; Pura, Mikulas; Gasperikova, Daniela; Smith, Steven R; Tkacova, Ruzena; Klimes, Iwar; Payer, Juraj; Wolfrum, Christian; Ukropec, Jozef

    2015-02-01

    Hypertrophic obesity is associated with impaired insulin sensitivity and lipid-mobilizing activity of zinc-α2-glycoprotein. Adipose tissue (AT) of growth hormone (GH) -deficient patients is characterized by extreme adipocyte hypertrophy due to defects in AT lipid metabolism. It was hypothesized that zinc-α2-glycoprotein is regulated by GH and mediates some of its beneficial effects in AT. AT from patients with GH deficiency and individuals with obesity-related GH deficit was obtained before and after 5-year and 24-month GH supplementation therapy. GH action was tested in primary human adipocytes. Relationships of GH and zinc-α2-glycoprotein with adipocyte size and insulin sensitivity were evaluated in nondiabetic patients with noncancerous cachexia and hypertrophic obesity. AT in GH-deficient adults displayed a substantial reduction of zinc-α2-glycoprotein. GH therapy normalized AT zinc-α2-glycoprotein. Obesity-related relative GH deficit was associated with almost 80% reduction of zinc-α2-glycoprotein mRNA in AT. GH increased zinc-α2-glycoprotein mRNA in both AT of obese men and primary human adipocytes. Interdependence of GH and zinc-α2-glycoprotein in regulating AT morphology and metabolic phenotype was evident from their relationship with adipocyte size and AT-specific and whole-body insulin sensitivity. The results demonstrate that GH is involved in regulation of AT zinc-α2-glycoprotein; however, the molecular mechanism linking GH and zinc-α2-glycoprotein in AT is yet unknown. © 2014 The Obesity Society.

  6. P-glycoprotein in proteoliposomes with low residual detergent: the effects of cholesterol.

    PubMed

    Bucher, Karsten; Belli, Sara; Wunderli-Allenspach, Heidi; Krämer, Stefanie D

    2007-11-01

    There is evidence that cholesterol affects the ATPase and transport functions of P-glycoprotein (P-gp). To study the influence of cholesterol on P-gp in a well defined lipid environment, we reconstituted P-gp in egg phosphatidylcholine (PhC) and PhC/cholesterol proteoliposomes with negligible residual amounts of detergents. P-gp proteoliposomes were prepared by continuous dialysis from micelles consisting of P-gp, lipids, sodium dodecyl sulfate and cholate. Basal and modulator-induced ATPase activities were studied in an established enzyme assay. Modulator affinities to P-gp and to the lipid bilayers were determined by equilibrium dialysis. In the absence of cholesterol the basal ATPase activity was six fold lower than in the presence of 20 or 40% cholesterol, and no P-gp binding and ATPase induction was detected for the tested modulators verapamil and progesterone. In proteoliposomes containing 20 and 40% cholesterol, respectively, the modulators showed significant P-gp binding and ATPase activation. The concentration of the modulators for half maximal activation of the ATPase was higher with 40% than with 20% cholesterol. Cholesterol influences P-gp in three ways: (a) it enhances its basal ATPase activity, (b) it renders P-gp sensitive towards the modulators verapamil and progesterone and (c) it affects the modulator concentration at half maximal ATPase activation.

  7. P-glycoprotein inhibitors of natural origin as potential tumor chemo-sensitizers: A review

    PubMed Central

    Abdallah, Hossam M.; Al-Abd, Ahmed M.; El-Dine, Riham Salah; El-Halawany, Ali M.

    2014-01-01

    Resistance of solid tumors to treatment is significantly attributed to pharmacokinetic reasons at both cellular and multi-cellular levels. Anticancer agent must be bio-available at the site of action in a cytotoxic concentration to exert its proposed activity. P-glycoprotein (P-gp) is a member of the ATP-dependent membrane transport proteins; it is known to pump substrates out of cells in ATP-dependent mechanism. The over-expression of P-gp in tumor cells reduces the intracellular drug concentrations, which decreases the cytotoxicity of a broad spectrum of antitumor drugs. Accordingly, P-gp inhibitors/blockers are potential enhancer for the cellular bioavailability of several clinically important anticancer drugs such as, anthracyclines, taxanes, vinca alkaloids, and podophyllotoxins. Besides several chemically synthesized P-gp inhibitors/blockers, some naturally occurring compounds and plant extracts were reported for their modulation of multidrug resistance; however, this review will focus only on major classes of naturally occurring inhibitors viz., flavonoids, coumarins, terpenoids, alkaloids and saponins. PMID:25685543

  8. Effects of polyoxyethylene (40) stearate on the activity of P-glycoprotein and cytochrome P450.

    PubMed

    Zhu, Saijie; Huang, Rongqin; Hong, Minghuang; Jiang, Yanyan; Hu, Zhuohan; Liu, Chen; Pei, Yuanying

    2009-07-12

    The present study was aimed to investigate the effects of polyoxyethylene (40) stearate (PS), a non-ionic surfactant, on the activity of P-glycoprotein (P-gp) and six major cytochrome P450 (CYP) isoforms. An in vitro diffusion chamber system was utilized to estimate the effects of PS concentration on the transport characteristics of Rhodamine 123 (R123) and Rhodamine 110 (R110), a standard P-gp substrate and nonsubstrate, respectively, across the excised intestinal segments of rat. Caco-2 cells were cultured to investigate the mechanisms by estimating the effects of PS on intracellular ATP levels, P-gp ATPase activity and membrane fluidity. The obtained results showed that PS inhibited P-gp mediated efflux in a concentration-dependent manner mainly by modulating substrate-stimulated P-gp ATPase activity. On the other hand, human liver microsomes were utilized to examine the inhibitive potential of PS on six major CYP isoforms. Inhibitive potential on two of these CYP2C9 and CYP2C19 was found to be clinically significant. In conclusion, PS is potentially useful as a pharmaceutical ingredient to improve the oral bioavailability of coadministered P-gp substrates and substrates for certain CYP isoforms.

  9. Moxidectin has a lower neurotoxic potential but comparable brain penetration in P-glycoprotein-deficient CF-1 mice compared to ivermectin.

    PubMed

    Janko, C; Geyer, J

    2013-06-01

    The anti-parasitic drugs ivermectin (IVM) and moxidectin (MOX) normally show limited brain penetration in vertebrates because of effective drug efflux at the blood-brain barrier by P-glycoprotein, encoded by the multi-drug resistance (MDR1) gene. However, dogs with homozygous nt230(del4) mutation in the MDR1 gene do not express a functionally active P-glycoprotein and show increased brain penetration of these drugs, resulting in neurological toxicity to different degrees. Thus, whereas IVM provokes neurological toxicity at 0.1 mg/kg, MOX is tolerated at this dosage. To investigate whether this difference is attributable to lower brain penetration of MOX in the absence of P-glycoprotein or to their neurotoxic potential, we applied IVM and MOX to P-glycoprotein-deficient CF-1 mice and comparatively analysed the absolute drug concentrations in the brain. Furthermore, we quantified drug-induced neurotoxicity by measuring the walking performance of the mice on a rotarod setup. We found that at a dosage of 0.2 mg/kg, representing 0.23 μmol/kg IVM and 0.31 μmol/kg MOX, the absolute drug concentrations in the brain were comparable with 100.8 pmol/g and 140.2 pmol/g, respectively. However, MOX induced the same degree of neurotoxicosis at the higher dosage of 1.09 μmol/kg (0.7 mg/kg) compared with IVM at 0.40 μmol/kg (0.35 mg/kg), demonstrating the 2.7-fold lower neurotoxic potential of MOX compared to IVM. This could be explained by a lower binding affinity or lower intrinsic activity of MOX at the relevant central nervous system receptors compared with IVM. © 2012 Blackwell Publishing Ltd.

  10. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein.

    PubMed

    Keck, Zhen-Yong; Enterlein, Sven G; Howell, Katie A; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L; Froude, Jeffrey W; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M; Wirchnianski, Ariel S; Lau, Patrick; Wang, Yong; Herbert, Andrew S; Dye, John M; Glass, Pamela J; Holtsberg, Frederick W; Foung, Steven K H; Aman, M Javad

    2016-01-01

    Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of

  11. Disulfide Bond Configuration of Human Cytomegalovirus Glycoprotein B

    PubMed Central

    Lopper, Matthew; Compton, Teresa

    2002-01-01

    Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically

  12. Disulfide bond configuration of human cytomegalovirus glycoprotein B.

    PubMed

    Lopper, Matthew; Compton, Teresa

    2002-06-01

    Glycoprotein B (gB) is the most highly conserved of the envelope glycoproteins of human herpesviruses. The gB protein of human cytomegalovirus (CMV) serves multiple roles in the life cycle of the virus. To investigate structural properties of gB that give rise to its function, we sought to determine the disulfide bond arrangement of gB. To this end, a recombinant form of gB (gB-S) comprising the entire ectodomain of the glycoprotein (amino acids 1 to 750) was constructed and expressed in insect cells. Proteolytic fragmentation and mass spectrometry were performed using purified gB-S, and the five disulfide bonds that link 10 of the 11 highly conserved cysteine residues of gB were mapped. These bonds are C94-C550, C111-C506, C246-C250, C344-C391, and C573-C610. This configuration closely parallels the disulfide bond configuration of herpes simplex type 2 (HSV-2) gB (N. Norais, D. Tang, S. Kaur, S. H. Chamberlain, F. R. Masiarz, R. L. Burke, and F. Markus, J. Virol. 70:7379-7387, 1996). However, despite the high degree of conservation of cysteine residues between CMV gB and HSV-2 gB, the disulfide bond arrangements of the two homologs are not identical. We detected a disulfide bond between the conserved cysteine residue 246 and the nonconserved cysteine residue 250 of CMV gB. We hypothesize that this disulfide bond stabilizes a tight loop in the amino-terminal fragment of CMV gB that does not exist in HSV-2 gB. We predicted that the cysteine residue not found in a disulfide bond of CMV gB, cysteine residue 185, would play a role in dimerization, but a cysteine substitution mutant in cysteine residue 185 showed no apparent defect in the ability to form dimers. These results indicate that gB oligomerization involves additional interactions other than a single disulfide bond. This work represents the second reported disulfide bond structure for a herpesvirus gB homolog, and the discovery that the two structures are not identical underscores the importance of empirically

  13. Interaction of macrocyclic lactones with a Dirofilaria immitis P-glycoprotein.

    PubMed

    Mani, Thangadurai; Bourguinat, Catherine; Keller, Kathy; Ashraf, Shoaib; Blagburn, Byron; Prichard, Roger K

    2016-09-01

    Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of

  14. Insights into the trimeric HIV-1 envelope glycoprotein structure.

    PubMed

    Ward, Andrew B; Wilson, Ian A

    2015-02-01

    The HIV-1 envelope glycoprotein (Env) trimer is responsible for receptor recognition and viral fusion with CD4(+) T cells, and is the sole target for neutralizing antibodies. Thus, understanding its molecular architecture is of significant interest. However, the Env trimer has proved to be a challenging target for 3D structure determination. Recent electron microscopy (EM) and X-ray structures have at last enabled us to decipher the structural complexity and unique features of the Env trimer, and how it is recognized by an ever-expanding arsenal of potent broadly neutralizing antibodies. We describe our current knowledge of the Env trimer structure in the context of exciting recent developments in the identification and characterization of HIV broadly neutralizing antibodies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

    NASA Astrophysics Data System (ADS)

    Schmidt, A. E.; Shvetsov, A. V.; Kuklin, A. I.; Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V.

    2016-01-01

    Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantial contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.

  16. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    PubMed Central

    Liu, Bo

    2013-01-01

    Abstract Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease. PMID:23697737

  17. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    SciTech Connect

    Kong, Leopold; Giang, Erick; Nieusma, Travis

    2014-08-26

    Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structuresmore » differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.« less

  18. Characterizing Glycoproteins by Mass Spectrometry in Campylobacter jejuni.

    PubMed

    Scott, Nichollas E

    2017-01-01

    The glycosylation systems of Campylobacter jejuni (C. jejuni) are considered archetypal examples of both N- and O-linked glycosylations in the field of bacterial glycosylation. The discovery and characterization of these systems both have revealed important biological insight into C. jejuni and have led to the refinement and enhancement of methodologies to characterize bacterial glycosylation. In general, mass spectrometry-based characterization has become the preferred methodology for the study of C. jejuni glycosylation because of its speed, sensitivity, and ability to enable both qualitative and quantitative assessments of glycosylation events. In these experiments the generation of insightful data requires the careful selection of experimental approaches and mass spectrometry (MS) instrumentation. As such, it is essential to have a deep understanding of the technologies and approaches used for characterization of glycosylation events. Here we describe protocols for the initial characterization of C. jejuni glycoproteins using protein-/peptide-centric approaches and discuss considerations that can enhance the generation of insightful data.

  19. Human milk glycoproteins protect infants against human pathogens.

    PubMed

    Liu, Bo; Newburg, David S

    2013-08-01

    Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease.

  20. Crystal Structure of the Human Cytomegalovirus Glycoprotein B

    PubMed Central

    Burke, Heidi G.; Heldwein, Ekaterina E.

    2015-01-01

    Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies. PMID:26484870

  1. N,N-bis(cyclohexanol)amine aryl esters inhibit P-glycoprotein as transport substrates.

    PubMed

    Neri, Annalisa; Frosini, Maria; Valoti, Massimo; Cacace, Marcello G; Teodori, Elisabetta; Sgaragli, Giampietro

    2011-12-15

    P-Glycoprotein (Pgp) inhibition by three sets of four isomers of N,N-bis(cyclohexanol)amine aryl esters was assessed on rhodamine 123 (R123) efflux in human MDR1-gene transfected mouse T-lymphoma L5178 cells and on Sf9 ATPase activity. The most active compounds inhibited Pgp with IC(50) values much lower than those of either cyclosporin A (CSA) or GF120918. As to R123 efflux inhibition, the role of the bond present in the second aryl moiety appeared important since the triple bond derivatives (3a-d) were the most powerful as compared to the double bond (2a-d) and the single bond (1a-d) counterparts. Concentration-inhibition curves of 2c and 3d exhibited a biphasic behaviour suggesting the existence of two binding sites in the recognition domain of Pgp. Persistence of inhibition by these compounds resulted to be intermediate between that caused by CSA and GF120918. R123 exhibited positive interaction with CSA, 1d, 1c, 2d, 2c and 3c, the concentration-inhibition curves being shifted leftward when R123 concentration was increased, while it exhibited negative interaction with 3d and no effect with GF120918. Sf9 ATPase activity was stimulated in an increasing order of potency by 2c, 3c, 2d, CSA, epirubicin and 3d. In a decreasing order of potency 3d, 2c, GF120918, CSA, 2d and 3c inhibited at sub-nanomolar concentrations epirubicin-stimulated ATPase activity. In conclusion, isomeric geometry and restriction of molecular flexibility of N,N-bis(cyclohexanol)amine aryl esters were crucial for their presentation to and inhibition of Pgp as transport substrates, R123 and epirubicin cooperating with them to this inhibition. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. The Remarkable Transport Mechanism of P-glycoprotein; a Multidrug Transporter

    PubMed Central

    Al-Shawi, Marwan K.; Omote, Hiroshi

    2006-01-01

    Human P-glycoprotein (ABCB1) is a primary multidrug transporter located in plasma membranes, that, utilizes the energy of ATP hydrolysis to pump toxic xenobiotics out of cells. P-glycoprotein employs a most unusual molecular mechanism to perform this drug transport function. Here we review our work to elucidate the molecular mechanism of drug transport by P-glycoprotein. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Development of novel spin-labeled transport substrates has allowed for quantitative and rigorous measurements of drug transport in real time by EPR spectroscopy. We have developed a new drug transport model of P-glycoprotein from the results of mutagenic, quantitative thermodynamic and kinetic studies. This model satisfactorily accounts for most of the unusual kinetic, coupling and physiological features of P-glycoprotein. Additionally, an atomic detail structural model of P-glycoprotein has been devised to place our results within a proper structural context. PMID:16691488

  3. Triflamp, a snake venom metalloproteinase, reduces neutrophil-platelet adhesion through proteolysis of PSGL-1 but not glycoprotein Ib alpha.

    PubMed

    Tseng, Yu-Lun; Lee, Chia-Jung; Hsu, Chun-Chieh; Huang, Tur-Fu

    2004-06-01

    Triflamp, a metalloproteinase isolated from Trimeresurus flavo-viridis, inhibits heterotypic adhesion between platelets and neutrophils. Coincubation studies demonstrate that direct interaction of triflamp with neutrophils is sufficient to inhibit the formation of neutrophil-platelet complexes. Its anti-adhesive effect is in a concentration- and incubation time-dependent manner. Triflamp reduces the expression of P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils and glycoprotein (GP) Ibalpha on platelets as probed by flow cytometry and Western blot. Moreover, triflamp disrupts P-selectin-mediated adhesion by cleaving PSGL-1 from the neutrophil surface. There are obvious differences regarding PSGL-1 proteolysis by triflamp and cathepsin G. Besides the NH2-terminus of PSGL-1, other sites are truncated by triflamp. The inhibitory effect of triflamp on PSGL-1 expression was prevented by pretreatment with a metalloproteinase inhibitor, phenanthroline. However, triflamp-treated platelets fully keep the ability for binding to PAF- or fMLP-stimulated neutrophils. Our results indicate that degradation of platelet GPIb alpha by triflamp does not interfere with neutrophil-platelet adhesion. Its effect on neutrophil PSGL-1 appears to be a critical factor for its inhibition on neutrophil-platelet interaction.

  4. Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study.

    PubMed Central

    Donald, A S; Feeney, J

    1986-01-01

    Treatment of Tamm-Horsfall urinary glycoprotein with Bacteroides fragilis endo-beta-galactosidase over a range of enzyme concentrations, pH and temperature resulted in the release of a small but constant proportion of the terminal sugars, which indicates the presence in the glycoprotein of relatively few enzyme-susceptible -GlcNAc beta 1-3Gal beta 1-4GlcNAc- units. Three oligosaccharides were isolated from the enzyme digest and characterized as Gal beta 1-4GlcNAc beta 1-3Gal, NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4GlcNAc beta 1-3Gal by methylation analysis and exo-glycosidase digestion. The alditols of these oligosaccharides and related structures were examined by 500 MHz 1H-n.m.r. spectroscopy aided by spin-spin decoupling and two-dimensional correlated spectroscopy. An almost complete assignment of proton shifts was possible, and significant differences between the signals of some of the protons in the blood-group-Sda-active oligosaccharide III and literature values for the corresponding signals in the structurally related Cad-blood-group determinant are noted. PMID:3098239

  5. Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay.

    PubMed

    Urbanowicz, Richard A; McClure, C Patrick; King, Barnabas; Mason, Christopher P; Ball, Jonathan K; Tarr, Alexander W

    2016-09-01

    Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, preclinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from quasispecies in individual patients were discovered to behave very differently in this entry model. Empirical optimization of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterized as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) was also sensitive to the amount and ratio of plasmids used, and that protocols for optimal production of these pseudoviruses are dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilizing pseudoviruses to conduct empirical optimization of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping.

  6. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion

    PubMed Central

    AlHajri, Salim M.; Nicola, Anthony V.; Aguilar, Hector C.; Li, Hong

    2017-01-01

    ABSTRACT Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism. IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of

  7. Dietary Hizikia fusiformis glycoprotein-induced IGF-I and IGFBP-3 associated to somatic growth, polyunsaturated fatty acid metabolism, and immunity in juvenile olive flounder Paralichthys olivaceus.

    PubMed

    Choi, Youn Hee; Kim, Kang-Woong; Han, Hyon-Sob; Nam, Taek Jeong; Lee, Bong-Joo

    2014-01-01

    This study was aimed to examine the effect of dietary glycoprotein extracted from the sea mustard Hizikia fusiformis (Phaeophyceae: Sargassaceae) as a dietary supplement on growth performance in association with somatotropin level, proximate compositions, and immunity in juvenile olive flounder Paralichthys olivaceus. Water-ethanol extracted glycoprotein from H. fusiformis was supplemented to three fishmeal-based diets at the concentration of 0, 5, and 10gkg(-1) diet (designated as H0, H5, and H10, respectively). After a 12week-long feeding trial, growth performance and biochemical responses were analyzed including proximate composition, and whole body amino acids and fatty acids. We also measured plasma insulin like growth factor (IGF), IGF-binding protein (IGFBP) and interleukin (IL). The fish fed H5 showed the greatest weight gain among the dietary treatments. In parallel with the growth, the fish fed the diets containing H. fusiformis glycoprotein showed an increased plasma IGF-I activity and increased expression of 43-kDa IGFBP-3 compared to that in the control, whereas an opposite trend was observed for 34-kDa IGFBP-1. Although no differences were found in the level of whole body linoleic acid (C18:2n-6) and linolenic acid (C18:3n-3) among treatments, increases in arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were observed in fish fed H5 compared to control. IL-2 and -6 levels increased significantly in fish fed H10 compared to those in the control indicating increased immunity. These results suggest that supplementation of H. fusiformis glycoprotein in fish diet may be beneficial for fish growth and immunity in juvenile olive flounder. © 2013.

  8. HDL Glycoprotein Composition and Site-Specific Glycosylation Differentiates Between Clinical Groups and Affects IL-6 Secretion in Lipopolysaccharide-Stimulated Monocytes

    PubMed Central

    Krishnan, Sridevi; Shimoda, Michiko; Sacchi, Romina; Kailemia, Muchena J.; Luxardi, Guillaume; Kaysen, George A.; Parikh, Atul N.; Ngassam, Viviane N.; Johansen, Kirsten; Chertow, Glenn M.; Grimes, Barbara; Smilowitz, Jennifer T.; Maverakis, Emanual; Lebrilla, Carlito B.; Zivkovic, Angela M.

    2017-01-01

    The goal of this pilot study was to determine whether HDL glycoprotein composition affects HDL’s immunomodulatory function. HDL were purified from healthy controls (n = 13), subjects with metabolic syndrome (MetS) (n = 13), and diabetic hemodialysis (HD) patients (n = 24). Concentrations of HDL-bound serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), apolipoprotein A-I (ApoA-I), apolipoprotein C-III (ApoC-III), α-1-antitrypsin (A1AT), and α-2-HS-glycoprotein (A2HSG); and the site-specific glycovariations of ApoC-III, A1AT, and A2HSG were measured. Secretion of interleukin 6 (IL-6) in lipopolysaccharide-stimulated monocytes was used as a prototypical assay of HDL’s immunomodulatory capacity. HDL from HD patients were enriched in SAA, LBP, ApoC-III, di-sialylated ApoC-III (ApoC-III2) and desialylated A2HSG. HDL that increased IL-6 secretion were enriched in ApoC-III, di-sialylated glycans at multiple A1AT glycosylation sites and desialylated A2HSG, and depleted in mono-sialylated ApoC-III (ApoC-III1). Subgroup analysis on HD patients who experienced an infectious hospitalization event within 60 days (HD+) (n = 12), vs. those with no event (HD−) (n = 12) showed that HDL from HD+ patients were enriched in SAA but had lower levels of sialylation across glycoproteins. Our results demonstrate that HDL glycoprotein composition, including the site-specific glycosylation, differentiate between clinical groups, correlate with HDL’s immunomodulatory capacity, and may be predictive of HDL’s ability to protect from infection. PMID:28287093

  9. Gefarnate stimulates mucin-like glycoprotein secretion in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models

    PubMed Central

    Dota, Atsuyoshi; Takaoka-Shichijo, Yuko; Nakamura, Masatsugu

    2013-01-01

    Purpose The aim of this study was to evaluate the effect of gefarnate on mucin-like glycoprotein secretion in isolated rabbit conjunctival tissue, and on corneal epithelial damage in rabbit and cat dry-eye models. Methods Conjunctival tissue isolated from rabbits was treated with gefarnate. Mucin-like glycoprotein was detected in the culture supernatant by an enzyme-linked lectin assay. Gefarnate ointment was topically applied to eyes once daily for 7 days in the rabbit dry-eye model, in which the lacrimal glands, Harderian gland, and nictitating membrane were removed, or for 4 weeks in the cat dry-eye model, in which the lacrimal gland and nictitating membrane were removed. Corneal epithelial damage was evaluated by measurement of corneal permeability by rose bengal in the rabbit model or by fluorescein staining in the cat model. Results Gefarnate stimulated mucin-like glycoprotein secretion in conjunctival tissue in a dose-dependent manner. In the rabbit dry-eye model, application of gefarnate ointment to the eyes resulted in a dose-dependent decrease in rose bengal permeability in the cornea, with the effect being significant at concentrations of ≥0.3%. In the cat dry-eye model, application of gefarnate ointment resulted in a significant decrease in the corneal fluorescein staining score. Conclusion These results suggest that gefarnate stimulates in vitro secretion of mucin-like glycoprotein in conjunctival tissue and ameliorates corneal epithelial damage in animal dry-eye models. Gefarnate may therefore be effective for treating dry eye. PMID:23386781

  10. Human cartilage glycoprotein 39--biomarker of joint damage in knee osteoarthritis.

    PubMed

    Zivanović, Sandra; Rackov, Ljiljana Petrović; Vojvodić, Danilo; Vucetić, Dusan

    2009-08-01

    Human glycoprotein of cartilage (YKL-40) synthesizes chondrocytes and synovial cells in inflammatory conditions or remodels the outer cell matrix in osteoarthritis. The aim of this study was to conduct a parallel analysis between thickness of cartilage and length of osteophytes, ultrasound indicators of joint destruction, with levels of YKL-40 in serum in patients with primary osteoarthritis. Ultrasound findings and concentration of YKL-40(ng/ml) were examined in 88 patients. The average value cartilage thickness measured on medial condyles of the femur was 1.30 +/- 0.23 mm and on lateral was 1.39 +/- 0.27 mm. Median YKL-40 in patients with shorter osteophytes was 62.0 (44.5-90) ng/ml, and with longer osteophytes was 119 (range 80-171) ng/ml (p = 0.000). YKL-40 can be a marker for the appearance of longer osteophytes (sensitivity = 79.1%; specificity = 61.9%; cut off = 75.0 ng/ml). The duration of illness is very much connected to values of YKL-40 (r = 0.651, p = 0.000). After an illness duration of five years, the concentration of YKL-40 was 83.68 +/- 33.65 ng/ml, after ten years it was 138.22 +/- 48.88 ng/ml, and after 15 and 20 years it was 209.30 +/- 79.36 ng/ml and 218.50 +/- 106.51 ng/ml, respectively. Higher concentrations of YKL-40 indicate the level of cartilage destruction and can be used for assessment of destruction.

  11. Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay

    PubMed Central

    Jouan, Elodie; Le Vée, Marc; Mayati, Abdullah; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-01-01

    In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug–drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context, the present study was designed to investigate the use of the fluorescent dye rhodamine 123 as a reference P-gp substrate probe for characterizing P-gp inhibitory potential of 16 structurally-unrelated drugs known to interact with P-gp. 14/16 of these P-gp inhibitors were found to increase rhodamine 123 accumulation in P-gp-overexpressing MCF7R cells, thus allowing the determination of their P-gp inhibitory potential, i.e., their half maximal inhibitor concentration (IC50) value towards P-gp-mediated transport of the dye. These IC50 values were in the range of variability of previously reported IC50 for P-gp and can be used for the prediction of clinical P-gp inhibition according to Food and Drug Administration (FDA) criteria, with notable sensitivity (80%). Therefore, the data demonstrated the feasibility of the use of rhodamine 123 for evaluating the P-gp inhibitory potential of drugs. PMID:27077878

  12. Inhibition of P-Glycoprotein Mediated Efflux in Caco-2 Cells by Phytic Acid.

    PubMed

    Li, Lujia; Fu, Qingxue; Xia, Mengxin; Xin, Lei; Shen, Hongyi; Li, Guowen; Ji, Guang; Meng, Qianchao; Xie, Yan

    2018-01-31

    Phytic acid (IP6) is a natural phosphorylated inositol, which is abundantly present in most cereal grains and seeds. This study investigated the effects of IP6 regulation on P-glycoprotein (P-gp) and its potential mechanisms using in situ and in vitro models. The effective permeability of the typical P-gp substrate rhodamine 123 (R123) in colon was significantly increased from (1.69 ± 0.22) × 10 -5 cm/s in the control group to (3.39 ± 0.417) × 10 -5 cm/s (p < 0.01) in the 3.5 mM IP6 group. Additionally, IP6 can concentration-dependently decrease the R123 efflux ratio in both Caco-2 and MDCK II-MDR1 cell monolayers and increase intracellular R123 accumulation in Caco-2 cells. Furthermore, IP6 noncompetitively inhibited P-gp by impacting R123 efflux kinetics. The noncompetitive inhibition of P-gp by IP6 was likely due to decreases in P-gp ATPase activity and P-gp molecular conformational changes induced by IP6. In summary, IP6 is a promising P-gp inhibitor candidate.

  13. Advances in Studies of P-Glycoprotein and Its Expression Regulators.

    PubMed

    Yano, Kentaro; Tomono, Takumi; Ogihara, Takuo

    2018-01-01

    This review deals with recent advances in studies on P-glycoprotein (P-gp) and its expression regulators, focusing especially on our own research. Firstly, we describe findings demonstrating that the distribution of P-gp along the small intestine is heterogeneous, which explains why orally administered P-gp substrate drugs often show bimodal changes of plasma concentration. Secondly, we discuss the post-translational regulation of P-gp localization and function by the scaffold proteins ezrin, radixin and moesin (ERM proteins), together with recent reports indicating that tissue-specific differences in regulation by ERM proteins in normal tissues might be retained in corresponding cancerous tissues. Thirdly, we review evidence that P-gp activity is enhanced in the process of epithelial-to-mesenchymal transition (EMT), which is associated with cancer progression, without any increase in expression of P-gp mRNA. Finally, we describe two examples in which P-gp critically influences the brain distribution of drugs, i.e., oseltamivir, where low levels of P-gp associated with early development allow oseltamivir to enter the brain, potentially resulting in neuropsychiatric side effects in children, and cilnidipine, where impairment of P-gp function in ischemia allows cilnidipine to enter the ischemic brain, where it exerts a neuroprotective action.

  14. Directly probing the antifreeze glycoprotein kinetics at the ice/solution interface

    NASA Astrophysics Data System (ADS)

    Zepeda, Salvador; Yokoyama, Etsuro; Furukawa, Yoshinor

    2009-03-01

    Antifreeze proteins (AFP) and glycoproteins (AFGP) help fish, plants, insects and bacteria survive sub-freezing environments. It is well known that these proteins function via some surface interaction, but the exact mechanism has eluded scientists. Aside from mutagenesis experiments directed towards examining the functional importance of specific residues, conclusions about the mechanism have been drawn from indirect studies or more precisely from studies that describe the proteins effects on the ice interface. Our work is aimed at directly studying the protein kinetics at the ice/solution interface. Fluorescent microscopy is used to determine interaction planes, surface concentrations as well as adsorption characteristics and the segregation constants, while fourier transform infra-red attenuated total reßectance (FTIR-ATR) is used to determine the protein structure vs. temperature in the liquid and solid states as well as the ice interface characteristics. All data show that AFGP do not function by the characteristic Gibbs-Thomson mechanism. While the surface coverage is similar for the AFPIII, segregation (amount in ice/amount in solution) is non-zero.

  15. Detection of glycoprotein using fiber optic surface plasmon resonance sensors with boronic acid

    NASA Astrophysics Data System (ADS)

    Wang, Fang; Zhang, Yang; Liu, Zigeng; Qian, Siyu; Gu, Yiying; Jing, Zhenguo; Sun, Changsen; Peng, Wei

    2017-04-01

    In this paper, we present a tilted fiber Bragg gratings (TFBG) based surface Plasmon resonance (SPR) label-free sensors with boronic acid derivative (ABA-PBA) as receptor molecule to detect glycoprotein with high sensitivity and selectivity. Tilted fiber Bragg gratings (TFBG) as a near infrared wavelengths detecting element can be able to excite a number of cladding modes whose properties can be detected accurately by measuring the variation of transmitted spectra. A 10° TFBG coated by 50nm gold film was manufactured to stimulate surface plasmon resonance on the surface of the sensor. The sensor was loaded with boronic acid derivative as the recognition molecule which has been widely used in various areas for the recognition matrix of diol-containing biomolecules. The proposed TFBG-SPR sensors exhibit good selectivity and repeatability with the protein concentration sensitivity up to 2.867dB/ (mg/ml) and the limit of detection was 2*10-5g/ml.

  16. Intercalated cell-specific Rh B glycoprotein deletion diminishes renal ammonia excretion response to hypokalemia

    PubMed Central

    Bishop, Jesse M.; Lee, Hyun-Wook; Handlogten, Mary E.; Han, Ki-Hwan; Verlander, Jill W.

    2013-01-01

    The ammonia transporter family member, Rh B Glycoprotein (Rhbg), is an ammonia-specific transporter heavily expressed in the kidney and is necessary for the normal increase in ammonia excretion in response to metabolic acidosis. Hypokalemia is a common clinical condition in which there is increased renal ammonia excretion despite the absence of metabolic acidosis. The purpose of this study was to examine Rhbg's role in this response through the use of mice with intercalated cell-specific Rhbg deletion (IC-Rhbg-KO). Hypokalemia induced by feeding a K+-free diet increased urinary ammonia excretion significantly. In mice with intact Rhbg expression, hypokalemia increased Rhbg protein expression in intercalated cells in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Deletion of Rhbg from intercalated cells inhibited hypokalemia-induced changes in urinary total ammonia excretion significantly and completely prevented hypokalemia-induced increases in urinary ammonia concentration, but did not alter urinary pH. We conclude that hypokalemia increases Rhbg expression in intercalated cells in the cortex and outer medulla and that intercalated cell Rhbg expression is necessary for the normal increase in renal ammonia excretion in response to hypokalemia. PMID:23220726

  17. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant

  18. Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae.

    PubMed

    Ciplys, Evaldas; Samuel, Dhanraj; Juozapaitis, Mindaugas; Sasnauskas, Kęstutis; Slibinskas, Rimantas

    2011-05-19

    The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic

  19. Immunoinformatic Analysis of Crimean Congo Hemorrhagic Fever Virus Glycoproteins and Epitope Prediction for Synthetic Peptide Vaccine.

    PubMed

    Tipu, Hamid Nawaz

    2016-02-01

    To determine the Crimean Congo Hemorrhagic Fever (CCHF) virus M segement glycoprotein's immunoinformatic parameters, and identify Human Leukocyte Antigen (HLA) class I binders as candidates for synthetic peptide vaccines. Cross-sectional study. Combined Military Hospital, Khuzdar Cantt, in May 2015. Data acquisition, antigenicity prediction, secondary and tertiary structure prediction, residue analysis were done using immunoinformatics tools. HLAclass I binders in glycoprotein's sequence were identified at nanomer length using NetMHC 3.4 and mapped onto tertiary structure. Docking was done for strongest binder against its corresponding allele with CABS-dock. HLAA*0101, 0201, 0301, 2402, 2601 and B*0702, 0801, 2705, 3901, 4001, 5801, 1501 were analyzed against two glycoprotein components of the virus. Atotal of 35 nanomers from GP1, and 3 from GP2 were identified. HLAB*0702 bound maximum number of peptides (6), while HLAB*4001 showed strongest binding affinity. HLAspecific glycoproteins epitope prediction can help identify synthetic peptide vaccine candidates.

  20. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

  1. Presynaptic neurones may contribute a unique glycoprotein to the extracellular matrix at the synapse

    NASA Astrophysics Data System (ADS)

    Caroni, Pico; Carlson, Steven S.; Schweitzer, Erik; Kelly, Regis B.

    1985-04-01

    As the extracellular matrix at the original site of a neuromuscular junction seems to play a major part in the specificity of synaptic regeneration1-5, considerable attention has been paid to unique molecules localized to this region6-11. Here we describe an extracellular matrix glycoprotein of the elasmobranch electric organ that is localized near the nerve endings. By immunological criteria, it is synthesized in the cell bodies, transported down the axons and is related to a glycoprotein in the synaptic vesicles of the neurones that innervate the electric organ. It is apparently specific for these neurones, as it cannot be detected elsewhere in the nervous system of the fish. Therefore, neurones seem to contribute unique extracellular matrix glycoproteins to the synaptic region. Synaptic vesicles could be involved in transporting these glycoproteins to or from the nerve terminal surface.

  2. Glycoproteins functionalized natural and synthetic polymers for prospective biomedical applications: A review.

    PubMed

    Tabasum, Shazia; Noreen, Aqdas; Kanwal, Arooj; Zuber, Mohammad; Anjum, Muhammad Naveed; Zia, Khalid Mahmood

    2017-05-01

    Glycoproteins have multidimensional properties such as biodegradability, biocompatibility, non-toxicity, antimicrobial and adsorption properties; therefore, they have wide range of applications. They are blended with different polymers such as chitosan, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone (PVP), polycaprolactone (PCL), heparin, polystyrene fluorescent nanoparticles (PS-NPs) and carboxyl pullulan (PC) to improve their properties like thermal stability, mechanical properties, resistance to pH, chemical stability and toughness. Considering the versatile charateristics of glycoprotein based polymers, this review sheds light on synthesis and characterization of blends and composites of glycoproteins, with natural and synthetic polymers and their potential applications in biomedical field such as drug delivery system, insulin delivery, antimicrobial wound dressing uses, targeting of cancer cells, development of anticancer vaccines, development of new biopolymers, glycoproteome research, food product and detection of dengue glycoproteins. All the technical scientific issues have been addressed; highlighting the recent advancement. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. A Method for Determining the Content of Glycoproteins in Biological Samples.

    PubMed

    Gao, Yang; Xu, Duoduo; Li, Hongyue; Yang, Xianling; Wang, Mingxing; Gao, Qipin

    2016-11-26

    The glycoprotein purified from the mycelium extract of Tremella fuciformis was marked with iodine through the iodine substitution reaction. The content of iodine, which is indicative of the amount of the marked tremella glycoprotein (ITG), was detected with Inductively coupled plasma mass spectrometry (ICP-MS). The method was found to be stable, sensitive, and accurate at detecting the content of iodine-substituted glycoprotein, and was used in the quantitative analysis of biological samples, including blood and organs. Different biological samples were collected from rats after oral administration of ITG, and were tested for iodine content by ICP-MS to calculate the amount of ITG in the samples. The results suggested that ICP-MS is a sensitive, stable, and accurate method for detection of iodinated glycoproteins in blood and organs.

  4. Glucocorticoid-Dependent Complementation of a Hepatoma Cell Variant Defective in Viral Glycoprotein Sorting

    NASA Astrophysics Data System (ADS)

    John, Nancy J.; Bravo, Deborah A.; Haffar, Omar K.; Firestone, Gary L.

    1988-02-01

    We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.

  5. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

  6. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

  7. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

  8. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification. Class...

  9. Characterization of Influenza Virus Pseudotyped with Ebolavirus Glycoprotein

    PubMed Central

    Xiao, Julie Huiyuan; Rijal, Pramila; Schimanski, Lisa; Tharkeshwar, Arun Kumar; Wright, Edward; Annaert, Wim

    2017-01-01

    ABSTRACT We have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC1280; Sigma) of 1,280 pharmacologically active compounds for inhibition of virus entry. A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies. IMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient

  10. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    SciTech Connect

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.

    2007-09-30

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed intomore » the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral

  11. Circulating N-Linked Glycoprotein Acetyls and Longitudinal Mortality Risk

    PubMed Central

    Lawler, Patrick R.; Akinkuolie, Akintunde O.; Chandler, Paulette D.; Moorthy, M. Vinayaga; Vandenburgh, Martin J.; Schaumberg, Debra A.; Lee, I-Min; Glynn, Robert J.; Ridker, Paul M; Buring, Julie E.; Mora, Samia

    2016-01-01

    Rationale Circulating glycoprotein N-acetyl glucosamine residues have recently been associated with incident cardiovascular disease (CVD) and diabetes mellitus. Objective Using a plasma glycan biosignature (GlycA) to identify circulating N-acetyl glycan groups, we examined the longitudinal association between GlycA and mortality among initially-healthy individuals. Methods and Results We quantified GlycA by 400 MHz 1H nuclear magnetic resonance (NMR) spectroscopy in 27,524 participants in the Women's Health Study (WHS; NCT00000479). The primary outcome was all-cause mortality. We replicated the findings in an independent cohort of 12,527 individuals in the JUPITER trial (NCT00239681). We also undertook secondary examination of CVD and cancer mortality in WHS. In WHS, during 524,515 person-years of follow-up (median 20.5 years) there were 3,523 deaths. Risk-factor adjusted multivariable Cox proportional hazard ratio (95% confidence interval) per standard deviation increment in GlycA for all-cause mortality was significantly increased at 5-years (1.21 [1.06, 1.40]) and during maximal follow-up (1.14 [1.09, 1.16]). Similar risk for all-cause mortality was observed in the replication cohort (1.33 [1.21, 1.45]). In WHS, risk of CVD mortality was increased at 5-years (1.43 [1.05, 1.95]) and during maximal follow-up (1.15 [1.04, 1.26]); and of cancer mortality at 5-years (1.23 [1.02, 1.47]) and during maximal follow-up (1.08 [1.01, 1.16]). Examination of correlations and mortality associations adjusted for hsCRP, fibrinogen, and ICAM-1, suggested that GlycA reflects summative risk related to multiple pathways of systemic inflammation. Conclusions Among initially-healthy individuals, elevated baseline circulating glycoprotein N-acetyl methyl groups were associated with longitudinal risk of all-cause, cardiovascular, and cancer mortality. PMID:26951635

  12. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    SciTech Connect

    Filipski, Elisabeth; Berland, Elodie; Univ Paris-Sud, UMR-S0776, Orsay F-91405

    2014-02-01

    The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F{sub 1} mice. A three-fold 24 h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p < 0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50 mg/kg/day i.v. × 4 days) as a single agent or combined with P-gp inhibitor PSC833 (6.25 mg/kg/day i.p. × 4 days) at ZT3 or ZT15, respectively corresponding tomore » the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40 mg/kg/day × 4 days) and +/− PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p < 0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p < 0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ∼ 60%. In tumor-bearing mice, body weight loss was ∼ halved in the mice on irinotecan or irinotecan–PSC833 combination at ZT15 as compared to ZT3 (p < 0.001). PSC833–irinotecan at ZT15 increased tumor inhibition by ∼ 40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan. - Highlights: • Irinotecan chronotolerance and chronoefficacy change as drug was applied with PSC833. • P-glycoprotein is an important player of the toxicity and efficacy of irinotecan. • Timing should be considered if chemotherapy is performed with a MDR1 inhibitor.« less

  13. Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

    PubMed

    Albani, J R; Plancke, Y D

    1999-05-31

    Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.

  14. Characterization of the envelope glycoproteins associated with infectious hepatitis C virus.

    PubMed

    Vieyres, Gabrielle; Thomas, Xavier; Descamps, Véronique; Duverlie, Gilles; Patel, Arvind H; Dubuisson, Jean

    2010-10-01

    Hepatitis C is caused by an enveloped virus whose entry is mediated by two glycoproteins, namely, E1 and E2, which have been shown to assemble as a noncovalent heterodimer. Despite extensive research in the field of such an important human pathogen, hepatitis C virus (HCV) glycoproteins have only been studied so far in heterologous expression systems, and their organization at the surfaces of infectious virions has not yet been described. Here, we characterized the envelope glycoproteins associated with cell-cultured infectious virions and compared them with their prebudding counterparts. Viral particles were analyzed by ultracentrifugation, and the envelope glycoproteins were characterized by coimmunoprecipitation and receptor pulldown assays. Furthermore, their oligomeric state was determined by sedimentation through sucrose gradients and by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In sucrose gradient analyses, HCV envelope glycoproteins were associated with fractions containing the most infectious viral particles. Importantly, besides maturation of some of their glycans, HCV envelope glycoproteins showed a dramatic change in their oligomeric state after incorporation into the viral particle. Indeed, virion-associated E1 and E2 envelope glycoproteins formed large covalent complexes stabilized by disulfide bridges, whereas the intracellular forms of these proteins assembled as noncovalent heterodimers. Furthermore, the virion-associated glycoprotein complexes were recognized by the large extracellular loop of CD81 as well as conformation-sensitive antibodies, indicating that these proteins are in a functional conformation. Overall, our study fills a gap in the description of HCV outer morphology and should guide further investigations into virus entry and assembly.

  15. Characterization of the Envelope Glycoproteins Associated with Infectious Hepatitis C Virus ▿

    PubMed Central

    Vieyres, Gabrielle; Thomas, Xavier; Descamps, Véronique; Duverlie, Gilles; Patel, Arvind H.; Dubuisson, Jean

    2010-01-01

    Hepatitis C is caused by an enveloped virus whose entry is mediated by two glycoproteins, namely, E1 and E2, which have been shown to assemble as a noncovalent heterodimer. Despite extensive research in the field of such an important human pathogen, hepatitis C virus (HCV) glycoproteins have only been studied so far in heterologous expression systems, and their organization at the surfaces of infectious virions has not yet been described. Here, we characterized the envelope glycoproteins associated with cell-cultured infectious virions and compared them with their prebudding counterparts. Viral particles were analyzed by ultracentrifugation, and the envelope glycoproteins were characterized by coimmunoprecipitation and receptor pulldown assays. Furthermore, their oligomeric state was determined by sedimentation through sucrose gradients and by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In sucrose gradient analyses, HCV envelope glycoproteins were associated with fractions containing the most infectious viral particles. Importantly, besides maturation of some of their glycans, HCV envelope glycoproteins showed a dramatic change in their oligomeric state after incorporation into the viral particle. Indeed, virion-associated E1 and E2 envelope glycoproteins formed large covalent complexes stabilized by disulfide bridges, whereas the intracellular forms of these proteins assembled as noncovalent heterodimers. Furthermore, the virion-associated glycoprotein complexes were recognized by the large extracellular loop of CD81 as well as conformation-sensitive antibodies, indicating that these proteins are in a functional conformation. Overall, our study fills a gap in the description of HCV outer morphology and should guide further investigations into virus entry and assembly. PMID:20668082

  16. Automated analysis of polyethylene glycol-induced inhibition of P-glycoprotein activity in vitro.

    PubMed

    Hugger, Erin D; Cole, Clayton J; Raub, Thomas J; Burton, Philip S; Borchardt, Ronald T

    2003-01-01

    Previous studies in our laboratories have shown that commonly used polyethoxylated pharmaceutical excipients inhibit P-glycoprotein activity in cell culture models of the intestinal mucosa. The results presented in this technical note show that the TECAN Genesis robotic workstation can be utilized to automate cellular transport studies for evaluating excipient effects on P-glycoprotein activity in vitro and for estimating the permeation of drug-like molecules across cell monolayers. Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

  17. Rabies virus (RV) glycoprotein expression levels are not critical for pathogenicity of RV.

    PubMed

    Wirblich, Christoph; Schnell, Matthias J

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain.

  18. Induction of Pro-Angiogenic Factors by Pregnancy-Specific Glycoproteins and Studies on Receptor Usage

    DTIC Science & Technology

    2008-01-01

    75. Dimitriadou, F., et al., Discordant secretion of pregnancy specific beta 1-glycoprotein and human chorionic gonadotropin by human pre-embryos...placental proteins human chorionic gonadotrophin, pregnancy-specific beta 1- glycoprotein, human placental lactogen and pregnancy-associated plasma...in the chorionic villi of the human placenta. Acta Anat (Basel), 1984. 119(1): p. 18-26. 224. Broxmeyer, H.E., et al., Myeloid progenitor cell

  19. Functional mutations in spike glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency.

    PubMed

    Ueda, Mahoko Takahashi; Kurosaki, Yohei; Izumi, Taisuke; Nakano, Yusuke; Oloniniyi, Olamide K; Yasuda, Jiro; Koyanagi, Yoshio; Sato, Kei; Nakagawa, So

    2017-02-01

    Ebola virus (EBOV) is extremely virulent, and its glycoprotein is necessary for viral entry. EBOV may adapt to its new host humans during outbreaks by acquiring mutations especially in glycoprotein, which allows EBOV to spread more efficiently. To identify these evolutionary selected mutations and examine their effects on viral infectivity, we used experimental-phylogenetic-structural interdisciplinary approaches. In evolutionary analysis of all available Zaire ebolavirus glycoprotein sequences, we detected two codon sites under positive selection, which are located near/within the region critical for the host-viral membrane fusion, namely alanine-to-valine and threonine-to-isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine-scale transmission dynamics of EBOV Makona variants that caused the 2014-2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, pseudotype assays for the Makona glycoprotein showed that the A82V mutation caused a small increase in viral infectivity compared with the T544I mutation. These findings suggest that mutation fixation in EBOV glycoprotein may be associated with their increased infectivity levels; the mutant with a moderate increase in infectivity will fix. Our findings showed that a driving force for Ebola virus evolution via glycoprotein may be a balance between costs and benefits of its virulence. © 2017 The Authors Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  20. Methods to study glycoproteins at the blood-brain barrier using mass spectrometry.

    PubMed

    Haqqani, Arsalan S; Hill, Jennifer J; Mullen, James; Stanimirovic, Danica B

    2011-01-01

    Glycosylation is the most common posttranslational modification of proteins in mammalian cells and is limited mainly to membrane and secreted proteins. Glycoproteins play several key roles in the physiology and pathophysiology of the blood-brain barrier (BBB) and are attractive as diagnostic markers and therapeutic targets for many neurological diseases. However, large-scale glycoproteomic studies of the BBB have been lacking, largely due to the complexity of analyzing glycoproteins and a lack of available tools for this analysis. Recent development of the hydrazide capture method and significant advances in mass spectrometry (MS)-based proteomics over the last few years have enabled selective enrichment of glycoproteins from complex biological samples and their quantitative comparisons in multiple conditions. In this chapter, we describe methods for: (1) isolating membrane and secreted proteins from BEC and other cells of the neurovascular unit, (2) enriching glycoproteins using hydrazide capture, and (3) performing label-free quantitative proteomics to identify differential glycoprotein expression in various biological conditions. Hydrazide capture, when coupled with label-free quantitative proteomics, is a reproducible and sensitive method that allows for quantitative profiling of a large number of glycoproteins from biological samples for the purposes of differential expression measurements and biomarker discovery.

  1. Development and validation of glycoprotein-based native-subunit vaccine for fish against Aeromonas hydrophila.

    PubMed

    Çiftci, A; Onuk, E E; Çiftci, G; Fındık, A; Söğüt, M Ü; Didinen, B I; Aksoy, A; Üstünakın, K; Gülhan, T; Balta, F; Altun, S

    2016-08-01

    Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein-based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA-specific lectin and size-exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)3 ] and Group IV [with Al(OH)3 and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish. © 2016 John Wiley & Sons Ltd.

  2. The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.

    PubMed

    Feldmann, H; Volchkov, V E; Volchkova, V A; Klenk, H D

    1999-01-01

    Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.

  3. [C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

    PubMed

    Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

    2017-01-01

    The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

  4. Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

    PubMed Central

    Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Tyagi, Charu; du Toit, Lisa C.

    2015-01-01

    The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

  5. Chemical Characterization and Surface Orientation of the Major Glycoprotein of the Human Erythrocyte Membrane

    PubMed Central

    Marchesi, V. T.; Tillack, T. W.; Jackson, R. L.; Segrest, J. P.; Scott, R. E.

    1972-01-01

    The major glycoprotein of the human erythrocyte membrane has been isolated by treatment with lithium di-iodosalicylate and found to be a single polypeptide chain with a molecular weight of about 50,000. This molecule, which is 60% carbohydrate and 40% protein, carries multiple blood-group antigens, the receptors for influenza viruses, and various plant agglutinins. Four unique carbohydrate-containing peptides (α-1, α-2, α-3, and β) are produced by tryptic digestion of the isolated glycoprotein; their order in the molecule has been determined by sequential tryptic digestion of intact erythrocyte membranes and partially digested glycoprotein fragments. Cleavage of the native protein with cyanogen bromide produces five fragments; two of these (C-5 and C-1) contain most of the carbohydrate in the molecule and are derived from the N-terminal half of the polypeptide chain. The nonpolar amino acids of this glycoprotein are located predominantly in the C-terminal fragment (C-2). Phytohemagglutinin conjugated to ferritin has been used to map the distribution of glycoprotein receptors over the surfaces of intact erythrocytes by freeze-etching and electron microscopy. This label localizes to sites on the membrane that overlie the intramembranous particles. These findings suggest that the glycoprotein is oriented at the cell surface with its oligosaccharide-rich N-terminal end exposed to the exterior, while its C-terminal segment interacts with other components in the interior of the membrane to form intramembranous particles. Images PMID:4504356

  6. A multigene family encoding surface glycoproteins inTrypanosoma congolense.

    PubMed

    Thonnus, Magali; Guérin, Amandine; Rivière, Loïc

    2017-03-02

    Trypanosoma congolense , the causative agent of the most important livestock disease in Africa, expresses specific surface proteins involved in its parasitic lifestyle. Unfortunately, the complete repertoire of such molecules is far from being deciphered. As these membrane components are exposed to the host environment, they could be used as therapeutic or diagnostic targets. By mining the T. congolense genome database, we identified a novel family of lectin-like glycoproteins (TcoClecs). These molecules are predicted to have a transmembrane domain, a tandem repeat amino acid motif, a signal peptide and a C-type lectin-like domain (CTLD). This paper depicts several experimental arguments in favor of a surface localization in bloodstream forms of T. congolense . A TcoClec gene was heterologously expressed in U-2 OS cells and the product could be partially found at the plasma membrane. TcoClecs were also localized at the surface of T. congolense bloodstream forms. The signal was suppressed when the cells were treated with a detergent to remove the plasma membrane or with trypsin to « shave » the parasites and remove their external proteins. This suggests that TcoClecs could be potential diagnostic or therapeutic antigens of African animal trypanosomiasis. The potential role of these proteins in T. congolense as well as in other trypanosomatids is discussed.

  7. Synonymous codon usage pattern in glycoprotein gene of rabies virus.

    PubMed

    Morla, Sudhir; Makhija, Aditi; Kumar, Sachin

    2016-06-10

    Rabies virus (RABV) is the causative agent of a fatal nervous system ailment. The disease is zoonotic and prevalent in many developing countries. The glycoprotein (G) of RABV is the major antigenic determinant of the virus and plays a pivotal role in its neurovirulence. Various aspects of 'G' protein biology have been explored, but the factors affecting the nucleotide choice and synonymous codon usage have never been reported. In the present study, we have analyzed the relative synonymous codon usage and effective number of codons (Nc) using 132 'G' protein genes of RABV. Corresponding analysis was used to calculate major trends in codon usage. The correlation between base composition and codon usage as well as the plot between Nc and GC3 suggest that mutational pressure is the major factor that influences the codon usage in the G gene of RABV. In addition, factors like aromaticity, aliphatic index and hydropathy have shown slight correlation suggesting that natural selection also contributes to the codon usage variations of the 'G' gene. In conclusion, codon usage bias in 'G' gene of RABV is mainly by mutational pressure and natural selection. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. The catabolism of plasma glycoproteins in normal and injured rats

    PubMed Central

    Robinson, G. B.

    1969-01-01

    The catabolism of 14C-labelled plasma glycoprotein in rats was studied after injecting homologous plasma protein labelled in the N-acetylglucosamine and sialic acid moieties. In normal animals the catabolism was approximately described by a four-compartment model. The fractional rate of catabolism of the plasma-protein amino sugar was found to be 0·0305hr.−1, corresponding to the degradation of 2·75μmoles/hr. The 14C label was eliminated from the animals largely as carbon dioxide with a small proportion appearing in the urine. Freely circulating amino sugars or glycopeptides did not appear in the plasma as a result of the catabolic processes, and there was no evidence that the protein-bound amino sugars were reutilized in biosynthetic processes. A study of the distribution of 14C label in the carcasses of animals 24hr. after injection provided evidence that the gastrointestinal tract accounted for 25–38% of the total catabolic pool; the lungs, kidneys, spleen and liver also appeared to contribute to catabolism. Studies were conducted with rats that had been treated with turpentine to induce an inflammatory reaction; the results could not be analysed kinetically, since the metabolism of plasma proteins in these animals did not appear to be in a steady state. The injected plasma protein disappeared from the intravascular pool more quickly than in normal animals, but there were no significant differences in the rates of excretion of the 14C label. PMID:5820648

  9. P-glycoprotein: a clue to vitamin K antagonist stabilization.

    PubMed

    Gschwind, Liliane; Rollason, Victoria; Boehlen, Françoise; Rebsamen, Michela; Combescure, Christophe; Matthey, Alain; Bonnabry, Pascal; Dayer, Pierre; Desmeules, Jules Alexandre

    2015-01-01

    Acenocoumarol is a vitamin K antagonist used in some European countries. As warfarin, this drug is characterized by a narrow therapeutic index and a large interindividual variability. The objective of this study was to assess the involvement of ABCB1 polymorphisms on acenocoumarol treatment. An observational cohort study was conducted to assess whether there is an association between the presence of the allelic variants of the ABCB1 gene coding for P-glycoprotein and acenocoumarol stabilization and daily doses during the first 35 days of treatment. One hundred and fifteen patients met the inclusion criteria. The results of the clinical study showed that carriers of ABCB1 c.3435TT were more rapidly stabilized than wild-type patients (HR: 2.97, 95% CI: 1.23-7.18; p = 0.02). The same tendency was observed for the ABCB1 c.2677GT and 2677TT genotypes compared with ABCB1 c.2677GG. The ABCB1 c.2677TT genotype was also associated with a significant increase in doses of acenocoumarol (p = 0.03), the same tendency was observed with the ABCB1 c.3435TT genotype compared with the wild-type patients. These data suggest that ABCB1 polymorphisms could be involved in the response to acenocoumarol treatment.

  10. Xanthone analogues as potent modulators of intestinal P-glycoprotein.

    PubMed

    Chae, Song Wha; Woo, Sangwook; Park, Jung Hyun; Kwon, Youngjoo; Na, Younghwa; Lee, Hwa Jeong

    2015-03-26

    Intestinal P-glycoprotein (P-gp) is a limiting step for oral absorption of drugs. Therefore, P-gp inhibitors have been studied as enhancers of oral absorption of drugs that are P-gp substrates. We investigated the in vitro and in vivo P-gp inhibitory activity of synthesized xanthone analogues. With 3-(3-chloro-2-hydroxypropoxy)-1-hydroxy-9H-thioxanthen-9-one, compound 13, accumulation of daunomycin (DNM) increased 707% and efflux of DNM decreased 66% compared to DNM alone. Relative bioavailability (RB) of paclitaxel (PTX, 25 mg/kg) increased 2.5-fold after oral administration with 13 (5 mg/kg). In a xenograft animal model, oral administration of PTX (40 mg/kg) with 13 (10 mg/kg) significantly inhibited tumour growth and was more effective than intravenously administered PTX (10 mg/kg) alone. Therefore, the synthesized xanthone analogue 13 might have therapeutic benefits for oral absorption of P-gp substrate anticancer drugs. Copyright © 2015. Published by Elsevier Masson SAS.

  11. Rabies virus glycoprotein as a carrier for anthrax protective antigen

    SciTech Connect

    Smith, Mary Ellen; Koser, Martin; Xiao Sa

    2006-09-30

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen wasmore » also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.« less

  12. Glycoprotein changes in tumours: a renaissance in clinical applications.

    PubMed

    Hounsell, E F; Young, M; Davies, M J

    1997-10-01

    1. Oligosaccharides linked to protein (glycoprotein) or lipid (glycolipid) are the major components at the outer surface of mammalian cells. Studies using antibodies and lectins have shown in the past that the oligosaccharides they recognize exhibit tumour-associated changes, i.e. they are carbohydrate tumour-associated antigens. 2. The oligosaccharides have been further characterized in recent years by structural analysis using high-resolution chromatographic techniques, MS and NMR. NMR gives an oligosaccharide finger-print that is characteristic of monosaccharide type and linkage and which can be correlated with magnetic resonance spectroscopic data on fine-needle tissue aspirates. 3. Also of relevance is the new understanding of the molecular biology of MUC genes, which code for mucin protein backbones, and of the glycosyltransferase genes, which determine oligosaccharide structure and immunological recognition. 4. For these reasons, we believe that tumour-associated oligosaccharide changes should be revisited in the context of what we now know about structure and expression. This review synopsizes the past data using the detection of carbohydrate tumour-associated antigens by binding of lectins and antibodies, and puts it into the context of NMR fingerprints or signatures.

  13. Four glycoproteins are expressed in the cat zona pellucida.

    PubMed

    Stetson, I; Avilés, M; Moros, C; García-Vázquez, F A; Gimeno, L; Torrecillas, A; Aliaga, C; Bernardo-Pisa, M V; Ballesta, J; Izquierdo-Rico, M J

    2015-04-15

    The mammalian oocyte is surrounded by a matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding and may be involved in speciation. In cat (Felis catus), this matrix is composed of at least three glycoproteins called ZP2, ZP3, and ZP4. However, recent studies have pointed to the presence of a fourth protein in several mammals (rat, human, hamster or rabbit), meaning that a reevaluation of cat ZP is needed. For this reason, the objective of this research was to analyze the protein composition of cat ZP by means of proteomic analysis. Using ZP from ovaries and oocytes, several peptides corresponding to four proteins were detected, yielding a coverage of 33.17%, 71.50%, 50.23%, and 49.64% for ZP1, ZP2, ZP3, and ZP4, respectively. Moreover, the expression of four genes was confirmed by molecular analysis. Using total RNA isolated from cat ovaries, the complementary deoxyribonucleic acids encoding cat ZP were partially amplified by reverse-transcribed polymerase chain reaction. Furthermore, ZP1 was totally amplified for the first time in this species. As far as we are aware, this is the first study that confirms the presence of four proteins in cat ZP. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

    PubMed

    Ben Azoun, Safa; Belhaj, Aicha Eya; Göngrich, Rebecca; Gasser, Brigitte; Kallel, Héla

    2016-05-01

    In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV-G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV-G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV-G gene was not tremendously increased as compared with the non-optimized one. In addition, similar levels of RABV-G were obtained when α-factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high-level expression of RABV-G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV-G expression cassette indicating that folding was the limiting step for RABV-G secretion. To circumvent this limitation, co-overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV-G reached 1230 ng ml(-1). Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  15. Functional characterization of P-glycoprotein in the intertidal copepod Tigriopus japonicus and its potential role in remediating metal pollution.

    PubMed

    Jeong, Chang-Bum; Kim, Bo-Mi; Kim, Rae-Kwon; Park, Heum Gi; Lee, Su-Jae; Shin, Kyung-Hoon; Leung, Kenneth Mei Yee; Rhee, Jae-Sung; Lee, Jae-Seong

    2014-11-01

    The intertidal copepod Tigriopus japonicus has been widely used in aquatic toxicity testing for diverse environmental pollutants including metals. Despite relatively well-characterized in vivo physiological modulations in response to aquatic pollutants, the molecular mechanisms due to toxicity and detoxification are still unclear. To better understand the mechanisms of metal transport and further detoxification, T. japonicus P-glycoprotein (TJ-P-gp) with conserved motifs/domains was cloned and measured for protein activity against the transcript and protein expression profiles in response to metal exposure. Specifically, we characterized the preliminary efflux activity and membrane topology of TJ-P-gp protein that supports a transport function for chemicals. To uncover whether the efflux activity of TJ-P-gp protein would be modulated by metal treatment, copepods were exposed to three metals (Cd, Cu, and Zn), and were observed for both dose- and time-dependency on the efflux activity of TJ-P-gp protein with or without 10μM of P-gp-specific inhibitors verapamil and zosuquidar (LY335979) for 24h over a wide range of metal concentrations. In particular, treatment with zosuquidar induced metal accumulation in the inner body of T. japonicus. In addition, three metals significantly induced the transporting activity of TJ-P-gp in a concentration-dependent manner in both transcript and protein levels within 24h. Together these data indicate that T. japonicus has a conserved P-gp-mediated metal defense system through the induction of transcriptional up-regulation of TJ-P-gp gene and TJ-P-gp protein activity. This finding provides further understanding of the molecular defense mechanisms involved in P-glycoprotein-mediated metal detoxification in copepods. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Differential effects of the enantiomers of tamsulosin and tolterodine on P-glycoprotein and cytochrome P450 3A4.

    PubMed

    Doricakova, Aneta; Theile, Dirk; Weiss, Johanna; Vrzal, Radim

    2017-01-01

    The pregnane X receptor (PXR) is a transcription factor regulating P-glycoprotein (P-gp; ABCB1)-mediated transport and cytochrome P450 3A4 (CYP3A4)-mediated metabolism of xenobiotics thereby affecting the pharmacokinetics of many drugs and potentially modulating clinical efficacy. Thus, pharmacokinetic drug-drug interactions can arise from PXR activation. Here, we examined whether the selective α1-adrenoreceptor blocker tamsulosin or the antagonist of muscarinic receptors tolterodine affect PXR-mediated regulation of CYP3A4 and of P-gp at the messenger RNA (mRNA) and protein level in an enantiomer-specific way. In addition, the effect of tamsulosin and tolterodine on P-gp activity was evaluated. We used quantitative real-time PCR, gene reporter assay, western blotting, rhodamine efflux assay, and calcein assay for determination of expression, activity, and inhibition of P-glycoprotein. The studied compounds significantly and concentration-dependently increased PXR activity in the ABCB1-driven luciferase-based reporter gene assay. We observed much stronger induction of ABCB1 mRNA by S-tamsulosin as compared to the R or racemic form. R or racemic form of tolterodine and R-tamsulosin concentration-dependently increased P-gp protein expression; the latter also enhanced P-gp efflux function in a rhodamine-based efflux assay. R-tamsulosin and all forms of tolderodine slightly inhibited P-gp. The effect on CYP3A4 expression followed the same pattern but was much weaker. Taken together, tamsulosin and tolterodine are demonstrated to interfere with P-gp and CYP3A4 regulation in an enantiomer-specific way.

  17. Biochemical mechanism of modulation of human P-glycoprotein by stemofoline.

    PubMed

    Chanmahasathien, Wisinee; Ohnuma, Shinobu; Ambudkar, Suresh V; Limtrakul, Pornngarm

    2011-12-01

    The resistance to chemotherapeutic drugs by cancer cells is considered to be one of the major obstacles for success in the treatment of cancer. A major mechanism underlying this multidrug resistance is the overexpression of P-glycoprotein (P-gp), resulting in insufficient drug delivery to the tumor sites. A previous study has shown that stemofoline, an alkaloid isolated from Stemona burkillii, could enhance the sensitivity of chemotherapeutics in a synergistic fashion. In the present study, we have focused on the effect of stemofoline on the modulation of P-gp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The effects of stemofoline on a radiolabeled drug, [(3)H]-vinblastine, and fluorescent P-gp substrates, rhodamine 123 and calcein-AM accumulation or retention were investigated to confirm this finding. Stemofoline could increase the accumulation or retention of radiolabeled drugs or fluorescent P-gp substrates in a dose-dependent manner. For additional studies on drug-P-gp binding, P-gp ATPase activity was stimulated by stemofoline in a concentration-dependent manner. More evidence was offered that stemofoline inhibits the effect on photoaffinity labeling of P-gp with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. These data indicate that stemofoline may interact directly with P-gp and inhibit P-gp activity, whereas stemofoline has no effect on P-gp expression. Taken together, the results exhibit that stemofoline possesses an effective MDR modulator, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells. © Georg Thieme Verlag KG Stuttgart · New York.

  18. Influence of P-glycoprotein on brucine transport at the in vitro blood-brain barrier.

    PubMed

    Xu, Dan-Hua; Yan, Miao; Li, Huan-De; Fang, Ping-Fei; Liu, Yan-Wen

    2012-09-05

    Brucine is a central agonist that can pass through the blood-brain barrier (BBB). The goal of this study is to examine whether brucine is one of the substrates of the drug transporter P-glycoprotein (P-gp) and to examine the effects of P-gp on the brucine transport at the in vitro BBB model. The P-gp ATPase assay was utilized to investigate the in vitro affinity of P-gp to brucine. Results suggested that K(m) of brucine (11.4 μmol/l) was smaller than the positive control, verapamil (16.4 μmol/l). In this study, we developed an in vitro BBB model, comprising a co-culture of primary rat brain microvessel endothelial cells and astrocytes for the transport study. The validated model was correct and available. Transendothelial electrical resistance reached (283.78 ± 18.85) Ω cm(2). The model displayed limited permeability to fluorescein sodium and [(125)I]albumin, with the apparent permeability coefficient Papp of (10.36 ± 0.86) × 10(-6) cm/s and (6.00 ± 0.78) × 10(-6)cm/s, respectively. The quantity of the bidirectional transport of brucine was determined by ultra-performance liquid chromatography-tandem mass spectrometry. In the absence of verapamil, the transport of brucine from basolateral compartment to apical compartment (BL-AP) was higher than from AP to BL at low, middle, and high concentrations (P<0.05). The excretion rate was 1.32, 1.56, and 1.54, respectively. However, following exposure to verapamil, the excretion rate at three different concentrations was decreased (P<0.05). All the results suggest that P-gp prevented brucine from passing through the in vitro BBB model. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Alpha 1-acid glycoprotein reverses cocaine-induced sodium channel blockade in cardiac myocytes.

    PubMed

    Ma, Yu-Ling; Peters, Nicholas S; Henry, John A

    2006-03-01

    Alpha 1-acid glycoprotein (AAG) is an acute phase protein capable of binding basic drugs. This action explains its reversal of sodium channel blockade by drugs such as amitriptyline and quinidine. We report here the reversal of cocaine-induced sodium channel blockade by AAG. The sodium channel blocking property of cocaine is a major mechanism behind cocaine-induced sudden cardiac death, since sodium channels play a key role in the initiation and regulation of the heart beat. Voltage-gated sodium current (I(Na)) was recorded using whole-cell patch-clamp techniques. Guinea-pig cardiac ventricular myocytes were isolated and continuously perfused at room temperature with physiological solutions. At concentrations ranging from 5 to 320 microM cocaine showed a dose-dependent and reversible blockade of I(Na) with an IC50 of 45.9 microM. The addition of equimolar amounts of AAG to cocaine produced almost complete reversal of cocaine's effects, suggesting a single binding site for cocaine on the AAG molecule. With changes of peak I(Na) normalized against control as 1, cocaine at 20 and 40 microM reduced I(Na) to 0.62+/-0.042 (n = 6) and 0.57+/-0.052 (n = 9), respectively, and the addition of an equimolar concentration of AAG reversed I(Na) to 0.86+/-0.022 and 0.91+/-0.060, respectively. AAG reverses cocaine-induced sodium channel blockade in a dose-dependent manner, indicating a therapeutic potential to reverse acute cocaine cardiac toxicity.

  20. Concentrating Radioactivity

    ERIC Educational Resources Information Center

    Herrmann, Richard A.

    1974-01-01

    By concentrating radioactivity contained on luminous dials, a teacher can make a high reading source for classroom experiments on radiation. The preparation of the source and its uses are described. (DT)

  1. Pre-staining of glycoprotein in SDS-PAGE by the synthesis of a new hydrazide derivative.

    PubMed

    Zhou, Ayi; Zhou, Tieli; Yu, Dongdong; Shen, Yingjie; Shen, Jiayi; Zhu, Zhongxin; Jin, Litai; Zhang, Huajie; Wang, Yang

    2015-11-01

    In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Chemical Synthesis of Glycoproteins with the Specific Installation of Gradient-Enriched15N-Labeled Amino Acids for Getting Insights into Glycoprotein Behavior.

    PubMed

    Minh Hien, Nguyen; Izumi, Masayuki; Sato, Hajime; Okamoto, Ryo; Kajihara, Yasuhiro

    2017-05-11

    Elucidating the effects of oligosaccharides on glycoprotein properties, such as local conformational changes, stability, and dynamics, has still been challenging. In this paper, a novel partial 15 N-labeling method for the amide backbone of a synthetic glycoprotein is proposed. Using solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL), thirteen 15 N-labeled amino acids were inserted at specific positions of the protein backbone, while intentionally varying the enrichment of 15 N atoms. This idea discriminated even the same type of amino acid based on the intensities of 1 H- 15 N HSQC signals, combined with classic homonuclear TOCSY and NOESY methods, thus allowing for understanding the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T 1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied circular dichroism (CD) spectra and T 1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure. This insight into oligosaccharide behavior suggests some further effects on binding affinity between a glycoprotein and its receptor. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The NS1 Glycoprotein Can Generate Dramatic Antibody-Enhanced Dengue Viral Replication in Normal Out-Bred Mice Resulting in Lethal Multi-Organ Disease

    PubMed Central

    Falconar, Andrew K. I.; Martinez, Fernando

    2011-01-01

    Antibody-enhanced replication (AER) of dengue type-2 virus (DENV-2) strains and production of antibody-enhanced disease (AED) was tested in out-bred mice. Polyclonal antibodies (PAbs) generated against the nonstructural-1 (NS1) glycoprotein candidate vaccine of the New Guinea-C (NG-C) or NSx strains reacted strongly and weakly with these antigens, respectively. These PAbs contained the IgG2a subclass, which cross-reacted with the virion-associated envelope (E) glycoprotein of the DENV-2 NSx strain, suggesting that they could generate its AER via all mouse Fcγ-receptor classes. Indeed, when these mice were challenged with a low dose (<0.5 LD50) of the DENV-2 NSx strain, but not the NG-C strain, they all generated dramatic and lethal DENV-2 AER/AED. These AER/AED mice developed life-threatening acute respiratory distress syndrome (ARDS), displayed by diffuse alveolar damage (DAD) resulting from i) dramatic interstitial alveolar septa-thickening with mononuclear cells, ii) some hyperplasia of alveolar type-II pneumocytes, iii) copious intra-alveolar protein secretion, iv) some hyaline membrane-covered alveolar walls, and v) DENV-2 antigen-positive alveolar macrophages. These mice also developed meningo-encephalitis, with greater than 90,000-fold DENV-2 AER titers in microglial cells located throughout their brain parenchyma, some of which formed nodules around dead neurons. Their spleens contained infiltrated megakaryocytes with DENV-2 antigen-positive red-pulp macrophages, while their livers displayed extensive necrosis, apoptosis and macro- and micro-steatosis, with DENV-2 antigen-positive Kuppfer cells and hepatocytes. Their infections were confirmed by DENV-2 isolations from their lungs, spleens and livers. These findings accord with those reported in fatal human “severe dengue” cases. This DENV-2 AER/AED was blocked by high concentrations of only the NG-C NS1 glycoprotein. These results imply a potential hazard of DENV NS1 glycoprotein-based vaccines

  4. Metabolic labeling and click chemistry detection of glycoprotein markers of mesenchymal stem cell differentiation.

    PubMed

    Hart, Courtenay; Chase, Lucas G; Hajivandi, Mahbod; Agnew, Brian

    2011-01-01

    Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into a variety of cell types in vitro including osteoblasts, adipocytes, and chondrocytes. Here we apply a metabolic labeling approach to characterize changes in cellular glycoprotein expression during hMSC differentiation and to identify glycoprotein markers unique to differentiated cell types. The two-step labeling method involves the metabolic incorporation of unnatural azido-modified sugars into protein glycans and subsequent ligation with fluorescent azide-reactive detection probes utilizing the copper (I)-catalyzed cycloaddition reaction between azides and alkynes, or "click" chemistry. Metabolic labeling of cell surface O-linked or sialic acid-containing glycoproteins, or intracellular O-GlcNAc-modified proteins was accomplished by feeding cells the tetraacetylated azide-modified sugar precursors, GalNAz, ManNAz, or GlcNAz, respectively, for 48-72 h prior to harvesting the cells. The cells were then lysed, and protein extracts were reacted with a fluorescent alkyne detection probe. Labeled glycoproteins were analyzed by 1D and 2D gel electrophoresis and detected by fluorescence imaging. Our results demonstrate highly sensitive labeling of O-linked, sialic acid-containing, and O-GlcNAc modified proteins in all cell types without affecting cell growth or morphology. Selective labeling of sialic acid-containing glycoproteins by ManNAz was validated by loss of labeling following digestion with sialidase A. Significant changes in cellular glycoprotein profiles were seen upon differentiation into different cell types, and several putative glycoprotein markers were identified by MALDI peptide fingerprinting. One of these identified proteins, Galectin 1, is validated and shown for the first time to be posttranslationally modified by O-glycosylation, most likely by O-linked N-acetylglucosamine (O-GlcNAc).

  5. The Domains of Glycoprotein D Required To Block Apoptosis Depend on Whether Glycoprotein D Is Present in the Virions Carrying Herpes Simplex Virus 1 Genome Lacking the Gene Encoding the Glycoprotein

    PubMed Central

    Zhou, Guoying; Roizman, Bernard

    2001-01-01

    An earlier report showed that viruses lacking the open reading frames encoding glycoproteins J and D but containing the glycoprotein D in their envelopes (gD−/+ stocks) and viruses lacking both the open reading frames and the glycoproteins in their envelopes (gD−/− stocks) induce apoptosis (G. Zhou, V. Galvan, G. Campadelli-Fiume, and B. Roizman, J. Virol. 74:11782–11791, 2000). Furthermore, apoptosis was blocked by delivery in trans of genes expressing glycoprotein D or J. Whereas gD−/− stocks attach but cannot initiate productive infection, gD−/+ stocks infect cells and produce gD−/− progeny virus. The difference in the infectivity of these two stocks suggested the possibility that the requirements for blocking apoptosis may be different. To test this hypothesis, we cloned into baculoviruses the entire wild-type glycoprotein D (Bac-gD-WT), the ectodomain only (Bac-gD-A), the ectodomain and the transmembrane domain (Bac-gD-B), the ectodomain and the cytoplasmic domain without the transmembrane domain (Bac-gD-C), or the transmembrane domain and the carboxyl-terminal cytoplasmic domain (Bac-gD-D). We report the following. Apoptosis induced by gD−/+ stocks was blocked by delivery in trans of recombinant baculovirus Bac-gD-WT, Bac-gD-A, Bac-gD-B, or Bac-gD-C but not of Bac-gD. Apoptosis induced by gD−/− stocks was blocked by Bac-gD-WT or by a mixture of Bac-gD-B and Bac-gD-D but not by any baculoviruses expressing truncated glycoprotein D alone or by the mixture of Bac-gD-A and Bac-gD-D. We conclude that the requirements to block apoptosis induced by the two virus stocks are different. The gD ectodomain is sufficient to block apoptosis induced by gD, whereas both the ectodomain and the cytoplasmic domain are required to block apoptosis induced by gD−/− stocks. The results indicate that in the case of gD−/− stocks, the transmembrane domain is required either to deliver the ectodomain to the appropriate intracellular compartment or to

  6. Myelin Oligodendrocyte Glycoprotein: Deciphering a Target in Inflammatory Demyelinating Diseases

    PubMed Central

    Peschl, Patrick; Bradl, Monika; Höftberger, Romana; Berger, Thomas; Reindl, Markus

    2017-01-01

    Myelin oligodendrocyte glycoprotein (MOG), a member of the immunoglobulin (Ig) superfamily, is a myelin protein solely expressed at the outermost surface of myelin sheaths and oligodendrocyte membranes. This makes MOG a potential target of cellular and humoral immune responses in inflammatory demyelinating diseases. Due to its late postnatal developmental expression, MOG is an important marker for oligodendrocyte maturation. Discovered about 30 years ago, it is one of the best-studied autoantigens for experimental autoimmune models for multiple sclerosis (MS). Human studies, however, have yielded controversial results on the role of MOG, especially MOG antibodies (Abs), as a biomarker in MS. But with improved detection methods using different expression systems to detect Abs in patients’ samples, this is meanwhile no longer the case. Using cell-based assays with recombinant full-length, conformationally intact MOG, several recent studies have revealed that MOG Abs can be found in a subset of predominantly pediatric patients with acute disseminated encephalomyelitis (ADEM), aquaporin-4 (AQP4) seronegative neuromyelitis optica spectrum disorders (NMOSD), monophasic or recurrent isolated optic neuritis (ON), or transverse myelitis, in atypical MS and in N-methyl-d-aspartate receptor-encephalitis with overlapping demyelinating syndromes. Whereas MOG Abs are only transiently observed in monophasic diseases such as ADEM and their decline is associated with a favorable outcome, they are persistent in multiphasic ADEM, NMOSD, recurrent ON, or myelitis. Due to distinct clinical features within these diseases it is controversially disputed to classify MOG Ab-positive cases as a new disease entity. Neuropathologically, the presence of MOG Abs is characterized by MS-typical demyelination and oligodendrocyte pathology associated with Abs and complement. However, it remains unclear whether MOG Abs are a mere inflammatory bystander effect or truly pathogenetic. This article

  7. Antifreeze Glycoproteins Bind Reversibly to Ice via Hydrophobic Groups.

    PubMed

    Mochizuki, Kenji; Molinero, Valeria

    2018-04-11

    Antifreeze molecules allow organisms to survive in subzero environments. Antifreeze glycoproteins (AFGPs), produced by polar fish, are the most potent inhibitors of ice recrystallization. To date, the molecular mechanism by which AFGPs bind to ice has not yet been elucidated. Mutation experiments cannot resolve whether the binding occurs through the peptide, the saccharides, or both. Here, we use molecular simulations to determine the mechanism and driving forces for binding of AFGP8 to ice, its selectivity for the primary prismatic plane, and the molecular origin of its exceptional ice recrystallization activity. Consistent with experiments, AFGP8 in simulations preferentially adopts the PPII helix secondary structure in solution. We show that the segregation of hydrophilic and hydrophobic groups in the PPII helix is vital for ice binding. Binding occurs through adsorption of methyl groups of the peptide and disaccharides to ice, driven by the entropy of dehydration of the hydrophobic groups as they nest in the cavities at the ice surface. The selectivity to the primary prismatic plane originates in the deeper cavities it has compared to the basal plane. We estimate the free energy of binding of AFGP8 and the longer AFGPs4-6, and find them to be consistent with the reversible binding demonstrated in experiments. The simulations reveal that AFGP8 binds to ice through a myriad of conformations that it uses to diffuse through the ice surface and find ice steps, to which it strongly adsorbs. We interpret that the existence of multiple, weak binding sites is the key for the exceptional ice recrystallization inhibition activity of AFGPs.

  8. Increasing nerve agent treatment efficacy by P-glycoprotein inhibition.

    PubMed

    Joosen, Marloes J A; Vester, Stefanie M; Hamelink, Jouk; Klaassen, Steven D; van den Berg, Roland M

    2016-11-25

    One of the shortcomings of current treatment of nerve agent poisoning is that not all drugs effectively penetrate the blood-brain barrier (BBB), whereas most nerve agents easily do. P-glycoprotein (Pgp) efflux transporters at the BBB may contribute to this aspect. It was previously shown that Pgp inhibition by tariquidar enhanced the efficacy of nerve agent treatment when administered as a pretreatment. In the present study soman-induced seizures were also substantially prevented when the animals were intravenously treated with tariquidar post-poisoning, in addition to HI-6 and atropine. In these animals, approximately twice as much AChE activity was present in their brain as compared to control rats. The finding that tariquidar did not affect distribution of soman to the brain indicates that the potentiating effects were a result of interactions of Pgp inhibition with drug distribution. In line with this, atropine appeared to be a substrate for Pgp in in vitro studies in a MDR1/MDCK cell model. This indicates that tariquidar might induce brain region specific effects on atropine distribution, which could contribute to the therapeutic efficacy increase found. Furthermore, the therapeutic enhancement by tariquidar was compared to that of the less specific and less potent Pgp inhibitor cyclosporine A. This compound appeared to induce a protective effect similar to tariquidar. In conclusion, treatment with a Pgp inhibitor resulted in enhanced therapeutic efficacy of HI-6 and atropine in a soman-induced seizure model in the rat. The mechanism underlying these effects should be further investigated. To that end, the potentiating effect of nerve agent treatment should be addressed against a broader range of nerve agents, for oximes and atropine separately, and for those at lower doses. In particular when efficacy against more nerve agents is shown, a Pgp inhibitor such as tariquidar might be a valid addition to nerve agent antidotes. Copyright © 2016 Elsevier Ireland

  9. Zinc alpha-2 glycoprotein is overproduced in Cushing's syndrome.

    PubMed

    Escoté, Xavier; Aranda, Gloria B; Mora, Mireia; Casals, Gregori; Enseñat, Joaquim; Vidal, Oscar; Esteban, Yaiza; Halperin, Irene; Hanzu, Felicia A

    2017-01-01

    Cushing syndrome (CS), an endogenous hypercortisolemic condition with increased cardiometabolic morbidity, leads to development of abdominal obesity, insulin resistance, diabetes and proatherogenic dyslipidemia. Zinc alpha-2 glycoprotein (ZAG) is a recently characterized lipolytic adipokine implicated in regulation of adipose tissue metabolism and fat distribution. In vitro and animal studies suggest that glucocorticoids interact with ZAG secretion and action. To assess the relationship between ZAG and glucocorticoids in a human model of hypercortisolism, circulating ZAG levels were tested in patients with CS and its counterpart controls. An observational, cross-sectional study on 39 women, 13 with active CS and 26 controls matched by age and body mass index. Plasma ZAG levels (μg/ml) were measured by ELISA and correlated with hypercortisolism, metabolic, and phenotypic parameters. Plasma ZAG levels were significantly higher in patients with CS compared to controls (64.3±16.6 vs. 44.0±16.1, p=0.002). In a univariate analysis, ZAG levels positively correlated to 24-h urinary free cortisol (p=0.001), body mass index (p=0.02), non-esterified fatty acids (p=0.05), glucose (p=0.003), LDL-C (p=0.028), and type 2 diabetes mellitus (p=0.016), and were inversely related to total adiponectin levels (p=0.035). In a multivariate analysis, after adjusting for CS, ZAG levels only correlated with body mass index (p=0.012), type 2 diabetes mellitus (p=0.004), and glucose (p<0.001). This study provides initial evidence that plasma ZAG levels are higher in patients with CS as compared to controls. The close relationship of ZAG with metabolic and phenotypic changes in CS suggests that ZAG may play a significant role in adipose tissue changes in hypercortisolism. Copyright © 2017 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.

  10. Molecular insight into conformational transmission of human P-glycoprotein

    NASA Astrophysics Data System (ADS)

    Chang, Shan-Yan; Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

    2013-12-01

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  11. Glycosylation Failure Extends to Glycoproteins in Gestational Diabetes Mellitus

    PubMed Central

    Lee, Cheuk-Lun; Chiu, Philip C.N.; Pang, Poh-Choo; Chu, Ivan K.; Lee, Kai-Fai; Koistinen, Riitta; Koistinen, Hannu; Seppälä, Markku; Morris, Howard R.; Tissot, Bérangère; Panico, Maria; Dell, Anne; Yeung, William S.B.

    2011-01-01

    OBJECTIVE Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities. RESEARCH DESIGN AND METHODS GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured. RESULTS GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6–sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcβ1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA. CONCLUSIONS Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients. PMID:21300843

  12. Measuring soluble platelet glycoprotein VI in human plasma by ELISA.

    PubMed

    Al-Tamimi, Mohammad; Mu, Fi-Tjen; Moroi, Masaaki; Gardiner, Elizabeth E; Berndt, Michael C; Andrews, Robert K

    2009-05-01

    Recent experimental evidence demonstrates that the platelet-specific collagen receptor, glycoprotein (GP)VI is essentially all uncleaved on normal circulating platelets, but is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligands (including collagen), anti-GPVI antibodies or activation at the platelet Fc receptor, FcgammaRIIa. This raises the question of whether shed ectodomain fragment in plasma could be a useful biomarker of thrombotic risk and/or autoimmune thrombocytopenia. In this study, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring soluble GPVI in human plasma, using rabbit anti-GPVI polyclonal antibody in the solid-phase, murine anti-GPVI monoclonal antibody (1A12) in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimized for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of approximately 1 ng/mL. Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. In ten healthy donors, soluble plasma GPVI levels were 18.9 +/- 4.1 ng/mL. Treating normal platelet-rich plasma with a GPVI ligand (collagen-related peptide, CRP), calmodulin inhibitor W7 (that induces GPVI shedding without platelet activation) or N-ethylmaleimide (that directly activates platelet sheddases), under conditions previously shown to induce GPVI shedding, also increased plasma GPVI levels by up to approximately 7-fold, compared to previously reported autoimmune (anti-GPVI) patient plasma where soluble GPVI was approximately 10-fold higher than normal. Characterization of this sensitive ELISA should facilitate analysis of functional/diagnostic role(s) for soluble GPVI in human plasma associated with thrombotic/immune dysfunction.

  13. Effects of Kampo medicines on P-glycoprotein.

    PubMed

    Satoh, Toshiyuki; Watanabe, Yuka; Ikarashi, Nobutomo; Ito, Kiyomi; Sugiyama, Kiyoshi

    2009-12-01

    The Kampo medicines are more and more often used in recent years, usually together with the western drugs. The need for the investigation of drug interactions between Kampo medicines and western drugs are, therefore, widely recognized. Among the various possible causes for the drug-drug interactions, those related to pharmacokinetics such as drug metabolism and transport are regarded as most frequent and clinically important. In the present study, the effects of Kampo medicines on the P-glycoprotein (P-gp), one of the major drug transporters, were investigated in in vitro studies using human P-gp membranes. The P-gp activity in the presence and absence of commonly used 50 Kampo medicines was evaluated by the ATPase assay detecting the inorganic phosphate produced by the ATP hydrolysis. The ATPase activity was inhibited by most of the Kampo medicines studied, indicating the possibility of their inhibiting the P-gp. The degree of inhibition in the presence of verapamil, a P-gp substrate, showed a significant correlation with that in the absence of verapamil. Furthermore, the inhibitory effect of the Kampo medicines on the ATPase activity correlated with their licorice root (kanzo) content, suggesting the contribution of licorice root in the P-gp inhibition. Because licorice root is one of the most common ingredients in the Kampo medicines and is also often used in the food as a sweetener, it might be necessary to pay attention on the interaction between the licorice root-containing drug/food and the number of drugs transported by P-gp.

  14. Exploring the Chemical Space of P-Glycoprotein Interacting Compounds.

    PubMed

    Prachayasittikul, Veda; Mandi, Prasit; Prachayasittikul, Supaluk; Prachayasittikul, Virapong; Nantasenamat, Chanin

    2017-01-01

    P-glycoprotein (Pgp) is well known for its clinical importance in the pharmacokinetics of drugs and its role in multidrug resistance. The promiscuity of Pgp that arises from its ability to extrude a wide range of lipophilic and structurally unrelated compounds from cells, render the classification and understanding of its interacting compounds a great challenge. In this study, a data set of Pgp-interacting compounds including 1463 inhibitors, 1085 noninhibitors, 308 substrates and 126 non-substrates was retrieved and subjected to a combination of analyses, including exploration of chemical space, statistical analysis of descriptor values and molecular fragment analysis, to obtain insight into distinct physicochemical properties and important chemical substructures which may govern the biological activity of investigated compounds toward Pgp. Statistical analysis of descriptor values and molecular fragment analysis indicated that particular size, shape, functional groups and molecular fragments may govern the classification of Pgp-interacting compounds by influencing their physicochemical properties and their interaction with Pgp. Overall, the interacting compounds (i.e., substrates and inhibitors) are larger in size, more flexible, more lipophilic, and less charged than non-interacting compounds (i.e., non-substrates and non-inhibitors). The fragment analysis suggested that methyl and amino groups may be involved in Pgp inhibition and/or transport. The 2-methoxyphenol fragment was noted to be a potential substructure for designing Pgp inhibitors, whereas the 2-sulfanylidene-1-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2- yl]-1,2-dihydropyridine-3-carbonitrile substructure was implied for avoiding transport by Pgp. Hence, this study could provide a comprehensive understanding of this drug transporter, which could benefit an early ADMET screening as well as drug design and development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Spatial Localization of the Ebola Virus Glycoprotein Mucin-Like Domain Determined by Cryo-Electron Tomography

    PubMed Central

    Tran, Erin E. H.; Simmons, James A.; Bartesaghi, Alberto; Shoemaker, Charles J.; Nelson, Elizabeth; White, Judith M.

    2014-01-01

    The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function. PMID:25008940

  16. Spatial localization of the Ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography.

    PubMed

    Tran, Erin E H; Simmons, James A; Bartesaghi, Alberto; Shoemaker, Charles J; Nelson, Elizabeth; White, Judith M; Subramaniam, Sriram

    2014-09-01

    The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Data Concentrator

    NASA Technical Reports Server (NTRS)

    Willett, Mike

    2015-01-01

    Orbital Research, Inc., developed, built, and tested three high-temperature components for use in the design of a data concentrator module in distributed turbine engine control. The concentrator receives analog and digital signals related to turbine engine control and communicates with a full authority digital engine control (FADEC) or high-level command processor. This data concentrator follows the Distributed Engine Controls Working Group (DECWG) roadmap for turbine engine distributed controls communication development that operates at temperatures at least up to 225 C. In Phase I, Orbital Research developed detailed specifications for each component needed for the system and defined the total system specifications. This entailed a combination of system design, compiling existing component specifications, laboratory testing, and simulation. The results showed the feasibility of the data concentrator. Phase II of this project focused on three key objectives. The first objective was to update the data concentrator design modifications from DECWG and prime contractors. Secondly, the project defined requirements for the three new high-temperature, application-specific integrated circuits (ASICs): one-time programmable (OTP), transient voltage suppression (TVS), and 3.3V. Finally, the project validated each design by testing over temperature and under load.

  18. Antibodies to the Glycoprotein GP2 Subunit Cross-React between Old and New World Arenaviruses.

    PubMed

    Amanat, Fatima; Duehr, James; Oestereich, Lisa; Hastie, Kathryn M; Ollmann Saphire, Erica; Krammer, Florian

    2018-06-27

    Arenaviruses pose a major public health threat and cause numerous infections in humans each year. Although most viruses belonging to this family do not cause disease in humans, some arenaviruses, such as Lassa virus and Machupo virus, are the etiological agents of lethal hemorrhagic fevers. The absence of a currently licensed vaccine and the highly pathogenic nature of these viruses both make the necessity of developing viable vaccines and therapeutics all the more urgent. Arenaviruses have a single glycoprotein on the surface of virions, the glycoprotein complex (GPC), and this protein can be used as a target for vaccine development. Here, we describe immunization strategies to generate monoclonal antibodies (MAbs) that cross-react between the glycoprotein complexes of both Old World and New World arenaviruses. Several monoclonal antibodies isolated from immunized mice were highly cross-reactive, binding a range of Old World arenavirus glycoproteins, including that of Lassa virus. One such monoclonal antibody, KL-AV-2A1, bound to GPCs of both New World and Old World viruses, including Lassa and Machupo viruses. These cross-reactive antibodies bound to epitopes present on the glycoprotein 2 subunit of the glycoprotein complex, which is relatively conserved among arenaviruses. Monoclonal antibodies binding to these epitopes, however, did not inhibit viral entry as they failed to neutralize a replication-competent vesicular stomatitis virus pseudotyped with the Lassa virus glycoprotein complex in vitro In addition, no protection from virus challenge was observed in in vivo mouse models. Even so, these monoclonal antibodies might still prove to be useful in the development of clinical and diagnostic assays. IMPORTANCE Several viruses in the Arenaviridae family infect humans and cause severe hemorrhagic fevers which lead to high case fatality rates. Due to their pathogenicity and geographic tropisms, these viruses remain very understudied. As a result, an effective

  19. Free diffusion to and from the inner nuclear membrane of newly synthesized plasma membrane glycoproteins

    PubMed Central

    1987-01-01

    Sindbis virus-infected baby hamster kidney (BHK) cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies was used in conjunction with colloidal gold- conjugated protein A. As we previously reported (Torrisi, M. R., and S. Bonatti, 1985, J. Cell Biol., 101:1300-1306), Sindbis transmembrane glycoproteins are present in the inner nuclear membrane as well as in the outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes. Viral glycoproteins located on the inner nuclear membrane resemble those present on the outer membrane in terms of amount, distribution, and preferential partition after fracture. We show in this paper that Sindbis glycoproteins after treatment with cycloheximide are removed from the inner nuclear membrane with the same kinetics as their counterparts present on the outer membrane. This finding strongly suggests that newly synthesized transmembrane glycoproteins may freely diffuse to and from the inner nuclear membrane before entering into the intracellular transport pathway to the plasma membrane. PMID:3818797

  20. Guidelines for cloning, expression, purification and functional characterization of primary HIV-1 envelope glycoproteins.

    PubMed

    Benureau, Yann; Colin, Philippe; Staropoli, Isabelle; Gonzalez, Nuria; Garcia-Perez, Javier; Alcami, Jose; Arenzana-Seisdedos, Fernando; Lagane, Bernard

    2016-10-01

    The trimeric HIV-1 envelope (Env) glycoproteins gp120 and gp41 mediate virus entry into target cells by engaging CD4 and the coreceptors CCR5 or CXCR4 at the cell surface and driving membrane fusion. Receptor/gp120 interactions regulate the virus life cycle, HIV infection transmission and pathogenesis. Env is also the target of neutralizing antibodies. Efforts have thus been made to produce soluble HIV-1 glycoproteins to develop vaccines and study the role and mechanisms of HIV/receptor interactions. However, production and purification of Env glycoproteins and their functional assessment has to cope with multiple obstacles. These include difficulties in amplifying and cloning env sequences and setting up receptor binding assays that are suitable for studies on large collections of glycoproteins, flexible enough to adapt to Env and receptor structural heterogeneities, and allow recapitulating the receptor binding properties of virion-associated Env trimers. Here we identify these difficulties and present protocols to produce primary gp120 and determination of their binding properties to receptors. The receptor binding assays confirmed that the produced glycoproteins are competent for binding CD4 and undergo proper CD4-induced conformational changes required for interaction with CCR5. These assays may help elucidate the role of gp120/receptor interactions in the pathophysiology of HIV infection and develop HIV-1 entry inhibitors. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Dynamics of glycoprotein charge in the evolutionary history of human influenza.

    PubMed

    Arinaminpathy, Nimalan; Grenfell, Bryan

    2010-12-30

    Influenza viruses show a significant capacity to evade host immunity; this is manifest both as large occasional jumps in the antigenic phenotype of viral surface molecules and in gradual antigenic changes leading to annual influenza epidemics in humans. Recent mouse studies show that avidity for host cells can play an important role in polyclonal antibody escape, and further that electrostatic charge of the hemagglutinin glycoprotein can contribute to such avidity. We test the role of glycoprotein charge on sequence data from the three major subtypes of influenza A in humans, using a simple method of calculating net glycoprotein charge. Of all subtypes, H3N2 in humans shows a striking pattern of increasing positive charge since its introduction in 1968. Notably, this trend applies to both hemagglutinin and neuraminidase glycoproteins. In the late 1980s hemagglutinin charge reached a plateau, while neuraminidase charge started to decline. We identify key groups of amino acid sites involved in this charge trend. To our knowledge these are the first indications that, for human H3N2, net glycoprotein charge covaries strongly with antigenic drift on a global scale. Further work is needed to elucidate how such charge interacts with other immune escape mechanisms, such as glycosylation, and we discuss important questions arising for future study.

  2. Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

    PubMed Central

    Thomas, Celestine J.; Shankar, Sundaresh; Casquilho-Gray, Hedi E.; York, Joanne; Sprang, Stephen R.; Nunberg, Jack H.

    2012-01-01

    The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein. PMID:23226473

  3. Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation

    SciTech Connect

    Piwnica-Worms, D.; Vallabhaneni, V.R.; Kronauge, J.F.

    1995-09-26

    Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an {approximately}170 integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis(2-methoxyisobutyl isonitrile) technetium(I) (Tc-SESTAMIBI), a {gamma}-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (T{sub 1/2} {approx} 6 min)more » of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. 70 refs., 7 figs., 2 tabs.« less

  4. Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis.

    PubMed

    Chen, Rui; Zou, Hanfa; Figeys, Daniel

    2016-06-03

    Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazide groups to oxidized glycoproteins, thus increasing the coverage of the N-glycoproteome. Compared with the conventional glycopeptide capture method, the detergent-assisted glycoprotein capture approach nearly doubled the number of N-glycosylation sites identified from HEK 293T cells with improved specificity. Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293T cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Furthermore, the application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immunodepletion or fractionation. Overall, the detergent-assisted glycoprotein capture method simplified the capture process, and it increased the number of sites observed on both hydrophobic membrane proteins and hydrophilic secreted proteins.

  5. Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation.

    PubMed

    Kim, Youngjin; Chen, Jue

    2018-02-23

    The multidrug transporter permeability (P)-glycoprotein is an adenosine triphosphate (ATP)-binding cassette exporter responsible for clinical resistance to chemotherapy. P-glycoprotein extrudes toxic molecules and drugs from cells through ATP-powered conformational changes. Despite decades of effort, only the structures of the inward-facing conformation of P-glycoprotein are available. Here we present the structure of human P-glycoprotein in the outward-facing conformation, determined by cryo-electron microscopy at 3.4-angstrom resolution. The two nucleotide-binding domains form a closed dimer occluding two ATP molecules. The drug-binding cavity observed in the inward-facing structures is reorientated toward the extracellular space and compressed to preclude substrate binding. This observation indicates that ATP binding, not hydrolysis, promotes substrate release. The structure evokes a model in which the dynamic nature of P-glycoprotein enables translocation of a large variety of substrates. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  6. MDR1-deficient genotype in Collie dogs hypersensitive to the P-glycoprotein substrate ivermectin.

    PubMed

    Roulet, Alain; Puel, Olivier; Gesta, Stéphane; Lepage, Jean-François; Drag, Marlene; Soll, Mark; Alvinerie, Michel; Pineau, Thierry

    2003-01-24

    Multidrug resistance (MDR) phenotypes in cancer cells are associated with overexpression of the drug carrier P-glycoprotein. The antiparasitic drug ivermectin, one of its substrates, abnormally accumulates in the brain of transgenic mice lacking the P-glycoprotein, resulting in neurotoxicity. Similarly, an enhanced sensitivity to ivermectin has been reported in certain dogs of the Collie breed. To explore the basis of this phenotype, we analyzed the canine P-glycoprotein-encoding MDR1 gene, and we report the first characterization of the cDNA for wild-type (Beagle) P-glycoprotein. The corresponding transcripts from ivermectin-sensitive Collies revealed a homozygous 4-bp exonic deletion. We established, by genetic testings, that the MDR1 frame shift is predictable. Accordingly, no P-glycoprotein was detected in the homozygote-deficient dogs. In conclusion, we characterized a unique case of naturally occurring gene invalidation. This provides a putative novel model that remains to be exploited in the field of human therapeutics and that might significantly affect tissue distribution and drug bioavailability studies.

  7. SYNTHESIS OF GLYCOPROTEIN, GLYCOLIPID, PROTEIN, AND LIPID IN SYNCHRONIZED L5178Y CELLS

    PubMed Central

    Bosmann, H. Bruce; Winston, R. Alan

    1970-01-01

    Synthesis of four macromolecular classes found in membranes—glycoprotein, glycolipid, protein, and lipid—was measured as a function of time of the cell cycle in synchronized L5178Y cells. Incorporation of leucine, choline, fucose, glucosamine, or thymidine into the cells, protein, nucleic acid, or lipid was measured by pulse-labeling for ½ hr at ½ hr intervals after release from the mitotic block. The amount of protein, lipid, glycoprotein, or glycolipid released or secreted into the medium by the L5178Y cells was also measured as a function of time of the cell cycle. Cellular protein was found to be synthesized throughout the cell cycle, with the highest synthesis occurring in the S period; synthesis was depressed in the M period. Cellular glycoprotein was synthesized at approximately the same times as protein, except that the rates of glycoprotein synthesis in the S period relative to other periods were much greater than for protein. Secreted protein was synthesized throughout the cell cycle without any general pattern, except that secretion was elevated in the late S and G2 periods. Secreted glycoprotein was similar to secreted protein. Cellular lipid and cellular glycolipid were synthesized almost exclusively in the G2 and M periods; there was no synthesis in the G1 and S periods. Release or secretion of glycolipid and lipid also occurred in the G2 and M periods. PMID:5458998

  8. Solar concentrator

    NASA Technical Reports Server (NTRS)

    Simpson, J. G. (Inventor)

    1979-01-01

    An improved solar concentrator is characterized by a number of elongated supporting members arranged in substantial horizontal parallelism with the axis and intersecting a common curve. A tensioned sheet of flexible reflective material is disposed in engaging relation with the supporting members in order to impart to the tensioned sheet a catenary configuration.

  9. Label-free detection of glycoproteins by the lectin biosensor down to attomolar level using gold nanoparticles

    PubMed Central

    Bertok, Tomas; Sediva, Alena; Katrlik, Jaroslav; Gemeiner, Pavol; Mikula, Milan; Nosko, Martin; Tkac, Jan

    2016-01-01

    We present here an ultrasensitive electrochemical biosensor based on a lectin biorecognition capable to detect concentrations of glycoproteins down to attomolar (aM) level by investigation of changes in the charge transfer resistance (Rct) using electrochemical impedance spectroscopy (EIS). On polycrystalline gold modified by an aminoalkanethiol linker layer, gold nanoparticles were attached. A Sambucus nigra agglutinin was covalently immobilised on a mixed self-assembled monolayer formed on gold nanoparticles and finally, the biosensor surface was blocked by poly(vinylalcohol). The lectin biosensor was applied for detection of sialic acid containing glycoproteins fetuin and asialofetuin. Building of a biosensing interface was carefully characterised by a broad range of techniques such as electrochemistry, EIS, atomic force microscopy, scanning electron microscopy and surface plasmon resonance with the best performance of the biosensor achieved by application of HS-(CH2)11-NH2 linker and gold nanoparticles with a diameter of 20 nm. The lectin biosensor responded to an addition of fetuin (8.7% of sialic acid) with sensitivity of (338 ± 11) Ω decade-1 and to asialofetuin (≤ 0.5% of sialic acid) with sensitivity of (109 ± 10) Ω decade-1 with a blank experiment with oxidised asialofetuin (without recognisable sialic acid) revealing sensitivity of detection of (79 ± 13) Ω decade-1. These results suggest the lectin biosensor responded to changes in the glycan amount in a quantitative way with a successful validation by a lectin microarray. Such a biosensor device has a great potential to be employed in early biomedical diagnostics of diseases such as arthritis or cancer, which are connected to aberrant glycosylation of protein biomarkers in biological fluids. PMID:23601864

  10. Label-free detection of glycoproteins by the lectin biosensor down to attomolar level using gold nanoparticles.

    PubMed

    Bertok, Tomas; Sediva, Alena; Katrlik, Jaroslav; Gemeiner, Pavol; Mikula, Milan; Nosko, Martin; Tkac, Jan

    2013-04-15

    We present here an ultrasensitive electrochemical biosensor based on a lectin biorecognition capable to detect concentrations of glycoproteins down to attomolar (aM) level by investigation of changes in the charge transfer resistance (Rct) using electrochemical impedance spectroscopy (EIS). On polycrystalline gold modified by an aminoalkanethiol linker layer, gold nanoparticles were attached. A Sambucus nigra agglutinin was covalently immobilised on a mixed self-assembled monolayer formed on gold nanoparticles and finally, the biosensor surface was blocked by poly(vinyl alcohol). The lectin biosensor was applied for detection of sialic acid containing glycoproteins fetuin and asialofetuin. Building of a biosensing interface was carefully characterised by a broad range of techniques such as electrochemistry, EIS, atomic force microscopy, scanning electron microscopy and surface plasmon resonance with the best performance of the biosensor achieved by application of HS-(CH2)11-NH2 linker and gold nanoparticles with a diameter of 20 nm. The lectin biosensor responded to an addition of fetuin (8.7% of sialic acid) with sensitivity of (338 ± 11) Ω decade(-1) and to asialofetuin (≤ 0.5% of sialic acid) with sensitivity of (109 ± 10) Ω decade(-1) with a blank experiment with oxidised asialofetuin (without recognisable sialic acid) revealing sensitivity of detection of (79 ± 13) Ω decade(-1). These results suggest the lectin biosensor responded to changes in the glycan amount in a quantitative way with a successful validation by a lectin microarray. Such a biosensor device has a great potential to be employed in early biomedical diagnostics of diseases such as arthritis or cancer, which are connected to aberrant glycosylation of protein biomarkers in biological fluids. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Infectious Mononucleosis Triggers Generation of IgG Auto-Antibodies against Native Myelin Oligodendrocyte Glycoprotein.

    PubMed

    Kakalacheva, Kristina; Regenass, Stephan; Wiesmayr, Silke; Azzi, Tarik; Berger, Christoph; Dale, Russell C; Brilot, Fabienne; Münz, Christian; Rostasy, Kevin; Nadal, David; Lünemann, Jan D

    2016-02-12

    A history of infectious mononucleosis (IM), symptomatic primary infection with the Epstein Barr virus, is associated with the development of autoimmune diseases and increases the risk to develop multiple sclerosis. Here, we hypothesized that immune activation during IM triggers autoreactive immune responses. Antibody responses towards cellular antigens using a HEp-2 based indirect immunofluorescence assay and native myelin oligodendrocyte glycoprotein (MOG) using a flow cytometry-based assay were determined in 35 patients with IM and in 23 control subjects. We detected frequent immunoglobulin M (IgM) reactivity to vimentin, a major constituent of the intermediate filament family of proteins, in IM patients (27/35; 77%) but rarely in control subjects (2/23; 9%). IgG autoantibodies binding to HEp-2 cells were absent in both groups. In contrast, IgG responses to native MOG, present in up to 40% of children with inflammatory demyelinating diseases of the central nervous system (CNS), were detectable in 7/35 (20%) patients with IM but not in control subjects. Normalization of anti-vimentin IgM levels to increased total IgM concentrations during IM resulted in loss of significant differences for anti-vimentin IgM titers. Anti-MOG specific IgG responses were still detectable in a subset of three out of 35 patients with IM (9%), even after normalization to increased total IgG levels. Vimentin-specific IgM and MOG-specific IgG responses decreased following clinical resolution of acute IM symptoms. We conclude from our data that MOG-specific memory B cells are activated in subset of patients with IM.

  12. Infectious Mononucleosis Triggers Generation of IgG Auto-Antibodies against Native Myelin Oligodendrocyte Glycoprotein

    PubMed Central

    Kakalacheva, Kristina; Regenass, Stephan; Wiesmayr, Silke; Azzi, Tarik; Berger, Christoph; Dale, Russell C.; Brilot, Fabienne; Münz, Christian; Rostasy, Kevin; Nadal, David; Lünemann, Jan D.

    2016-01-01

    A history of infectious mononucleosis (IM), symptomatic primary infection with the Epstein Barr virus, is associated with the development of autoimmune diseases and increases the risk to develop multiple sclerosis. Here, we hypothesized that immune activation during IM triggers autoreactive immune responses. Antibody responses towards cellular antigens using a HEp-2 based indirect immunofluorescence assay and native myelin oligodendrocyte glycoprotein (MOG) using a flow cytometry-based assay were determined in 35 patients with IM and in 23 control subjects. We detected frequent immunoglobulin M (IgM) reactivity to vimentin, a major constituent of the intermediate filament family of proteins, in IM patients (27/35; 77%) but rarely in control subjects (2/23; 9%). IgG autoantibodies binding to HEp-2 cells were absent in both groups. In contrast, IgG responses to native MOG, present in up to 40% of children with inflammatory demyelinating diseases of the central nervous system (CNS), were detectable in 7/35 (20%) patients with IM but not in control subjects. Normalization of anti-vimentin IgM levels to increased total IgM concentrations during IM resulted in loss of significant differences for anti-vimentin IgM titers. Anti-MOG specific IgG responses were still detectable in a subset of three out of 35 patients with IM (9%), even after normalization to increased total IgG levels. Vimentin-specific IgM and MOG-specific IgG responses decreased following clinical resolution of acute IM symptoms. We conclude from our data that MOG-specific memory B cells are activated in subset of patients with IM. PMID:26907324

  13. Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.

    PubMed

    Schuster, M; Wasserbauer, E; Aversa, G; Jungbauer, A

    2001-02-01

    Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain. The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand. Therefore several fragments of this region have been expressed as Flag-fusions in S. cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region. By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway. All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence. Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted. High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases. After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody. The fusion protein concentration in the supernatant was 30 microg/ml. The

  14. Inhibition of P-glycoprotein functionality by vandetanib may reverse cancer cell resistance to doxorubicin.

    PubMed

    Jovelet, C; Bénard, J; Forestier, F; Farinotti, R; Bidart, J M; Gil, S

    2012-08-15

    P-glycoprotein belongs to the ATP binding cassette transporters, responsible for the multidrug resistance of cancer cells. These transporters efflux hydrophobic drugs outside cells and decrease their therapeutic efficacy. The aim of this study was to investigate the effect of vandetanib, an oral tyrosine kinase inhibitor of EGFR, VEGFR 2 and RET kinases, on the functionality of P-gp after a 24h-treatment at therapeutic concentration (2μM), and its ability to increase the cytotoxicity of chemotherapeutic agents in multidrug resistance cancer cells. In this study we found that IGROV1-DXR and IGROV1-CDDP cells were resistant to doxorubicin and cisplatin respectively, compare to parental cell line IGROV1. The parental sensitive and the two resistant cell lines similarly expressed MRP1 and did not express BCRP. Moreover, in contrast to the IGROV1 and IGROV1-CDDP cells, IGROV1-DXR cell line overexpressed P-gp. Functional activity studies demonstrated that MRP1 was not functional and the MDR phenotype in IGROV1-DXR cells was linked to P-gp functionality. Results also showed that vandetanib reversed resistance to doxorubicin in IGROV1-DXR cells, but not to cisplatin in IGROV1-CDDP cells. After 24h of treatment, vandetanib increased the accumulation of rhodamine 123 and calcein AM, demonstrating a functional inhibition of the transporter. In IGROV1-DXR cell line, vandetanib reverse resistance to doxorubicin by inhibiting the functionality of P-gp. In conclusion, vandetanib should be an option for drug combination in patients already developing a P-gp mediated multidrug resistance. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella

    PubMed Central

    1991-01-01

    The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half- time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport. PMID:1874785

  16. Novel mutations in Marburg virus glycoprotein associated with viral evasion from antibody mediated immune pressure.

    PubMed

    Kajihara, Masahiro; Nakayama, Eri; Marzi, Andrea; Igarashi, Manabu; Feldmann, Heinz; Takada, Ayato

    2013-04-01

    Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.

  17. Novel mutations in Marburg virus glycoprotein associated with viral evasion from antibody mediated immune pressure

    PubMed Central

    Kajihara, Masahiro; Nakayama, Eri; Marzi, Andrea; Igarashi, Manabu; Feldmann, Heinz

    2013-01-01

    Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability. PMID:23288419

  18. Jatrophane diterpenoids from Euphorbia sororia as potent modulators against P-glycoprotein-based multidrug resistance.

    PubMed

    Hu, Rui; Gao, Jie; Rozimamat, Rushangul; Aisa, Haji Akber

    2018-02-25

    Five new (1-5) and ten known (6-15) jatrophane diterpenoids were isolated from the fructus of Euphorbia sororia and their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1 and 4 were confirmed by X-ray crystallographic analysis. Cytotoxicity and anti-multidrug resistance effects of these jatrophane diterpenoids were evaluated in multidrug-resistant MCF-7/ADR breast cancer cells with an overexpression of P-glycoprotein (P-gp). Eight compounds (1, 2, 4, 6, 8, 10, 11, and 15) showed promising chemoreversal abilities compared to verapamil (VRP). The most potent compound, Euphosorophane A (1), possessed many advantages, including (1) high potency (EC 50  = 92.68 ± 18.28 nM) in reversing P-gp-mediated resistance to doxorubicin (DOX), low cytotoxicity, and a high therapeutic index, (2) potency in reversing resistance to other cytotoxic agents associated with MDR, and (3) inhibition of P-gp-mediated Rhodamine123 (Rh123) efflux function in MCF-7/ADR cells. The results of the Western blot analysis indicated that the multidrug resistance (MDR) reversal induced by 1 was not due to the inhibiton of P-gp expression. Compound 1 stimulated P-gp-ATPase activity and caused the dose-dependent inhibition of DOX transport activity. Lineweaver-Burk and Dixon plots implied that 1 was a competitive inhibitor to DOX in the binding site of P-gp with a Ki of 0.49-0.50 μM. Our data suggested that 1 had a high binding affinity toward the DOX recognition site of P-gp. This resulted in inhibiting DOX transport, increasing intracellular DOX concentration, and finally resensitizing MCF-7/ADR to DOX. In addition, we discussed some added contents in the structure-activity relationship (SAR) of jatrophane diterpenoids. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  19. Design, synthesis, and biological evaluation of 6-methoxy-2-arylquinolines as potential P-glycoprotein inhibitors.

    PubMed

    Aboutorabzadeh, Sayyed Mohammad; Mosaffa, Fatemeh; Hadizadeh, Farzin; Ghodsi, Razieh

    2018-01-01

    In the present study, a new series of 6-methoxy-2-arylquinoline analogues was designed and synthesized as P-glycoprotein (P-gp) inhibitors using quinine and flavones as the lead compounds. The cytotoxic activity of the synthesized compounds was evaluated against two human cancer cell lines including EPG85-257RDB, multidrug-resistant gastric carcinoma cells (P-gp-positive gastric carcinoma cell line), and EPG85-257P, drug-sensitive gastric carcinoma cells. Compounds showing low to moderate toxicity in the MTT test were selected to investigate their P-gp inhibition activity. Moreover, trying to explain the results of biological experiments, docking studies of the selected compounds into the homology-modeled human P-gp, were carried out. The physicochemical and ADME properties of the compounds as drug candidate were also predicted. Most of our compounds exhibited negligible or much lower cytotoxic effect in both cancer cells. Among the series, 5a and 5b, alcoholic quinoline derivatives were found to inhibit the efflux of rhodamine 123 at the concentration of 10 μM significantly. Among the tested quinolines, 5a and 5b showed the most potent P-gp inhibitory activity in the series and were 1.3-fold and 2.1-fold stronger than verapamil, respectively. SAR data revealed that hydroxyl methyl in position 4 of quinolines has a key role in P-gp efflux inhibition of our compounds. ADME studies suggested that all of the compounds included in this study may have a good human intestinal absorption.

  20. Kuguacin J isolated from Momordica charantia leaves inhibits P-glycoprotein (ABCB1)-mediated multidrug resistance

    PubMed Central

    Pitchakarn, Pornsiri; Ohnuma, Shinobu; Pintha, Komsak; Pompimon, Wilart; Ambudkar, Suresh V.; Limtrakul, Pornngarm

    2012-01-01

    Multidrug-resistance (MDR), a phenomenon in which cancer cells exhibit simultaneous resistance to chemically unrelated drugs, is a major factor in the failure of chemotherapy in cancer patients. Resistance to chemotherapy has been correlated to the overexpression of ABC drug transporters including P-glycoprotein (P-gp) that actively efflux chemotherapeutic drugs from cancer cells. Our previous study showed that bitter melon (Momordica charantia) leaf extract (BMLE) was able to reverse the MDR phenotype by increasing the intracellular accumulation of chemotherapeutic drugs. In the present study, bioguided fractionation was used to identify the active component(s) of BMLE, which is able to modulate the function of P-gp and the MDR phenotype in a human cervical carcinoma cell line (KB-V1). We found that kuguacin J, one of the active components in BMLE, increased sensitivity to vinblastine and paclitaxel in KB-V1 cells. A flow cytometry assay indicated that kuguacin J inhibits the transport function of P-gp and thereby significantly increase the accumulation of rhodamine123 and calcein AM in the cells. These results were confirmed by [3H]-vinblastine transport assay. Kuguacin J significantly increases intracellular [3H]-vinblastine accumulation and decreased the [3H]-vinblastine efflux in the cells. Kuguacin J also inhibited the incorporation of [125I]-iodoarylazidoprazosin into P-gp in a concentration-dependent manner, indicating that kuguacin J directly interacts with the drug-substrate-binding site on P-gp. These results indicate that kuguacin J modulates the function of P-gp by directly interacting at the drug-substrate-binding site and it appears to be an effective inhibitor of P-gp activity in vitro, and thus could be developed as an effective chemosensitizer to treat multidrug-resistant cancers. PMID:21414769

  1. Influence of the multidrug transporter P-glycoprotein on the intracellular pharmacokinetics of vandetanib.

    PubMed

    Jovelet, C; Deroussent, A; Broutin, S; Paci, A; Farinotti, R; Bidart, J M; Gil, S

    2013-09-01

    Efflux transporters play an important role in the resistance of tumor cells against anticancer agents. Interaction between these transporters, including P-glycoprotein (P-gp), and drugs might influence their pharmacological properties and toxicities. The aim of this study was to investigate whether vandetanib (Caprelsa(®)), a small tyrosine kinase inhibitor, could interact with the multidrug transporter P-gp. Interaction of vandetanib with the P-gp was investigated using the parental cell line (IGROV1) and the P-gp doxorubicin-resistant (IGROV1-DXR) cell line, derived from the parental drug-sensitive IGROV1 cells. Cytotoxicity tests were assessed in both cell lines to examine the impact of P-gp on the cell survival after a vandetanib treatment. The effects of P-gp on vandetanib intracellular pharmacokinetics were investigated. To this aim, we developed a quantitative liquid chromatography tandem mass spectrometry to quantify vandetanib in cell medium. Results showed that overexpression of P-gp confers resistance to vandetanib in the IGROV1-DXR cell line. Using a LC-MS/MS assay validated in cell medium, cellular pharmacokinetic studies revealed that in cells overexpressing the P-gp intracellular concentrations of vandetanib were decreased compared to parental cell line. For the first time, vandetanib is described as a substrate of P-gp. In tumor cells, P-gp could be responsible for cellular resistance to vandetanib. It may be relevant to the clinical efficacy of vandetanib. Moreover, interaction of vandetanib with P-gp could modify the pharmacodynamics of other conventional chemotherapeutics, substrates of P-gp. It could impact on the overall response to anticancer therapy.

  2. In Vitro Comparison of the Role of P-Glycoprotein and Breast Cancer Resistance Protein on Direct Oral Anticoagulants Disposition.

    PubMed

    Hodin, Sophie; Basset, Thierry; Jacqueroux, Elodie; Delezay, Olivier; Clotagatide, Anthony; Perek, Nathalie; Mismetti, Patrick; Delavenne, Xavier

    2018-04-01

    Pharmacokinetics of direct oral anticoagulants (DOACs) are influenced by ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). To better understand the role of transporters in DOAC disposition, we evaluated and compared the permeabilities and transport properties of these drugs. Bidirectional permeabilities of DOACs were investigated across Caco-2 cells monolayer. Transport assays were performed using different concentrations of DOAC and specific inhibitors of ABC transporters. Cell model functionality was evaluated by transport assay of two positive control substrates. The results of transport assays suggest a concentration-dependent efflux of apixaban, dabigatran etexilate and edoxaban, whereas the efflux transport of rivaroxaban did not seem to depend on concentration. Verapamil, a strong inhibitor of P-gp, decreased DOAC efflux in the Caco-2 cell model by 12-87%, depending on the drug tested. Ko143 reduced BCRP-mediated DOAC efflux in Caco-2 cells by 46-76%. This study allowed identification of three different profiles of ABC carrier-mediated transport: predominantly P-gp-dependent transport (dabigatran), preferential BCRP-dependent transport (apixaban) and approximately equivalent P-gp and BCRP-mediated transport (edoxaban and rivaroxaban).

  3. Reversal of P-glycoprotein-mediated multidrug resistance by 5,6,7,3',4'-pentamethoxyflavone (Sinensetin).

    PubMed

    Choi, Cheol Hee; Sun, Kyung Hoon; An, Chun San; Yoo, Jin Cheol; Hahm, Kyung Soo; Lee, In Hwa; Sohng, Jae Kyung; Kim, Youn Chul

    2002-07-26

    Multidrug resistance (MDR) cells can be sensitized to anticancer drugs when treated concomitantly with chemosensitizers. In this study, chemosensitizing effects of 5,6,7,3',4'-pentamethoxyflavone (sinensetin) and its analogs were investigated with respect to in vitro efficacy and structure-activity relationship. Sinensetin reversed the resistance of P-glycoprotein (Pgp)-overexpressing AML-2/D100 to vincristine in a concentration-dependent manner. Chemosensitizing effect of sinensetin was 10- and 18-fold higher than those of 5,7,3',4'-tetramethoxyflavone and 3,7-dihydroxy-3',4'-dimethoxyflavone, respectively. Sinensetin cytotoxicity in AML-2/D100 was not changed by the complete inhibition of Pgp, suggesting that it is not a substrate for Pgp. Flow cytometry showed that sinensetin increased drug accumulation in the AML-2/D100 in a concentration-dependent manner. Unlike verapamil and cyclosporin A, the maximum non-cytotoxic concentrations of sinensetin were found to decrease the Pgp levels. Azidopine-binding assay showed that cyclosporin A or verapamil inhibited azidopine binding on Pgp partially but sinensetin did not. Taken together, these results suggest that sinensetin has a chemosensitizing effect in reversing Pgp-mediated MDR by increasing the intracellular accumulation of drugs without competition in a binding site of azidopine. Thus, sinensetin is anticipated as a novel and highly potent second-generation flavonoid chemosensitizer, since sinensetin has significant advantages of having a high therapeutic index, of being a non-transportable inhibitor, and of effecting no induction of Pgp.

  4. Classification analysis of P-glycoprotein substrate specificity.

    PubMed

    Didziapetris, Remigijus; Japertas, Pranas; Avdeef, Alex; Petrauskas, Alanas

    2003-08-01

    Prediction of P-glycoprotein substrate specificity (S(PGP)) can be viewed as a constituent part of a compound's "pharmaceutical profiling" in drug design. This task is difficult to achieve due to several factors that raised many contradictory opinions: (i) the disparity between the S(PGP) values obtained in different assays, (ii) the confusion between Pgp substrates and inhibitors, (iii) the confusion between lipophilicity and amphiphilicity of Pgp substrates, and (iv) the dilemma of describing class-specific relationships when Pgp has no binding sites of high ligand specificity. In this work, we compiled S(PGP) data for 1000 compounds. All data were represented in a binary format, assigning S(PGP) = 1 for substrates and S(PGP) = 0 for non-substrates. Each value was ranked according to the reliability of experimental assay. Two data sets were considered. Set 1 included 220 compounds with S(PGP) from polarized transport across MDR1 transfected cell monolayers. Set 2 included the entire list of 1000 compounds, with S(PGP) values of generally lower reliability. Both sets were analysed using a stepwise classification structure-activity relationship (C-SAR) method, leading to derivation of simple rules for crude estimation of S(PGP) values. The obtained rules are based on the following factors: (i) compound's size expressed through molar weight or volume, (ii) H-accepting given by the Abraham's beta (that can be crudely approximated by the sum of O and N atoms), and (iii) ionization given by the acid and base pKa values. Very roughly, S(PGP) can be estimated by the "rule of fours". Compounds with (N + O) > or = 8, MW > 400 and acid pKa > 4 are likely to be Pgp substrates, whereas compounds with (N + O) < or = 4, MW < 400 and base pKa < 8 are likely to be non-substrates. The obtained results support the view that Pgp functioning can be compared to a complex "mini-pharmacokinetic" system with fuzzy specificity. This system can be described by a probabilistic version of

  5. Contribution of the attachment G glycoprotein to pathogenicity and immunogenicity of avian metapneumovirus subgroup C.

    PubMed

    Govindarajan, Dhanasekaran; Kim, Shin-Hee; Samal, Siba K

    2010-03-01

    Avian metapneumovirus (AMPV) causes an upper respiratory tract infection in turkeys leading to serious economic losses to the turkey industry. The G glycoprotein of AMPV is known to be associated with viral attachment and pathogenesis. In this study, we determined the role of the G glycoprotein in the pathogenicity and immunogenicity of AMPV strain Colorado (AMPV/CO). Recombinant AMPV/CO lacking the G protein (rAMPV/CO-deltaG) was generated using a reverse-genetics system. The recovered rAMPV/CO-deltaG replicated slightly better than did wild-type AMPV in Vero cells. However, deletion of the G gene in AMPV resulted in attenuation of the virus in turkeys. The mutant virus induced less-severe clinical signs and a weaker immune response in turkeys than did the wild-type AMPV. Our results suggest that the G glycoprotein is an important determinant for the pathogenicity and immunogenicity of AMPV.

  6. The fluorescence scanning of microgels stained for glycoproteins with fluorescein isothiocyanate-labelled concanavalin A.

    PubMed

    Lane, J D; Zimmer, H G; Neuhoff, V

    1979-10-01

    A technique is presented which allows one to label and quantitate glycoproteins. Small amounts of protein from biological samples (0.5-2.5 microgram for mixtures and less for individual proteins) are separated by sodium dodecyl sulfate gel electrophoresis on 1-30% polyacrylamide gradient microgels. The gels are stained with Co-omassie Brillant Blue R250 to evaluate relative migration or fixed in 2-propanol/acetic acid and stained with fluorescein isothiocyanate-labelled concanavalin A. The microgels are then scanned using a fluorescence microscope controlled by a computer, although simpler configurations are possible. Standards of known carbohydrate composition (e.g., glucose oxidase) are used for comparative purposes. Glycoproteins in the order of 5-30 ng protein (or 1-5 ng carbohydrate) can be detected without difficulty. This technique may prove valuable in evaluating glycoproteins when the available material is limited.

  7. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    PubMed Central

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  8. Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

    PubMed

    Cao, Xueyan; Song, Dahe; Yang, Mei; Yang, Ning; Ye, Qing; Tao, Dongbing; Liu, Biao; Wu, Rina; Yue, Xiqing

    2017-11-29

    Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

  9. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism.

  10. Use of an informed search space maximizes confidence of site-specific assignment of glycoprotein glycosylation.

    PubMed

    Khatri, Kshitij; Klein, Joshua A; Zaia, Joseph

    2017-01-01

    In order to interpret glycopeptide tandem mass spectra, it is necessary to estimate the theoretical glycan compositions and peptide sequences, known as the search space. The simplest way to do this is to build a naïve search space from sets of glycan compositions from public databases and to assume that the target glycoprotein is pure. Often, however, purified glycoproteins contain co-purified glycoprotein contaminants that have the potential to confound assignment of tandem mass spectra based on naïve assumptions. In addition, there is increasing need to characterize glycopeptides from complex biological mixtures. Fortunately, liquid chromatography-mass spectrometry (LC-MS) methods for glycomics and proteomics are now mature and accessible. We demonstrate the value of using an informed search space built from measured glycomes and proteomes to define the search space for interpretation of glycoproteomics data. We show this using α-1-acid glycoprotein (AGP) mixed into a set of increasingly complex matrices. As the mixture complexity increases, the naïve search space balloons and the ability to assign glycopeptides with acceptable confidence diminishes. In addition, it is not possible to identify glycopeptides not foreseen as part of the naïve search space. A search space built from released glycan glycomics and proteomics data is smaller than its naïve counterpart while including the full range of proteins detected in the mixture. This maximizes the ability to assign glycopeptide tandem mass spectra with confidence. As the mixture complexity increases, the number of tandem mass spectra per glycopeptide precursor ion decreases, resulting in lower overall scores and reduced depth of coverage for the target glycoprotein. We suggest use of α-1-acid glycoprotein as a standard to gauge effectiveness of analytical methods and bioinformatics search parameters for glycoproteomics studies. Graphical Abstract Assignment of site specific glycosylation from LC

  11. Modulation of P-glycoprotein at the Blood-Brain Barrier: Opportunities to Improve CNS Pharmacotherapy

    PubMed Central

    Miller, David S.; Bauer, Björn; Hartz, Anika M.S.

    2009-01-01

    Pharmacotherapy of CNS disorders, e.g., neurodegenerative diseases, epilepsy, brain cancer, and neuro-AIDS, is limited by the blood-brain barrier. P-glycoprotein, an ATP-driven, drug efflux transporter, is a critical element of that barrier. High level of expression, luminal membrane location, specificity and high transport potency make P-glycoprotein a selective gate-keeper of the blood-brain barrier and thus a primary obstacle to drug delivery into the brain. As such, P-glycoprotein limits entry into the CNS for a large number of prescribed drugs, contributes to the poor success rate of CNS drug candidates and likely contributes to patient-to-patient variability in response to CNS pharmacotherapy. Modulating P-glycoprotein could therefore improve drug delivery into the brain. Here we review the current understanding of signaling mechanisms responsible for the modulation of P-glycoprotein activity/expression at the blood-brain barrier with an emphasis on recent studies from our laboratories. Using intact brain capillaries from rats and mice, we have identified multiple extracellular and intracellular signals that regulate this transporter; several signaling pathways have been mapped. Three pathways are triggered by elements of the brain's innate immune response, one by glutamate, one by xenobiotic-nuclear receptor (PXR) interactions and one by elevated β-amyloid levels. Signaling is complex, with several pathways sharing common signaling elements (TNF-R1, ETB receptor, PKC, NOS), suggesting a regulatory network. Several pathways utilize autocrine/paracrine elements, involving release of the proinflammatory cytokine, TNF-α, and the polypeptide hormone, ET-1. Finally, several steps in signaling are potential therapeutic targets that could be used to modulate P-glycoprotein activity in the clinic. PMID:18560012

  12. Sphingolipid signaling reduces basal P-glycoprotein activity in renal proximal tubule.

    PubMed

    Miller, David S

    2014-03-01

    P-glycoprotein is an ATP-driven xenobiotic export pump that is highly expressed in barrier and excretory tissues, where it greatly influences drug pharmacokinetics. Recent studies in the blood-brain and spinal cord barriers identified a sphingolipid-based signaling pathway that regulates basal activity of P-glycoprotein. Here we use an established comparative renal model that permits direct measurement of P-glycoprotein activity to determine whether such signaling occurs in another tissue, killifish renal proximal tubule. Isolated killifish tubules exposed to 0.01-1.0 μM sphingosine-1-phosphate (S1P) exhibited a profound decrease in P-glycoprotein transport activity, measured as specific accumulation of a fluorescent cyclosporine A derivative in the tubule lumen. Loss of activity had a rapid onset and was fully reversible when the S1P was removed. Transport mediated by multidrug resistance-associated protein 2 (Mrp2) or a teleost fish organic anion transporter (Oat) was not affected. S1P effects were blocked by a specific S1P receptor 1 (S1PR1) antagonist and mimicked by a S1PR agonist. Sphingosine also reduced P-glycoprotein transport activity and those effects were blocked by an inhibitor of sphingosine kinase and by the S1PR1 antagonist. These results for a comparative renal model suggest that sphingolipid signaling to P-glycoprotein is not just restricted to the blood-brain and blood-spinal cord barriers, but occurs in other excretory and barrier tissues.

  13. Monitoring binding affinity between drug and α1-acid glycoprotein in real time by Venturi easy ambient sonic-spray ionization mass spectrometry.

    PubMed

    Liu, Ning; Lu, Xin; Yang, YuHan; Yao, Chen Xi; Ning, BaoMing; He, Dacheng; He, Lan; Ouyang, Jin

    2015-10-01

    A new approach for monitoring the binding affinity between drugs and alpha 1-acid glycoprotein in real time was developed based on a combination of drug-protein reaction followed by Venturi easy ambient sonic-spray ionization mass spectrometry determination of the free drug concentrations. A known basic drug, propranolol was used to validate the new built method. Binding constant values calculated by venturi easy ambient sonic-spray ionization mass spectrometry was in good accordance with a traditional ultrafiltration combined with high performance liquid chromatography method. Then six types of basic drugs were used as the samples to conduct the real time analysis. Upon injection of alpha 1-acid glycoprotein to the drug mixture, the ion chromatograms were extracted to show the changes in the free drug concentrations in real time. By observing the drop-out of six types of drugs during the whole binding reaction, the binding affinities of different drugs were distinguished. A volume shift validating experiment and an injection delay correcting experiment were also performed to eliminate extraneous factors and verify the reliability of our experiment. Therefore, the features of Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS) and the experimental results indicate that our technique is likely to become a powerful tool for monitoring drug-AGP binding affinity in real time. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Diagnosis of Herpes Simplex Encephalitis by ELISA Using Antipeptide Antibodies Against Type-Common Epitopes of Glycoprotein B of Herpes Simplex Viruses.

    PubMed

    Bhullar, Shradha S; Chandak, Nitin H; Baheti, Neeraj N; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

    2016-01-01

    Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system (CNS). As effective antiviral drugs are available, an early, rapid, and reliable diagnosis has become important. The objective of this article was to develop a sensitive ELISA protocol for herpes simplex viruses (HSV) antigen detection and quantitation by assessing the usefulness of antipeptide antibodies against potential peptides of HSV glycoprotein B (gB). A total of 180 cerebrospinal fluid (CSF) samples of HSE and non-HSE patients were analyzed using a panel of antipeptide antibodies against synthetic peptides of HSV glycoprotein gB. The cases of confirmed and suspected HSE showed 80% and 51% positivity for antipeptide against synthetic peptide QLHDLRF and 77% and 53% positivity for antipeptide against synthetic peptide MKALYPLTT, respectively for the detection of HSV antigen in CSF. The concentration of HSV antigen was found to be higher in confirmed HSE as compared to suspected HSE group and the viral load correlated well with antigen concentration obtained using the two antipeptides in CSF of confirmed HSE group. This is the first article describing the use of antibodies obtained against synthetic peptides derived from HSV in diagnostics of HSE using patients' CSF samples.

  15. Molecular Study of Interactions Between P-Glycoprotein and Anticancer Drugs.

    DTIC Science & Technology

    1996-09-01

    Denicoff, A., Le , T.B., Tucker, E., Steinberg, S.M., Elwood, L.J. (1995) Pharma. 35:457-463. Bradley, G., Naik, M. and Ling, V. (1989) Cancer Res. 49...and Joh-nson, K. (1988) Gene 67:31-40. Sugawara, I., Kataoka, I., Morishita, Y., Hamada, H., Tsuruo, T., Itoyama, S and Mori, S. (1988) Cancer Res...therapy, P-glycoprotein, topology, CFTR Abbreviations: Pgp - P-glycoprotein; MDR - multidrug resistance; ABC - ATP-binding cassette; RRL - rabbit

  16. High P-glycoprotein-mediated export observed in patients with a history of idiopathic thrombocytopenic purpura.

    PubMed

    Levy, Adam S; Cunningham-Rundles, Susanna; Mazza, BethAnne; Simm, Maciej; Gorlick, Richard; Bussel, James

    2002-09-01

    Studies have suggested that high P-glycoprotein expression in lymphocytes from patients with autoimmune disorders may affect disease outcome. Idiopathic thrombocytopenic purpura (ITP) and Evans' syndrome are widely thought to be autoimmune processes, however, the precise mechanisms remain unknown. Peripheral blood mononuclear cells from patients with refractory or recurrent ITP or Evans' syndrome were studied using the rhodamine 123 flow cytometric assay to investigate functional export levels. Lymphocytes from ITP and Evans' syndrome patients showed a significantly decreased ability to retain rhodamine, suggesting increased export protein function. Reverse transcription polymerase chain reaction distinguished P-glycoprotein as the likely export protein.

  17. Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein.

    PubMed

    Favier, Anne-Laure; Gout, Evelyne; Reynard, Olivier; Ferraris, Olivier; Kleman, Jean-Philippe; Volchkov, Viktor; Peyrefitte, Christophe; Thielens, Nicole M

    2016-06-01

    Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the

  18. ICP-MS-Based Multiplex Profiling of Glycoproteins Using Lectins Conjugated to Lanthanide-Chelating Polymers

    PubMed Central

    Leipold, Michael D.; Herrera, Isaac; Ornatsky, Olga; Baranov, Vladimir; Nitz, Mark

    2009-01-01

    Lectins have been increasingly important in the study of glycoproteins. Here we report a glycoprofiling method based on the covalent attachment of metal-chelating polymers to lectins for use in an ICP-MS-based assays. The labeled lectins are able to distinguish between glycoproteins covalently attached to a microtiter plate and their binding can be directly quantified by ICP-MS. Since each conjugate contains a different lanthanide, the assays can be conducted in a single or multiplex fashion, and may be readily elaborated to many different assay formats. PMID:19072657

  19. Localization of Epstein-Barr virus envelope glycoproteins on the inner nuclear membrane of virus-producing cells.

    PubMed Central

    Torrisi, M R; Cirone, M; Pavan, A; Zompetta, C; Barile, G; Frati, L; Faggioni, A

    1989-01-01

    Epstein-Barr virus-producing cells were used as a model to analyze, with a fracture-immunolabel technique, the distribution, behavior on fracture, and extent of glycosylation of viral transmembrane glycoproteins at the inner nuclear membrane. Surface and fracture immunolabeling with two monoclonal antibodies directed against the carbohydrate or polypeptide portions of the major viral envelope glycoproteins gp350/220 showed the following. (i) The glycoproteins present on the inner and outer nuclear membranes were labeled only with the monoclonal antibody directed against the polypeptide chain, whereas over the surface of virus-producing cells and on mature virions the labeling was dense and uniformly distributed with both monoclonal antibodies. (ii) The glycoproteins were nonuniformly distributed only over the inner nuclear membranes; at the sites of viral budding, the glycoproteins showed a preferential partition with the protoplasmic face. Since fully glycosylated glycoproteins were not present on the nuclear membranes, our observations support the proposed model of herpesvirus maturation. The peculiar distribution and partition on fracture of the envelope glycoproteins on the inner nuclear membrane are similar to those of Sindbis virus envelope glycoproteins on the plasma membrane of infected cells. Therefore, our results suggest that inner nuclear membranes may behave like plasma membranes during viral assembly. Images PMID:2536106

  20. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

    SciTech Connect

    Follis, Kathryn E.; York, Joanne; Nunberg, Jack H.

    2006-07-05

    The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctionalmore » region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.« less

  1. Synthesis of peptide-immunogens corresponding to amino acid sequences from human histocompatibility class II membrane glycoproteins.

    PubMed

    Chillemi, F; Cappelletti, S; Francescato, P; Chersi, A

    1990-03-01

    Six peptides with amino acid sequences of human histocompatibility Class II membrane glycoproteins were synthesized by conventional solution methods. Five peptides were prepared by stepwise procedures from the carboxyterminus. The sixth was synthesized by fragment condensation (5 + 10 coupling). Antibodies to synthetic peptides were then used to locate exposed and buried regions in the membrane glycoproteins.

  2. Determination of N-linked glycosylation in viral glycoproteins by negative ion mass spectrometry and ion mobility

    PubMed Central

    Bitto, David; Harvey, David J.; Halldorsson, Steinar; Doores, Katie J.; Pritchard, Laura K.; Huiskonen, Juha T.; Bowden, Thomas A.; Crispin, Max

    2016-01-01

    Summary Glycan analysis of virion-derived glycoproteins is challenging due to the difficulties in glycoprotein isolation and low sample abundance. Here, we describe how ion mobility mass spectrometry can be used to obtain spectra from virion samples. We also describe how negative ion fragmentation of glycans can be used to probe structural features of virion glycans. PMID:26169737

  3. A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate-protein interaction profiling and cell surface carbohydrate expression evaluation

    NASA Astrophysics Data System (ADS)

    Wang, Yangzhong; Chen, Zhuhai; Liu, Yang; Li, Jinghong

    2013-07-01

    infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes. Electronic supplementary information (ESI) available: Experimental details; characterization of probes; the influence of electrolyte pH; probe concentration and glucose concentration on the electrode ECL effect. See DOI: 10.1039/c3nr01598j

  4. Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    SciTech Connect

    Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

    1986-10-01

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX;more » gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.« less

  5. THE RELATIONSHIP OF THE INFLUENZA VIRUS INHIBITORY ACTIVITY OF GLYCOPROTEINS TO THEIR MOLECULAR SIZE AND SIALIC ACID CONTENT

    PubMed Central

    Springer, Georg F.; Schwick, H. Gerhard; Fletcher, Mary Ann

    1969-01-01

    Twenty-four different glycoproteins were investigated for their ability to inhibit hemagglutination by the A/PRS and the B/Md influenza virus strains. A relationship between activity, the molecular size, and sialic acid content was found. This relationship was readily shown for the A/PR8 virus if the properties of the glycoproteins were compared with one another on a per cent basis. A proportion of approximately 1:1:1 for activity (weight basis) to moles sialic acid content to molecular weight existed for each inhibitory glycoprotein with more than 3 per cent sialic acid, on comparison with any other active glycoprotein. A 1:3 correspondence between viral subunit and sialic acid residues of the inhibitor ovine submaxillary mucin was found experimentally and confirmed by calculation on a molecular model. The most potent inhibitors, were the antigens of the human blood-group MN system and the Tamm-Horsfall urinary glycoprotein. PMID:5261039

  6. Chemoenzymatic Site-Specific Labeling of Influenza Glycoproteins as a Tool to Observe Virus Budding in Real Time

    PubMed Central

    Ploegh, Hidde L.

    2012-01-01

    The influenza virus uses the hemagglutinin (HA) and neuraminidase (NA) glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging. PMID:22457626

  7. Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

    PubMed Central

    Saw, Wan Ting; Eisenberg, Roselyn J.; Cohen, Gary H.

    2016-01-01

    ABSTRACT Receptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH2/gL2), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD1) and gH2/gL2 can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion. IMPORTANCE Cell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing the activity of the mutants. Under low-pH conditions or when

  8. Enhanced Brain Disposition and Effects of Δ9-Tetrahydrocannabinol in P-Glycoprotein and Breast Cancer Resistance Protein Knockout Mice

    PubMed Central

    Spiro, Adena S.; Wong, Alexander; Boucher, Aurélie A.; Arnold, Jonathon C.

    2012-01-01

    The ABC transporters P-glycoprotein (P-gp, Abcb1) and breast cancer resistance protein (Bcrp, Abcg2) regulate the CNS disposition of many drugs. The main psychoactive constituent of cannabis Δ9-tetrahydrocannabinol (THC) has affinity for P-gp and Bcrp, however it is unknown whether these transporters modulate the brain accumulation of THC and its functional effects on the CNS. Here we aim to show that mice devoid of Abcb1 and Abcg2 retain higher brain THC levels and are more sensitive to cannabinoid-induced hypothermia than wild-type (WT) mice. Abcb1a/b (−/−), Abcg2 (−/−) and wild-type (WT) mice were injected with THC before brain and blood were collected and THC concentrations determined. Another cohort of mice was examined for THC-induced hypothermia by measuring rectal body temperature. Brain THC concentrations were higher in both Abcb1a/b (−/−) and Abcg2 (−/−) mice than WT mice. ABC transporter knockout mice exhibited delayed elimination of THC from the brain with the effect being more prominent in Abcg2 (−/−) mice. ABC transporter knockout mice were more sensitive to THC-induced hypothermia compared to WT mice. These results show P-gp and Bcrp prolong the brain disposition and hypothermic effects of THC and offer a novel mechanism for both genetic vulnerability to the psychoactive effects of cannabis and drug interactions between CNS therapies and cannabis. PMID:22536451

  9. Effects of borneol on the intestinal transport and absorption of two P-glycoprotein substrates in rats.

    PubMed

    He, Huijuan; Shen, Qi; Li, Jian

    2011-07-01

    As the most prevalent route of delivery, oral administration has the challenge of potentially low bioavailability in part because P-glycoprotein (P-gp) in the intestinal tract affects absorption. Therefore, absorption enhancers or P-gp inhibitors are strategies to solve this problem. The aim of the present study was to investigate the effects of borneol on transportation of colchicine and rhodamine123, two P-gp substrates, in rats. In vitro transportation was assessed with a diffusion chamber system with isolated rat intestines. Different concentrations of borneol (10, 40 and 80 μg/mL) were prepared in solutions with two P-gp substrates compared with blank solutions. The in vivo effects on colchicine were assessed by a pharmacokinetic study. Borneol enhanced the absorptive transport of two P-gp substrates, which was relevant to the concentration. A pharmacokinetic study showed that in the presence of borneol, a significant increase in C(max) and AUC(0→8) of colchicine occurred when compared to colchicine alone. The study showed that borneol affected two P-gp substrates in the intestine, possibly by inhibiting the effects of P-gp and enhancing intestinal absorption of drugs. Therefore, borneol could be developed as a P-gp inhibitor and absorptive enhancer.

  10. Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

    PubMed Central

    El Omari, Kamel; Iourin, Oleg; Harlos, Karl; Grimes, Jonathan M.; Stuart, David I.

    2013-01-01

    Summary Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed. PMID:23273918

  11. Rapid N-glycan release from glycoproteins using immobilized PNGase F microcolumns.

    PubMed

    Szigeti, Marton; Bondar, Judit; Gjerde, Douglas; Keresztessy, Zsolt; Szekrenyes, Akos; Guttman, Andras

    2016-10-01

    N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection, hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators (glycobiomarkers) for various pathological conditions in the biomedical field. Therefore, there is a growing demand for rapid and easy to automate sample preparation methods for N-glycosylation analysis. In this paper, we report on the design and implementation of immobilized recombinant glutathione-S-transferase (GST) tagged PNGase F enzyme microcolumns for rapid and efficient removal of N-linked carbohydrates from glycoproteins. Digestion speed and efficiency were compared to conventional in-solution based protocols. The use of PNGase F functionalized microcolumns resulted in efficient N-glycan removal in 10min from all major N-linked glycoprotein types of: (i) neutral (IgG), (ii) highly sialylated (fetuin), and (iii) high mannose (ribonuclease B) carbohydrate containing glycoprotein standards. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Production of homogeneous glycoprotein with multisite modifications by an engineered N-glycosyltransferase mutant.

    PubMed

    Song, Qitao; Wu, Zhigang; Fan, Yueyuan; Song, Woran; Zhang, Peiru; Wang, Li; Wang, Faxing; Xu, Yangyang; Wang, Peng G; Cheng, Jiansong

    2017-05-26

    Naturally occurring N -glycoproteins exhibit glycoform heterogeneity with respect to N -glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties of N -glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmic N -glycosyltransferase from Actinobacillus pleuropneumoniae , has strict selectivity toward the residues around the Asn of N -glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10) N -glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Plasma Krebs von den Lungen glycoprotein, lung injury, and noninvasive ventilation in Duchenne muscular dystrophy.

    PubMed

    Hamada, Satoshi; Ishikawa, Yuka; Aoyagi, Tomoyuki; Ishikawa, Yukitoshi; Minami, Ryoji; Bach, John R

    2012-10-01

    There have been few reports of ventilator-induced lung injury associated with noninvasive ventilation (NIV), but many with invasive mechanical ventilation. The purpose of this study was to detect subclinical NIV-associated lung injury by monitoring Krebs von den Lungen glycoprotein plasma levels. Forty-one Duchenne muscular dystrophy patients were divided into three categories: group 1, asymptomatic and not using ventilators; group 2, NIV use less than 24 hrs/day at full ventilatory support settings; and group 3, continuous NIV dependence. Plasma Krebs von den Lungen glycoprotein level was measured by electrochemical luminescent immunoassay using Krebs von den Lungen glycoprotein antibodies. One-way analysis of variance, followed by the Tukey-Kramer test, was used as appropriate to compare intergroup differences. Extent of ventilator dependence correlated with age (P < 0.05). Intergroup plasma Krebs von den Lungen glycoprotein levels were not significantly different. NIV used at volumes and pressures of full (invasive) ventilatory support may not induce the alveolar septal barrier injury commonly seen with invasive mechanical ventilation.

  14. Chemical composition and tissue distribution of the human CDw44 glycoprotein.

    PubMed Central

    Flanagan, B F; Dalchau, R; Allen, A K; Daar, A S; Fabre, J W

    1989-01-01

    The CDw44 glycoprotein was purified from 2.3 x 10(11) CD3+ CD4+ CD8- T-chronic lymphocytic leukaemia (CLL) cells using F10-44-2 monoclonal antibody affinity chromatography, DEAE-Sepharose anion-exchange chromatography, passage down carboxymethyl (CM)-Sepharose cation-exchange columns, wheat germ lectin affinity chromatography and gel-permeation chromatography. On elution in non-ionic detergents from the DEAE column, two distinct peaks of antigen activity were obtained. The CDw44 glycoprotein in each peak was a glycoprotein of 85,000 MW, but the amino acid composition of the peaks was noticeably different. Carbohydrate compositions showed that each peak contained approximately 30% (w/w) carbohydrate, the composition suggesting both O-linked and complex N-linked glycans. Modulation studies with the F10-44-2 antibody on normal peripheral blood mononuclear cells (PBMC) demonstrated that the CDw44 glycoprotein of T cells consisted of one fraction that was readily modulated, and the other which was resistant to modulation. Detailed tissue distribution studies for CDw44 were performed using the F10-44-2 antibody on frozen sections of human tissues. CDw44 has a restricted tissue distribution, but is found on many highly diverse cell types (e.g. T lymphocytes, smooth muscle cells, some secretory glands, skin epithelial cells). Images Figure 3 Figure 5 PMID:2666306

  15. Self-organizing maps for identification of new inhibitors of P-glycoprotein.

    PubMed

    Kaiser, Dominik; Terfloth, Lothar; Kopp, Stephan; Schulz, Jan; de Laet, Randolf; Chiba, Peter; Ecker, Gerhard F; Gasteiger, Johann

    2007-04-05

    Self-organizing maps were trained to separate high- and low-active propafenone-type inhibitors of P-glycoprotein. The trained maps were subsequently used to identify highly active compounds in a virtual screen of the SPECS compound library.

  16. Analysis of codon usage pattern of infectious laryngotracheitis virus immunogenic glycoproteins and its biological implications.

    PubMed

    Das, Moushumee; Kumar, Sachin

    2018-04-11

    Infectious laryngotracheitis virus (ILTV) is a highly contagious acute respiratory poultry pathogen. Modified live ILTV vaccines are the only control against ILT infections. Reversions and establishment of latent infections are the major concerns imparting the need to develop safer vaccines against ILTV infection. ILTV glycoprotein B and D (gB and gD) are major protective immunogens. The factors shaping synonymous codon usage bias and nucleotide composition in ILTV glycoprotein genes have not yet been reported. In the present study, we have analyzed the synonymous codon usage indices of ILTV gB and gD genes. Variation in the codon usage was seen in both the glycoproteins majorly by mutational pressure. The pattern was determined using the correspondence analysis, effective number of codon (Nc), GC3 plot and correlation analyses among different indices. The study is a comprehensive analysis of the codon usage patterns of ILTV glycoprotein genes. This will be helpful in understanding the codon usage bias of ILTV and related DNA viruses which could further explore its biology. Copyright © 2018. Published by Elsevier B.V.

  17. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

    PubMed

    Gardner, Thomas J; Tortorella, Domenico

    2016-09-01

    The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Development of Recombinant Newcastle Disease Viruses Expressing the Glycoprotein (G) of Avian Metapneumovirus as Bivalent Vaccines

    USDA-ARS?s Scientific Manuscript database

    Using reverse genetics technology, Newcastle disease virus (NDV) LaSota strain-based recombinant viruses were engineered to express the glycoprotein (G) of avian metapneumovirus (aMPV), subtype A, B or C, as bivalent vaccines. These recombinant viruses were slightly attenuated in vivo, yet maintaine...

  19. Mass Spectrometric Analysis of N-Glycoforms of Soybean Allergenic Glycoproteins Separated by SDS-PAGE.

    PubMed

    Li, Lingmei; Wang, Chengjian; Qiang, Shan; Zhao, Jixiang; Song, Shuang; Jin, Wanjun; Wang, Bo; Zhang, Ying; Huang, Linjuan; Wang, Zhongfu

    2016-10-05

    Glycosylation of many proteins has been revealed to be closely related with food allergies, and screening and structural analysis of related glycoproteins and glycoallergens are essential for studies in this field. Herein, we describe detailed N-glycoform analysis of all glycoprotein fractions of soybean protein isolate (SPI) separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to disclose structural features of the glycan moieties of more soybean glycoproteins. SPI was fractionated by SDS-PAGE, and the generated protein bands were recovered and subjected to in-gel N-glycan release and labeling using a one-pot method newly developed by our group, followed by detailed analysis by electrospray ionization mass spectrometry (ESI-MS) and online hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). As a result, we found seven bands mainly containing oligomannose-type glycans; two mainly contain core α1,3-fucosylated glycans, and six have no glycans. This study is the first report that discovers core α1,3-fucosylated N-glycans in bands 1, 2, and 6 and discloses bands 3, 4, 5, and 7 as glycoproteins and their N-glycoforms. Therefore, it can expand our knowledge about soybean protein glycosylation and provide significant structural reference for research of soybean allergens.

  20. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

    SciTech Connect

    Backovic, Marija; Longnecker, Richard; Jardetzky, Theodore S

    2009-03-16

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which formsmore » 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.« less

  1. Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015.

    PubMed

    Kim, Dae-Won; Kim, You-Jin; Park, Sung Han; Yun, Mi-Ran; Yang, Jeong-Sun; Kang, Hae Ji; Han, Young Woo; Lee, Han Saem; Kim, Heui Man; Kim, Hak; Kim, A-Reum; Heo, Deok Rim; Kim, Su Jin; Jeon, Jun Ho; Park, Deokbum; Kim, Joo Ae; Cheong, Hyang-Min; Nam, Jeong-Gu; Kim, Kisoon; Kim, Sung Soon

    2016-01-01

    An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

  2. Intracellular transport and stability of varicella-zoster virus glycoprotein K

    SciTech Connect

    Hall, Susan L.; Govero, Jennifer L.; Heineman, Thomas C.

    2007-02-20

    VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established thatmore » gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.« less

  3. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  4. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

    PubMed Central

    Gardner, Thomas J.

    2016-01-01

    SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

  5. Splice variation in the cytoplasmic domains of myelin oligodendrocyte glycoprotein affects its cellular localisation and transport.

    PubMed

    Boyle, Louise H; Traherne, James A; Plotnek, Gemma; Ward, Rosemary; Trowsdale, John

    2007-09-01

    Although myelin oligodendrocyte glycoprotein is a candidate autoantigen in multiple sclerosis, its function remains unknown. In humans, mRNA expressed by the myelin oligodendrocyte glycoprotein gene is alternatively spliced resulting in at least nine unique protein isoforms. In this study, we investigated the sub-cellular localisation and membrane trafficking of six isoforms by cloning them into mammalian expression vectors. Confocal microscopy revealed that these protein products are expressed in different cellular compartments. While two full-length isoforms (25.6 and 25.1) are expressed at the cell surface, three alternatively spliced forms (22.7, 21.0 and 20.5) have a more intracellular distribution, localising to the endoplasmic reticulum and/or endosomes. Isoform 16.3, which lacks a transmembrane domain, is secreted. A switch in the sub-cellular localisation of myelin oligodendrocyte glycoprotein may have profound effects on receptor:ligand interactions and consequently the function of the protein. The structural features of the alternative isoforms and their differential, sub-cellular expression patterns could dictate the exposure of major immunogenic determinants within the central nervous system. Our findings highlight myelin oligodendrocyte glycoprotein splicing as a factor that could be critical to the phenotypic expression of multiple sclerosis.

  6. Averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using Jsubtomo.

    PubMed

    Huiskonen, Juha T; Parsy, Marie-Laure; Li, Sai; Bitto, David; Renner, Max; Bowden, Thomas A

    2014-10-21

    Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein 'spikes' is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

  7. Refining the Mechanisms of Heniparvirus-Mediated Membrane Fusion Through Mutagenesis of Hendra virus Envelope Glycoproteins

    DTIC Science & Technology

    2007-09-06

    receptors are Measles virus (MeV), Rinderpest virus, and Canine Distemper virus (CDV) (reviewed in (91, 92)). There is currently no solved structure...parainfluenza virus-1 (hPIV-1) and hPIV-3, and the H glycoprotein of MeV and Canine Distemper Virus. An amino acid sequence alignment of the stalk region

  8. The combination of simple MALDI matrices for the improvement of intact glycoproteins and glycans analysis

    NASA Astrophysics Data System (ADS)

    Lastovicková, Markéta; Chmelik, Josef; Bobalova, Janette

    2009-03-01

    The choice of matrix has fundamental importance in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of glycoproteins. Therefore, a set of selected matrices has been tested. The attention was focused on new binary matrices, that were a combination of matrices commonly used for carbohydrates or proteins analysis such as 2,5-dihydroxybenzoic acid (2,5-DHB), 2,5-dihydroxyacetophenone (DHAP), 2,4,6-trihydroxyacetophenone (THAP), [alpha]-cyano-4-hydroxycinnamic acid (CHCA), and sinapinic acid (SA). The binary matrices 2,5-DHB/CHCA and 2,5-DHB/SA have been proved the mass spectra with the best quality and showed an universal applicability. Application of these matrices enabled to quickly screen the microheterogeneity of glycan moieties for either intact glycoproteins, or a mixture of deglycosylated glycoproteins together with free, underivatized glycans. Moreover, these binary matrices were more tolerant to the presence of salts in studied samples. Our results suggest that a combination of two matrix compounds could be useful for an improved determination of the molecular mass values of analytes coming from complex biological samples, especially for the structural characterization of glycoproteins of pharmaceutical interest.

  9. Location of the Spike Glycoproteins in the Semliki Forest Virus Membrane

    PubMed Central

    Garoff, Henrik; Simons, Kai

    1974-01-01

    Labeling experiments with formyl-[35S]-methionyl sulfone methylphosphate and crosslinking studies with dimethylsuberimidate suggest that the spike glycoproteins of Semliki Forest virus extend through the viral membrane into close contact with the nucleocapsid. Based on this finding, we present a mechanism for the formation of virus-specific patches in the host cell plasma membrane during virus assembly. Images PMID:4530279

  10. Alpha 1-acid glycoprotein has immunomodulatory effects in neonatal swine adipose tissue

    USDA-ARS?s Scientific Manuscript database

    Alpha 1-acid glycoprotein (AGP) is the most abundant protein in serum of neonatal swine. This protein functions as an immunomodulator in the pig. Recent work has demonstrated that adipose tissue can express AGP mRNA, as well as numerous cytokine mRNA. The present study was designed to determine i...

  11. Lectin-binding proteins in central-nervous-system myelin. Binding of glycoproteins in purified myelin to immobilized lectins

    PubMed Central

    Quarles, Richard H.; McIntyre, Laurence J.; Pasnak, Carol F.

    1979-01-01

    The capacities of immature and mature rat brain myelin, bovine myelin and human myelin to be agglutinated by soya-bean agglutinin, Ricinus communis agglutinin, wheatgerm agglutinin, and Lotus tetragonolobus agglutinin were examined. The first two lectins, which are specific for galactose and N-acetylgalactosamine, strongly agglutinated immature and mature rat myelin, weakly agglutinated bovine myelin, but did not affect human myelin. The other myelin and lectin combinations resulted in very weak or no agglutination. [3H]Fucose-labelled glycoproteins of purified adult rat brain myelin were solubilized with sodium dodecyl sulphate and allowed to bind to concanavalin A–Sepharose and each of the other lectins mentioned above, which had been immobilized on agarose. About 60% of the radioactive fucose was in glycoproteins that bound to concanavalin A–Sepharose and these glycoproteins could be eluted with solutions containing methyl α-d-mannoside and sodium dodecyl sulphate. Periodate/Schiff staining or radioactive counting of analytical gels showed that most of the major myelin-associated glycoprotein (apparent mol.wt. approx. 100000) bound to the concanavalin A, whereas the glycoproteins that did not bind were mostly of lower molecular weight. Preparative polyacrylamide-gel electrophoresis of the glycoprotein fraction that was eluted with methyl α-d-mannoside yielded a relatively pure preparation of the myelin-associated glycoprotein. Similar results were obtained with each of the other lectins, i.e. the myelin-associated glycoprotein was in the fraction that bound to the immobilized lectin. Double-labelling experiments utilizing [3H]fucose-labelled glycoproteins from adult myelin and [14C]fucose-labelled glycoproteins from 14-day-old rat brain myelin did not reveal any difference in the binding of the mature and immature glycoproteins to any of the immobilized lectins. The results in this and the preceding paper [McIntyre, Quarles & Brady (1979) Biochem. J. 183

  12. Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

    PubMed

    Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Rajendran, Madhusudan; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R; García-Sastre, Adolfo; Basler, Christopher F; Munoz-Fontela, Cesar; Krammer, Florian

    2017-08-15

    Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 -/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro , suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two

  13. HIV-1 ENVELOPE GLYCOPROTEIN-MEDIATED FUSION AND PATHOGENESIS: IMPLICATIONS FOR THERAPY AND VACCINE DEVELOPMENT

    PubMed Central

    Jacobs, Amy; Garg, Himanshu; Viard, Mathias; Raviv, Yossef; Puri, Anu; Blumenthal, Robert

    2008-01-01

    Our overall goal is to understand how viral envelope proteins mediate membrane fusion and pathogenesis. Membrane fusion is a crucial step in the delivery of the viral genome into the cell resulting in infection. On the other hand, fusion activity of viral envelope glycoproteins expressed in infected cells may cause the demise of uninfected bystander cells by apoptosis. Our general approach is to kinetically resolve steps in the pathway of viral envelope glycoprotein-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. Since HIV fusion involves a complex cascade of interactions of the envelope glycoprotein with two receptors, membrane organization plays an important role and interfering with it may modulate entry. To study this phenomenon, we have either examined cell lines with differential expression of sphingolipids (such as GM3), or altered membrane organization by modifying levels of cholesterol, ceramides, or glycosphingolipids. We show that the localized plasma membrane lipid microenvironment (and not the specific membrane lipids) in the vicinity of CD4 controls receptor mobility and HIV-1 fusion. The complex cascade of conformational changes that must occur to allow virus entry is also a very important target for therapy and vaccine development. We have recently designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to promote permanent attachment. Using a temperature-arrested state in vitro assay we show evidence for the trapping of a pre-six helix bundle fusion intermediate by a covalent reaction with the inhibitory reactive peptide. Also, using photo-reactive hydrophobic probes we have found ways to inactivate viral envelope glycoproteins while leaving their overall structures intact. Finally, in order to study the envelope glycoprotein effects on pathogenesis, we

  14. Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus.

    PubMed

    Coleman, Stewart; Hornig, Julia; Maddux, Sarah; Choi, K Yeon; McGregor, Alistair

    2015-01-01

    Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene

  15. Identification of New Functional Regions in Hepatitis C Virus Envelope Glycoprotein E2▿

    PubMed Central

    Albecka, Anna; Montserret, Roland; Krey, Thomas; Tarr, Alexander W.; Diesis, Eric; Ball, Jonathan K.; Descamps, Véronique; Duverlie, Gilles; Rey, Felix; Penin, François; Dubuisson, Jean

    2011-01-01

    Little is known about the structure of the envelope glycoproteins of hepatitis C virus (HCV). To identify new regions essential for the function of these glycoproteins, we generated HCV pseudoparticles (HCVpp) containing HCV envelope glycoproteins, E1 and E2, from different genotypes in order to detect intergenotypic incompatibilities between these two proteins. Several genotype combinations were nonfunctional for HCV entry. Of interest, a combination of E1 from genotype 2a and E2 from genotype 1a was nonfunctional in the HCVpp system. We therefore used this nonfunctional complex and the recently described structural model of E2 to identify new functional regions in E2 by exchanging protein regions between these two genotypes. The functionality of these chimeric envelope proteins in the HCVpp system and/or the cell-cultured infectious virus (HCVcc) was analyzed. We showed that the intergenotypic variable region (IgVR), hypervariable region 2 (HVR2), and another segment in domain II play a role in E1E2 assembly. We also demonstrated intradomain interactions within domain I. Importantly, we also identified a segment (amino acids [aa] 705 to 715 [segment 705-715]) in the stem region of E2, which is essential for HCVcc entry. Circular dichroism and nuclear magnetic resonance structural analyses of the synthetic peptide E2-SC containing this segment revealed the presence of a central amphipathic helix, which likely folds upon membrane binding. Due to its location in the stem region, segment 705-715 is likely involved in the reorganization of the glycoprotein complexes taking place during the fusion process. In conclusion, our study highlights new functional and structural regions in HCV envelope glycoprotein E2. PMID:21147916

  16. Identification of new functional regions in hepatitis C virus envelope glycoprotein E2.

    PubMed

    Albecka, Anna; Montserret, Roland; Krey, Thomas; Tarr, Alexander W; Diesis, Eric; Ball, Jonathan K; Descamps, Véronique; Duverlie, Gilles; Rey, Felix; Penin, François; Dubuisson, Jean

    2011-02-01

    Little is known about the structure of the envelope glycoproteins of hepatitis C virus (HCV). To identify new regions essential for the function of these glycoproteins, we generated HCV pseudoparticles (HCVpp) containing HCV envelope glycoproteins, E1 and E2, from different genotypes in order to detect intergenotypic incompatibilities between these two proteins. Several genotype combinations were nonfunctional for HCV entry. Of interest, a combination of E1 from genotype 2a and E2 from genotype 1a was nonfunctional in the HCVpp system. We therefore used this nonfunctional complex and the recently described structural model of E2 to identify new functional regions in E2 by exchanging protein regions between these two genotypes. The functionality of these chimeric envelope proteins in the HCVpp system and/or the cell-cultured infectious virus (HCVcc) was analyzed. We showed that the intergenotypic variable region (IgVR), hypervariable region 2 (HVR2), and another segment in domain II play a role in E1E2 assembly. We also demonstrated intradomain interactions within domain I. Importantly, we also identified a segment (amino acids [aa] 705 to 715 [segment 705-715]) in the stem region of E2, which is essential for HCVcc entry. Circular dichroism and nuclear magnetic resonance structural analyses of the synthetic peptide E2-SC containing this segment revealed the presence of a central amphipathic helix, which likely folds upon membrane binding. Due to its location in the stem region, segment 705-715 is likely involved in the reorganization of the glycoprotein complexes taking place during the fusion process. In conclusion, our study highlights new functional and structural regions in HCV envelope glycoprotein E2.

  17. Role of P-glycoprotein in the disposition of macrocyclic lactones: A comparison between ivermectin, eprinomectin, and moxidectin in mice.

    PubMed

    Kiki-Mvouaka, Solange; Ménez, Cécile; Borin, Christiane; Lyazrhi, Faouri; Foucaud-Vignault, Magali; Dupuy, Jacques; Collet, Xavier; Alvinerie, Michel; Lespine, Anne

    2010-04-01

    Macrocyclic lactones (MLs) are lipophilic anthelmintics and substrates for P-glycoprotein (P-gp), an ATP-binding cassette transporter involved in drug efflux out of both host and parasites. To evaluate the contribution of P-gp to the in vivo kinetic disposition of MLs, the plasma kinetics, brain concentration, and intestinal excretion of three structurally different MLs (ivermectin, eprinomectin, and moxidectin) were compared in wild-type and P-gp-deficient [mdr1ab(-/-)] mice. Each drug (0.2 mg/kg) was administered orally, intravenously, or subcutaneously to the mice. Plasma, brain, and intestinal tissue concentrations were measured by high-performance liquid chromatography. The intestinal excretion rate after intravenous administration was determined at different levels of the small intestine by using an in situ intestinal perfusion model. P-gp deficiency led to a significant increase in the area under the plasma concentration-time curve (AUC) of ivermectin (1.5-fold) and eprinomectin (3.3-fold), whereas the moxidectin AUC was unchanged. Ivermectin and to a greater extent eprinomectin were both excreted by the intestine via a P-gp-dependent pathway, whereas moxidectin excretion was weaker and mostly P-gp-independent. The three drugs accumulated in the brains of the mdr1ab(-/-) mice, but eprinomectin concentrations were significantly lower. We concluded that eprinomectin disposition in mice is controlled mainly by P-gp efflux, more so than that of ivermectin, whereas moxidectin disposition appears to be mostly P-gp-independent. Given that eprinomectin and ivermectin have higher affinity for P-gp than moxidectin, these findings demonstrated that the relative affinity of MLs for P-gp could be predictive of the in vivo kinetic behavior of these drugs.

  18. Unravelling the complex drug–drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein

    PubMed Central

    Ledwitch, Kaitlyn V.; Barnes, Robert W.; Roberts, Arthur G.

    2016-01-01

    Drug–drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug–Pgp interactions, verapamil was found to have little effect on digoxin–Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin–Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil–digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes. PMID:26823559

  19. Unravelling the complex drug-drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein.

    PubMed

    Ledwitch, Kaitlyn V; Barnes, Robert W; Roberts, Arthur G

    2016-01-28

    Drug-drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug-Pgp interactions, verapamil was found to have little effect on digoxin-Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin-Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil-digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes. © 2016 Authors.

  20. Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα

    PubMed Central

    Gitz, Eelo; Koopman, Charlotte D.; Giannas, Alèkos; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan-Willem N.; Gerritsen, Hans C.; Urbanus, Rolf T.

    2013-01-01

    Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s−1 induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s−1. Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation. PMID:23753027

  1. Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells.

    PubMed

    Chang, Haigang; Song, Shanshan; Chen, Zhongcan; Wang, Yaxiao; Yang, Lujun; Du, Mouxuan; Ke, Yiquan; Xu, Ruxiang; Jin, Baozhe; Jiang, Xiaodan

    2014-03-01

    Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

  2. What Do Chaotrope-Based Avidity Assays for Antibodies to HIV-1 Envelope Glycoproteins Measure?

    PubMed

    Alexander, Marina R; Ringe, Rajesh; Sanders, Rogier W; Voss, James E; Moore, John P; Klasse, Per Johan

    2015-06-01

    When HIV-1 vaccine candidates that include soluble envelope glycoproteins (Env) are tested in humans and other species, the resulting antibody responses to Env are sifted for correlates of protection or risk. One frequently used assay measures the reduction in antibody binding to Env antigens by an added chaotrope (such as thiocyanate). Based on that assay, an avidity index was devised for assessing the affinity maturation of antibodies of unknown concentration in polyclonal sera. Since a high avidity index was linked to protection in animal models of HIV-1 infection, it has become a criterion for evaluating antibody responses to vaccine candidates. But what does the assay measure and what does an avidity index mean? Here, we have used a panel of monoclonal antibodies to well-defined epitopes on Env (gp120, gp41, and SOSIP.664 trimers) to explore how the chaotrope acts. We conclude that the chaotrope sensitivity of antibody binding to Env depends on several properties of the epitopes (continuity versus tertiary- and quaternary-structural dependence) and that the avidity index has no simple relationship to antibody affinity for functional Env spikes on virions. We show that the binding of broadly neutralizing antibodies against quaternary-structural epitopes is particularly sensitive to chaotrope treatment, whereas antibody binding to epitopes in variable loops and to nonneutralization epitopes in gp41 is generally resistant. As a result of such biases, the avidity index may at best be a mere surrogate for undefined antibody or other immune responses that correlate weakly with protection. An effective HIV-1 vaccine is an important goal. Such a vaccine will probably need to induce antibodies that neutralize typically transmitted variants of HIV-1, preventing them from infecting target cells. Vaccine candidates have so far failed to induce such antibody responses, although some do protect weakly against infection in animals and, possibly, humans. In the search for

  3. α 1-acid glycoprotein inhibits lipogenesis in neonatal swine adipose tissue.

    PubMed

    Ramsay, T G; Blomberg, L; Caperna, T J

    2016-05-01

    Serum α1-acid glycoprotein (AGP) is elevated during late gestation and at birth in the pig and rapidly declines postnatally. In contrast, the pig is born with minimal lipid stores in the adipose tissue, but rapidly accumulates lipid during the first week. The present study examined if AGP can affect adipose tissue metabolism in the neonatal pig. Isolated cell cultures or tissue explants were prepared from dorsal subcutaneous adipose tissue of preweaning piglets. Porcine AGP was used at concentrations of 0, 100, 1000 and 5000 ng/ml medium in 24 h incubations. AGP reduced the messenger RNA (mRNA) abundance of the lipogenic enzymes, malic enzyme (ME), fatty acid synthase and acetyl coA carboxylase by at least 40% (P<0.001). The activity of ME and citrate lyase were also reduced by AGP (P<0.05). Glucose oxidation was reduced by treatment with 5000 ng AGP/ml medium (P<0.05). The 14C-glucose incorporation into fatty acids was reduced by ~25% by AGP treatment for 24 h with 1000 ng AGP/ml medium (P<0.05). The decrease in glucose metabolism by AGP appears to function through an inhibition in insulin-mediated glucose oxidation and incorporation into fatty acids. This was supported by the analysis of the mRNA abundance for sterol regulatory element-binding protein (SREBP), carbohydrate regulatory element-binding protein (ChREBP) and insulin receptor substrate 1 (IRS1), which all demonstrated reductions of at least 23% in response to AGP treatment (P<0.05). These data demonstrate an overall suppression of lipogenesis due to AGP inhibition of lipogenic gene expression in vitro, which the metabolic data and SREBP, ChREBP and IRS1 gene expression analysis suggest is through an inhibition in insulin-mediated events. Second, these data suggest that AGP may contribute to limiting lipogenesis within adipose tissue during the perinatal period, as AGP levels are highest for any serum protein at birth.

  4. P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with an ALK rearrangement.

    PubMed

    Kim, Hee Joung; Lee, Kye Young; Kim, Young Whan; Choi, Yun Jung; Lee, Jung-Eun; Choi, Chang Min; Baek, In-Jeoung; Rho, Jin Kyung; Lee, Jae Cheol

    2015-07-29

    Because anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement. Resistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123. The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp. P-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement.

  5. Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus

    PubMed Central

    Kropff, Barbara; Reuter, Nina; Sticht, Heinrich; Winkler, Thomas H.; Britt, William J.

    2017-01-01

    Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in

  6. Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus.

    PubMed

    Bootz, Anna; Karbach, Astrid; Spindler, Johannes; Kropff, Barbara; Reuter, Nina; Sticht, Heinrich; Winkler, Thomas H; Britt, William J; Mach, Michael

    2017-08-01

    Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in

  7. Activation and Inactivation of Primary Human Immunodeficiency Virus Envelope Glycoprotein Trimers by CD4-Mimetic Compounds

    PubMed Central

    Madani, Navid; Princiotto, Amy M.; Zhao, Connie; Jahanbakhshsefidi, Fatemeh; Mertens, Max; Herschhorn, Alon; Melillo, Bruno; Smith, Amos B.

    2016-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the viral envelope glycoproteins (Env), a trimer of three gp120 exterior glycoproteins, and three gp41 transmembrane glycoproteins. The metastable Env is triggered to undergo entry-related conformational changes when gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell. Small-molecule CD4-mimetic compounds (CD4mc) bind gp120 and act as competitive inhibitors of gp120-CD4 engagement. Some CD4mc have been shown to trigger Env prematurely, initially activating Env function, followed by rapid and irreversible inactivation. Here, we study CD4mc with a wide range of anti-HIV-1 potencies and demonstrate that all tested CD4mc are capable of activating as well as inactivating Env function. Biphasic dose-response curves indicated that the occupancy of the protomers in the Env trimer governs viral activation versus inactivation. One CD4mc bound per Env trimer activated HIV-1 infection. Envs with two CD4mc bound were activated for infection of CD4-negative, CCR5-positive cells, but the infection of CD4-positive, CCR5-positive cells was inhibited. Virus was inactivated when all three Env protomers were occupied by the CD4mc, and gp120 shedding from the Env trimer was increased in the presence of some CD4mc. Env reactivity and the on rates of CD4mc binding to the Env trimer were found to be important determinants of the potency of activation and entry inhibition. Cross-sensitization of Env protomers that do not bind the CD4mc to neutralization by an anti-V3 antibody was not evident. These insights into the mechanism of antiviral activity of CD4mc should assist efforts to optimize their potency and utility. IMPORTANCE The trimeric envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) mediate virus entry into host cells. Binding to the host cell receptors, CD4 and CCR5, triggers changes in the conformation of the HIV-1 envelope glycoprotein trimer important

  8. P-glycoprotein epitope mapping. II. The murine monoclonal antibody MM6.15 to human multidrug-resistant cells binds with three distinct loops in the MDR1-P-glycoprotein extracellular domain.

    PubMed

    Cianfriglia, M; Romagnoli, G; Tombesi, M; Poloni, F; Falasca, G; Di Modugno, F; Castagna, M; Chersi, A

    1995-03-29

    A new murine monoclonal antibody (MAb), MM6.15, to human MDR1 P-glycoprotein was found to be reactive in ELISA with synthetic peptides selected from the predicted sequences of the first, fourth and sixth extracellular loop of MDR1-P-glycoprotein. In order to precisely define the MM6.15-binding site, a peptide library of overlapping 5- to 9-mer residues covering the entire sixth extracellular loop of both human and rodent class-1 P-glycoproteins was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MAb MM6.15 reacts only with human synthetic peptides and that the critical component of the MAb recognition is made up of the amino-acid sequence LVAHKL (residues 963-968 of the MDR1-P-glycoprotein) with histidine (H), lysine (K) and possibly leucine (L), key residues of this immunogenic domain.

  9. Glycoproteins and Gal-GalNAc cause Cryptosporidium to switch from an invasive sporozoite to a replicative trophozoite.

    PubMed

    Edwinson, Adam; Widmer, Giovanni; McEvoy, John

    2016-01-01

    The apicomplexan parasite Cryptosporidium causes cryptosporidiosis, a diarrheal disease that can become chronic and life threatening in immunocompromised and malnourished people. There is no effective drug treatment for those most at risk of severe cryptosporidiosis. The disease pathology is due to a repeated cycle of host cell invasion and parasite replication that amplifies parasite numbers and destroys the intestinal epithelium. This study aimed to better understand the Cryptosporidium replication cycle by identifying molecules that trigger the switch from invasive sporozoite to replicative trophozoite. Our approach was to treat sporozoites of Cryptosporidium parvum and Cryptosporidium hominis, the species causing most human cryptosporidiosis, with various media under axenic conditions and examine the parasites for rounding and nuclear division as markers of trophozoite development and replication, respectively. FBS had a concentration-dependent effect on trophozoite development in both species. Trophozoite development in C. parvum, but not C. hominis, was enhanced when RPMI supplemented with 10% FBS (RPMI-FBS) was conditioned by HCT-8 cells for 3h. The effect of non-conditioned and HCT-8 conditioned RPMI-FBS on trophozoite development was abrogated by proteinase K and sodium metaperiodate pretreatment, indicating a glycoprotein trigger. Cryptosporidium parvum and C. hominis trophozoite development also was triggered by Gal-GalNAc in a concentration-dependent manner. Cryptosporidium parvum replication was greatest following treatments with Gal-GalNAc, followed by conditioned RPMI-FBS and non-conditioned RPMI-FBS (P<0.05). Cryptosporidium hominis replication was significantly less than that in C. parvum for all treatments (P<0.05), and was greatest at the highest tested concentration of Gal-GalNAc (1mM). Copyright © 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  10. Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the HIV-1 Envelope Glycoproteins

    PubMed Central

    Ma, Xiaochu; Gu, Christopher; Ventura, John D.; Castillo-Menendez, Luis; Melillo, Bruno; Terry, Daniel S.; Smith, Amos B.; Blanchard, Scott C.; Munro, James B.; Mothes, Walther; Finzi, Andrés

    2016-01-01

    ABSTRACT Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. PMID:27795397

  11. Structural stability of hepatitis C virus envelope glycoprotein E1: effect of pH and dissociative detergents.

    PubMed

    He, Feng; Joshi, Sangeeta B; Bosman, Fons; Verhaeghe, Marijke; Middaugh, C Russell

    2009-09-01

    The hepatitis C virus (HCV) envelope glycoprotein E1 has been widely employed as a potential vaccine antigen in clinical research. A truncated form (amino acids 192-326) of the E1 protein (E1y) was expressed in the yeast Hansenula polymorpha and purified from the cell lysate. E1y forms protein particles in the absence of detergent and remains monomeric when detergent concentration is high. In this work, a variety of spectroscopic and hydrodynamic techniques including circular dichroism, intrinsic and ANS fluorescence as well as static and dynamic light scattering are employed to evaluate E1y structural stability. The effect of two dissociative detergents, Empigen BB and Zwittergent 3-12 on E1y stability, is investigated and the results are summarized using the empirical phase diagram (EPD)-based approach. The EPDs reveal that when temperature is increased, E1y particles are more thermally stable than monomers at both pH 5 and 7. A more detailed biophysical characterization of the E1y particles is also performed including pH and temperature as variables. The EPD indicates that E1y particles are most stable at pH 7 and 8 under the given experimental conditions. The results from this study provide detailed information that will help guide the future development of E1-based HCV vaccines.

  12. Mitochondrial P-glycoprotein ATPase contributes to insecticide resistance in the cotton bollworm, Helicoverpa armigera (Noctuidae: Lepidoptera).

    PubMed

    Akbar, S Md; Aurade, Ravindra M; Sharma, H C; Sreeramulu, K

    2014-09-01

    Cotton bollworm, Helicoverpa armigera, is one of the most damaging polyphagous pests worldwide, which has developed high levels of resistance to commonly applied insecticides. Mitochondrial P-glycoprotein (Pgp) was detected in the insecticide-resistant strain of H. armigera using C219 antibodies, and its possible role was demonstrated in the efflux of xenobiotic compounds using spectrofluorometer. The TMR accumulated in mitochondria in the absence of ATP, and effluxed out in presence of ATP; the process of efflux was inhibited in the presence of ortho-vandate, an inhibitor of Pgp, in insecticide-resistant larvae of H. armigera. The mitochondria isolated from insecticide-resistant larvae were resistant to insecticide-induced inhibition of oxygen consumption and cytochrome c release. Membrane potential decreased in a dose-dependent manner in the presence of higher concentration of insecticides (>50 µM) in mitochondria of insecticide-resistant larvae. In conclusion, mitochondrial Pgp ATPase detected in the insecticide-resistant larvae influenced the efflux of xenobiotic compounds. Pgp might be involved in protecting the mitochondrial DNA and the components of the electron transport chain from damage due to insecticides, and contributing to the resistance to the deleterious effects of insecticides on the growth of insecticide-resistant H. armigera larvae.

  13. Binding of thalidomide to alpha1-acid glycoprotein may be involved in its inhibition of tumor necrosis factor alpha production.

    PubMed Central

    Turk, B E; Jiang, H; Liu, J O

    1996-01-01

    In addition to its well known sedative and teratogenic effects, thalidomide also possesses potent immunomodulatory and antiinflammatory activities, being most effective against leprosy and chronic graft-versus-host disease. The immunomodulatory activity of thalidomide has been ascribed to the selective inhibition of tumor necrosis factor alpha from monocytes. The molecular mechanism for the immunomodulatory effect of thalidomide remains unknown. To elucidate this mechanism, we synthesized an active photoaffinity label of thalidomide as a probe to identify the molecular target of the drug. Using the probe, we specifically labeled a pair of proteins of 43-45 kDa with high acidity from bovine thymus extract. Purification of these proteins and partial peptide sequence determination revealed them to be alpha1-acid glycoprotein (AGP). We show that the binding of thalidomide photoaffinity label to authentic human AGP is competed with both thalidomide and the nonradioactive photoaffinity label at concentrations comparable to those required for inhibition of production of tumor necrosis factor alpha from human monocytes, suggesting that AGP may be involved in the immunomodulatory activity of thalidomide. Images Fig. 3 Fig. 4 Fig. 5 Fig. 7 PMID:8755512

  14. The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alpha-syntrophin in glia.

    PubMed

    Connors, Nathan C; Adams, Marvin E; Froehner, Stanley C; Kofuji, Paulo

    2004-07-02

    One of the major physiological roles of potassium channels in glial cells is to promote "potassium spatial buffering" in the central nervous system, a process necessary to maintain an optimal potassium concentration in the extracellular environment. This process requires the precise distribution of potassium channels accumulated at high density in discrete subdomains of glial cell membranes. To obtain a better understanding of how glial cells selectively target potassium channels to discrete membrane subdomains, we addressed the question of whether the glial inwardly rectifying potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex (DGC). Immunoprecipitation experiments revealed that Kir4.1 is associated with the DGC in mouse brain and cultured cortical astrocytes. In vitro immunoprecipitation and pull-down assays demonstrated that Kir4.1 can bind directly to alpha-syntrophin, requiring the presence of the last three amino acids of the channel (SNV), a consensus PDZ domain-binding motif. Furthermore, Kir4.1 failed to associate with the DGC in brains from alpha-syntrophin knockout mice. These results suggest that Kir4.1 is localized in glial cells by its association with the DGC through a PDZ domain-mediated interaction with alpha-syntrophin and suggest an important role for the DGC in central nervous system physiology.

  15. P-glycoprotein Inhibition Increases the Brain Distribution and Antidepressant-Like Activity of Escitalopram in Rodents

    PubMed Central

    O'Brien, Fionn E; O'Connor, Richard M; Clarke, Gerard; Dinan, Timothy G; Griffin, Brendan T; Cryan, John F

    2013-01-01

    Despite the clinical prevalence of the antidepressant escitalopram, over 30% of escitalopram-treated patients fail to respond to treatment. Recent gene association studies have highlighted a potential link between the drug efflux transporter P-glycoprotein (P-gp) and response to escitalopram. The present studies investigated pharmacokinetic and pharmacodynamic interactions between P-gp and escitalopram. In vitro bidirectional transport studies revealed that escitalopram is a transported substrate of human P-gp. Microdialysis-based pharmacokinetic studies demonstrated that administration of the P-gp inhibitor cyclosporin A resulted in increased brain levels of escitalopram without altering plasma escitalopram levels in the rat, thereby showing that P-gp restricts escitalopram transport across the blood–brain barrier (BBB) in vivo. The tail suspension test (TST) was carried out to elucidate the pharmacodynamic impact of P-gp inhibition on escitalopram effect in a mouse model of antidepressant activity. Pre-treatment with the P-gp inhibitor verapamil enhanced the response to escitalopram in the TST. Taken together, these data indicate that P-gp may restrict the BBB transport of escitalopram in humans, potentially resulting in subtherapeutic brain concentrations in certain patients. Moreover, by verifying that increasing escitalopram delivery to the brain by P-gp inhibition results in enhanced antidepressant-like activity, we suggest that adjunctive treatment with a P-gp inhibitor may represent a beneficial approach to augment escitalopram therapy in depression. PMID:23670590

  16. Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

    PubMed

    McInerney, Mitchell P; Volitakis, Irene; Bush, Ashley I; Banks, William A; Short, Jennifer L; Nicolazzo, Joseph A

    2018-03-05

    Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

  17. Sucrose esters increase drug penetration, but do not inhibit p-glycoprotein in caco-2 intestinal epithelial cells.

    PubMed

    Kiss, Lóránd; Hellinger, Éva; Pilbat, Ana-Maria; Kittel, Ágnes; Török, Zsolt; Füredi, András; Szakács, Gergely; Veszelka, Szilvia; Sipos, Péter; Ózsvári, Béla; Puskás, László G; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A

    2014-10-01

    Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and β-catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P-gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  18. Cytochrome P450 and P-Glycoprotein-Mediated Interactions Involving African Herbs Indicated for Common Noncommunicable Diseases

    PubMed Central

    Kikete, Siambi; Liang, Rongjia; Wang, Lili

    2017-01-01

    Herbal remedies are regularly used to complement conventional therapies in the treatment of various illnesses in Africa. This may be because they are relatively cheap and easily accessible and are believed by many to be safe, cause fewer side effects, and are less likely to cause dependency. On the contrary, many herbs have been shown to alter the pharmacokinetics of coadministered allopathic medicines and can either synergize or antagonize therapeutic effects as well as altering the toxicity profiles of these drugs. Current disease burden data point towards epidemiological transitions characterised by increasing urbanization and changing lifestyles, risk factors for chronic diseases like hypertension, diabetes, and cancer which often present as multimorbidities. As a result, we highlight African herb-drug interactions (HDIs) modulated via cytochrome P450 enzyme family (CYP) and P-glycoprotein (P-gp) and the consequences thereof in relation to antihypertensive, antidiabetic, and anticancer drugs. CYPs are enzymes which account for to up to 70% of drug metabolism while P-gp is an efflux pump that extrudes drug substrates out of cells. Consequently, regulation of the relative activity of both CYP and P-gp by African herbs influences the effective drug concentration at the site of action and modifies therapeutic outcomes. PMID:28250793

  19. Opioid Analgesics and P-glycoprotein Efflux Transporters: A Potential Systems-Level Contribution to Analgesic Tolerance

    PubMed Central

    Mercer, Susan L.; Coop, Andrew

    2012-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationship development to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation. PMID:21050174

  20. Opioid analgesics and P-glycoprotein efflux transporters: a potential systems-level contribution to analgesic tolerance.

    PubMed

    Mercer, Susan L; Coop, Andrew

    2011-01-01

    Chronic clinical pain remains poorly treated. Despite attempts to develop novel analgesic agents, opioids remain the standard analgesics of choice in the clinical management of chronic and severe pain. However, mu opioid analgesics have undesired side effects including, but not limited to, respiratory depression, physical dependence and tolerance. A growing body of evidence suggests that P-glycoprotein (P-gp), an efflux transporter, may contribute a systems-level approach to the development of opioid tolerance. Herein, we describe current in vitro and in vivo methodology available to analyze interactions between opioids and P-gp and critically analyze P-gp data associated with six commonly used mu opioids to include morphine, methadone, loperamide, meperidine, oxycodone, and fentanyl. Recent studies focused on the development of opioids lacking P-gp substrate activity are explored, concentrating on structure-activity relationships to develop an optimal opioid analgesic lacking this systems-level contribution to tolerance development. Continued work in this area will potentially allow for delineation of the mechanism responsible for opioid-related P-gp up-regulation and provide further support for evidence based medicine supporting clinical opioid rotation.

  1. P-Glycoprotein inhibitory activity of lipophilic constituents of Echinacea pallida roots in a human proximal tubular cell line.

    PubMed

    Romiti, Nadia; Pellati, Federica; Nieri, Paola; Benvenuti, Stefania; Adinolfi, Barbara; Chieli, Elisabetta

    2008-02-01

    The N-hexane root extracts from Echinacea pallida, Echinacea angustifolia and Echinacea purpurea were evaluated for inhibition of the multidrug transporter P-glycoprotein (Pgp) activity, the product of the ABCB1 gene, involved in cancer multidrug resistance (MDR) and in herb-drug or drug-drug interactions. The biological assay was performed using the human proximal tubule HK-2 cell line that constitutively expresses ABCB1. The N-hexane extracts of all three species reduced the efflux of the Pgp probe calcein-AM from HK-2 cells two-fold in a concentration-dependent manner, and E. pallida was found to be the most active species. For the first time, two polyacetylenes and three polyenes, isolated from the N-hexane extract of E. pallida roots by a bioassay-guided fractionation, were found to be able to reduce Pgp activity. Pentadeca-(8 Z,13 Z)-dien-11-yn-2-one was the most efficient compound, being able to decrease the calcein-AM efflux about three-fold with respect to the control at 30 microg/mL.

  2. [Expression and purification of a secreted form of fusion glycoprotein of human respiratory syncytial virus encoded by recombinant baculovirus].

    PubMed

    Fu, Yuan-hui; Wei, Wei; He, Jin-sheng; Zheng, Xian-xian; Wang, Xiao-bo; Tang, Qian; Zhang, Mei; Qu, Jian-guo; Hong, Tao

    2009-10-01

    To study the expression and purification of a secreted form of fusion glycoprotein (sF) of human respiratory syncytial virus (RSV) encoded by recombinant baculovirus. According the ORF of F protein, a pair of specific primers was designed and PCR technique was exploited to amplify the gene of sF in which the gene sequence of the transmembrane and cytoplasmic tail domains were replaced by a C-terminal six-histidine tag. Then, a recombinant baculovirus encoding sF-His was constructed, and transfected into sf9 insect cells by Lipofectamine cellfectine reagent. Finally, the expressed sF was purified by Ni2+ -affinity chromatograph. The gene encoding sF-His was obtained. The resulting construct of recombinant baculovirus is capable of expressing sF protein. The concentration of Ni2+ -affinity chromatograph purified sF is 1.084 mg/ml with the purity of no less than 90%. Baculovirus expression system is a good method for large scale of preparation of sF. The purified F paves the way for the development of potential RSV vaccine and diagnostic kit, etc.

  3. P-glycoprotein (P-gp)-mediated efflux limits intestinal absorption of the Hsp90 inhibitor SNX-2112 in rats.

    PubMed

    Liu, Hongming; Sun, Hua; Wu, Zhufeng; Zhang, Xingwang; Wu, Baojian

    2014-08-01

    1. The promising anticancer agent SNX-2112 (a novel Hsp90 inhibitor) is poorly bioavailable after oral administration. Here, we aim to determine the role of P-glycoprotein (P-gp) in the intestinal absorption of SNX-2112. 2. We found that SNX-2112 significantly stimulated P-gp ATPase activity in in vitro ATPase assay with a small EC50 (the half-maximal effective concentration) value of 0.32 µM. 3. In the single-pass perfused rat intestine model, absorption of SNX-2112 was not favored in the small intestine with a [Formula: see text] (the wall permeability) value of 0.38-0.64. By contrast, the compound was well absorbed in the colon with a [Formula: see text] value of 1.19. The P-gp inhibitors cyclosporine and elacridar (i.e. GF120918A) markedly enhanced SNX-2112 absorption in all four intestinal segments (i.e. duodenum, jejunum, ileum and colon) and the fold change ranged from 3.1 to 14.1. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of SNX-2112 by a 2.5-fold after oral administration. 4. This is the first report that P-gp-mediated efflux is a limiting factor for intestinal absorption of SNX-2112 in rats.

  4. A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics.

    PubMed

    Saaby, Lasse; Brodin, Birger

    2017-09-01

    Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate kinetic parameters, such as K m and V max , for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC 50 ) and the disassociation constant for an inhibitor/P-gp complex (K i ) have been determined to estimate P-gp inhibition. This review addresses in vitro methods commonly used to study P-gp transport kinetics and aims at providing a critical evaluation of the application of steady-state Michaelis-Menten analysis of kinetic parameters for substrate/P-gp interactions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. Schistosoma mansoni P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility

    PubMed Central

    Messerli, Shanta M.; Kasinathan, Ravi S.; Morgan, William; Spranger, Stefani; Greenberg, Robert M.

    2009-01-01

    One potential physiological target for new antischistosomals is the parasite’s system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, increased expression of Pgp is associated with multidrug resistance in tumor cells. Pgp may also play a role in drug resistance in helminths. In this report, we examine the relationship between praziquantel (PZQ), the current drug of choice against schistosomiasis, and Pgp expression in Schistosoma mansoni. We show that levels of RNA for SMDR2, a Pgp homolog from S. mansoni, increase transiently in adult male worms following exposure to sublethal concentrations (100 – 500 nM) of PZQ. A corresponding, though delayed, increase in anti-Pgp immunoreactive protein expression occurs in adult males following exposure to PZQ. The level of anti-Pgp immunoreactivity in particular regions of adult worms also increases in response to PZQ. Adult worms from an Egyptian S. mansoni isolate with reduced sensitivity to PZQ express increased levels of SMDR2 RNA and anti-Pgp-immunoreactive protein, perhaps indicating a role for multidrug resistance proteins in development or maintenance of PZQ resistance. PMID:19406169

  6. Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

    SciTech Connect

    Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.

    1985-08-01

    It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. (/sup 14/C)Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium inducedmore » a concentration-dependent inhibition of (/sup 14/C)proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.« less

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

    Garcia-Campos, Andres; Cwiklinski, Krystyna; Dalton, John P.; Hokke, Cornelis H.; O’Neill, Sandra; Mulcahy, Grace

    2016-01-01

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

  9. P-glycoprotein epitope mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells.

    PubMed

    Cianfriglia, M; Willingham, M C; Tombesi, M; Scagliotti, G V; Frasca, G; Chersi, A

    1994-01-02

    A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.

  10. Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

    PubMed Central

    Affranchino, José L.; González, Silvia A.

    2014-01-01

    The lentiviral envelope glycoproteins (Env) mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV) infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively) are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles. PMID:24441862

  11. A novel mechanism of immune evasion mediated by Ebola virus soluble glycoprotein.

    PubMed

    Basler, Christopher F

    2013-05-01

    Ebola viruses encode two glycoproteins (GPs): a membrane-associated GP that is present in the viral membrane and mediates viral attachment and entry into host cells; and a secreted, nonstructural glycoprotein (sGP) that is identical to GP over approximately 90% of its length. A recent study by Mohan and colleagues attributes a novel immune evasion mechanism dubbed 'antigenic subversion' to sGP. Using DNA immunization in mice, the authors demonstrate that sGP elicits antibodies that crossreact with GP, but these antibodies are non-neutralizing. Coimmunization with sGP plus GP or sequential immunizations with GP and sGP direct the host antibody response toward non-neutralizing epitopes. Therefore, the production of sGP may prevent effective neutralization of the virus during Ebola virus infection, and may reduce the effectiveness of vaccines that rely upon neutralizing antibody responses.

  12. Overcoming human P-glycoprotein-dependent multidrug resistance with novel dihydro-β-agarofuran sesquiterpenes.

    PubMed

    Perestelo, Nayra R; Sánchez-Cañete, María P; Gamarro, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Bazzocchi, Isabel L

    2011-10-01

    Sixteen (1-16) dihydro-β-agarofuran sesquiterpenes were isolated from the fruits of Maytenus jelskii and evaluated against mammalian cells with a multidrug resistance phenotype mediated by the overexpression of the human P-glycoprotein. Their stereostructures have been elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies, chemical correlations and biogenetic means. Eight compounds from this series were discovered as potent chemosensitizers (1, 2, 4, 6, 8, 9, 11 and 14), showing similar effectiveness to or higher than the classical P-glycoprotein reversal agent verapamil, a first-generation chemosensitizer, when reversing resistance to daunomycin and vinblastine. Detailed structure-activity relationships revealed that aromatic substituents at the 6 and 9-position of the sesquiterpene scaffold were able to modulate the intensity of inhibition. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  13. Similar diagnostic performance for neurocysticercosis of three glycoprotein preparations from Taenia solium metacestodes.

    PubMed

    Villota, Guido E; Gomez, Diana I; Volcy, Michel; Franco, Andrés F; Cardona, Edgar A; Isaza, Rodrigo; Sanzón, Fernando; Teale, Judy M; Restrepo, Blanca I

    2003-03-01

    The detection of antibodies to Taenia solium metacestodes is very important in the differential diagnosis of neurocysticercosis (NCC). In this study, an electroimmunotransfer blot (EITB) assay that uses an elaborate protocol with metacestode glycoproteins as antigens was compared with two other Western blots that use glycoproteins obtained using simpler methods, including an eluate from a lectin column, or the vesicular fluid (VF) of the parasite. The concordance between the three assays was 91% in patients with active NCC and 100% in patients with suspected NCC and previous documentation of negative serology. The specificities for the Western blots and the EITB assay were 98% and 100%, respectively (98% concordance). These data suggest that the simplest of these immunoassays, the one that uses the VF of T. solium metacestodes in a Western blot format, can be reliably used for the serologic diagnosis of NCC in developing countries where access to the EITB assay is difficult.

  14. Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses.

    PubMed

    Zimmer, Gert; Locher, Samira; Berger Rentsch, Marianne; Halbherr, Stefan J

    2014-08-01

    Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment. © 2014 The Authors.

  15. Cyclophosphamide metabolite inducing apoptosis in RLS mouse lymphosarcoma cells is a substrate for P-glycoprotein.

    PubMed

    Patutina, O A; Mironova, N L; Logashenko, E B; Popova, N A; Nikolin, V P; Vasil'ev, G V; Kaledin, V I; Zenkova, M A; Vlasov, V V

    2012-01-01

    RLS lymphosarcoma characterized by enhanced expression of mdr1a and mdr1b genes encoding P-glycoprotein is insensitive to low doses of cyclophosphamide, but is susceptible to its high doses approximating the maximum tolerated doses. Induction of apoptotic death of RLS cells by high doses of cyclophosphamide was demonstrated by cytofluorometry and electrophoresis. Experiments on RLS(40) tumor cells derived from RLS lymphosarcoma and characterized by more intensive expression of mdr1a/1b genes showed that the therapeutic effects of cyclophosphamide increased under conditions of simultaneous suppression of these genes by specific small interfering RNA (siRNA). These findings suggest that active cyclophosphamide metabolite can be a substrate for P-glycoprotein.

  16. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  17. Differential expression of cell surface glycoproteins on various organ-derived microvascular endothelia and endothelial cell cultures.

    PubMed

    Belloni, P N; Nicolson, G L

    1988-09-01

    Glycoproteins expressed on the luminal surfaces of microvascular endothelium derived from various murine organs were analyzed and compared with those expressed by cultured vascular endothelial cells. Cell-surface vascular proteins were radiolabeled in situ via intracardiac perfusion with lactoperoxidase/Na125I. Autoradiography confirmed that the radiolabel was restricted to the vessel lumen in most tissues. Controls contained 125I-labeled serum proteins to identify adsorbed serum components. Glycoproteins were analyzed by western enzyme-linked lectin analysis using detergent extracts of 125I-labeled microvessels isolated from different organs. The western transfers were probed with a panel of lectin-peroxidase conjugates to determine differences in protein glycosylation. The same transfers were also screened for exposed 125I-labeled cell-surface proteins by autoradiography. This dual analysis detected glycoprotein patterns unique for each organ. At least seven major proteins (Mr approximately 180 K, 130 K, 95 K, 80 K, 75 K, 60 K, 12 K) were common to microvessels derived from each organ; however, certain glycoproteins appeared to be expressed differentially in particular organs. For example, a Mr approximately 135 K WGA-binding glycoprotein was detected in brain microvessels, whereas another WGA-binding glycoprotein of Mr approximately 40 K was detected only in kidney. In lung microvessels, a Mr approximately 140 K WGA binding glycoprotein and a Mr approximately 55 K RCA-I-binding galactoprotein were expressed preferentially, and liver microvessels displayed Mr approximately 220 K protein and a Mr approximately 35 K PNA-binding galactoprotein. The cell-surface-iodinated protein profiles from in situ labeled microvessels were similar to profiles derived from cultured bovine aortic endothelial cells and several short-term endothelial cell cultures isolated from different organs. The results from this study suggest that organ-associated endothelia express glycoprotein

  18. Purification of Recombinant Ebola Virus Glycoprotein and VP40 from a Human Cell Line

    DTIC Science & Technology

    2017-01-01

    this report, we describe procedures for the recombinant expression and purification of the Ebola Zaire structural glycoprotein and matrix protein VP40...from a human cell line. Plasmids coding for the expression of these proteins were transiently transfected into human embryonic kidney cells 293 and...typical yield for each of these proteins was approximately 1 mg/30 mL of cell culture with >90% purity. The procedures described herein may be useful

  19. Extracellular Matrix Glycoprotein-Derived Synthetic Peptides Differentially Modulate Glioma and Sarcoma Cell Migration.

    PubMed

    Brösicke, Nicole; Sallouh, Muhammad; Prior, Lisa-Marie; Job, Albert; Weberskirch, Ralf; Faissner, Andreas

    2015-07-01

    Glycoproteins of the extracellular matrix (ECM) regulate proliferation, migration, and differentiation in numerous cell lineages. ECM functions are initiated by small peptide sequences embedded in large constituents that are recognized by specific cellular receptors. In this study, we have investigated the biological effects of peptides derived from collagen type IV and tenascin-C compared to the well-known RGD peptide originally discovered in fibronectin. The influence of glycoproteins and corresponding peptides on the migration of the glioma cell lines U-251-MG and U-373-MG and the sarcoma line S-117 was studied. When the cell lines were tested in a modified Boyden chamber assay on filters coated with the ECM glycoproteins, glioma cells showed a strong migration response on tenascin-C and the basal lamina constituent collagen IV, in contrast to S-117 cells. In order to identify relevant stimulatory motifs, peptides derived from fibronectin (6NHX-GRGDSF), tenascin-C (TN-C, VSWRAPTA), and collagen type IV (MNYYSNS) were compared, either applied in solution in combination with ECM glycoprotein substrates, in solution in the presence of untreated membranes, or coated on the filters of the Boyden chambers. Using this strategy, we could identify the novel tenascin-C-derived peptide motif VSWRAPTA as a migration stimulus for glioma cells. Furthermore, while kin peptides generally blocked the effects of the respective homologous ECM proteins, unexpected effects were observed in heterologous situations. There, in several cases, addition of soluble peptides strongly boosted the response to the coated ECM proteins. We propose that peptides may synergize or antagonize each other by stimulating different signaling pathways.

  20. Messenger RNA of the Histidine-Rich Glycoprotein in Breast Tumors.

    PubMed

    Autenshlyus, A I; Brusentsov, I I; Marinkin, I O; Smirnova, S A; Rukavishnikov, M Yu; Lyakhovich, V V

    2018-01-01

    The content of mRNA of the histidine-rich glycoprotein (HRG), a potential marker of malignant neoplasia, which can be used in differential diagnosis of breast tumors, was determined in 110 breast tumor biopsy samples. The presence of HRG mRNA did not depend on the cancer type, on the preoperative treatment or its absence, as well as on the tumor progression stage and the presence of metastases.

  1. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells

    PubMed Central

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

    2014-01-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5′-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd+ baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation. PMID:24362443

  2. The Cloning, Characterization, and Functional Analysis of Murine Pregnancy Specific Glycoproteins

    DTIC Science & Technology

    1999-07-23

    such as spontaneous abortion (8, 9), intrauterine growth retardation (10), fetal hypoxia (11), and pre - eclampsia (12). Funhennore, 1 2 treatment...complications such as pre - eclampsia (73) and intrauterine growth retardation (74) are associated with shallow cytotrophoblast invasion. One possible cause of...specific beta 1 glycoprotein (SP-l) in maternal sClUm and amniotic fluid; pre - eclampsia , small for gestational age fetus and fetal distress. Placenta 14

  3. Trypanosoma cruzi serinecarboxipeptidase is a sulfated glycoprotein and a minor antigen in human Chagas disease infection.

    PubMed

    Soprano, Luciana L; Parente, Juliana E; Landoni, Malena; Couto, Alicia S; Duschak, Vilma G

    2018-04-01

    In this work, the presence of sulfated N-glycans was studied in a high-mannose-type glycoprotein of Trypanosoma cruzi with serinecarboxipeptidase (TcSCP) activity. The immune cross-reactivity between purified SCP and Cruzipain (Cz) was evidenced using rabbit sera specific for both glycoproteins. Taking advantage that SCP co-purifies with Cz from Concanavalin-A affinity columns, the Cz-SCP mixture was desulfated, ascribing the cross-reactivity to the presence of sulfate groups in both molecules. Therefore, knowing that Cz is a sulfated glycoprotein, with antigenic sulfated epitopes (sulfotopes), SCP was excised from SDS-PAGE and the N-glycosydic chains were analyzed by UV-MALDI-TOF-MS, confirming the presence of short-sulfated high-mannose-type oligosaccharidic chains. Besides, the presence of sulfotopes was analyzed in lysates of the different parasite stages demonstrating that a band with apparent molecular weight similar to SCP was highly recognized in trypomastigotes. In addition, SCP was confronted with sera of infected people with different degrees of cardiac dysfunction. Although most sera recognized it in different groups, no statistical association was found between sera antibodies specific for SCP and the severity of the disease. In summary, our findings demonstrate (1) the presence of sulfate groups in the N-glycosidic short chains of native TcSCP, (2) the existence of immune cross-reactivity between Cz and SCP, purified from epimastigotes, (3) the presence of common sulfotopes between both parasite glycoproteins, and (4) the enhanced presence of sulfotopes in trypomastigotes, probably involved in parasite-host relationship and/or infection. Interestingly, we show for the first time that SCP is a minor antigen recognized by most of chronic Chagas disease patient's sera.

  4. Aspirin resistance in cerebrovascular disease and the role of glycoprotein IIIa polymorphism in Turkish stroke patients.

    PubMed

    Derle, Eda; Öcal, Ruhsen; Kibaroğlu, Seda; Çelikkol, Ceyda; Bayraktar, Nilüfer; Verdi, Hasibe; Ataç, Belgin F; Can, Ufuk

    2016-03-01

    Aspirin resistance occurs in 5-45% of high-risk patients, with various mechanisms proposed for its development. This study aimed to determine the relationships among aspirin resistance, aspirin dosage, type of aspirin and glycoprotein IIIa P1A1/A2 polymorphism in patients with vascular risk factors. Two hundred and eight (75 symptomatic, 133 asymptomatic) patients with vascular risk factors who were using aspirin for primary or secondary prevention were prospectively included. The symptomatic group was further classified into two groups according to aspirin use at the time of stroke. Aspirin resistance was measured by the PFA-100 system (collagen/epinephrine cartridge) and glycoprotein IIIa P1A1/A2 polymorphism was determined by PCR. The overall prevalence of aspirin resistance was 32.2%. The mean age of patients with aspirin resistance was significantly higher than that in those who did not have resistance (P = 0.009). The prevalence of aspirin resistance was similar for the symptomatic and asymptomatic under aspirin therapy groups. The resistance rate was found to be highest with 100 mg enteric-coated preparation use (39.3%). Increasing the aspirin dosage and/or shifting to uncoated preparations caused a change in aspirin sensitivity of 36-60%. Repeated measurements showed development of aspirin resistance in 14% of patients who were sensitive to aspirin in previous measurements. Glycoprotein IIIaP1A1/A2 polymorphism, aspirin resistance and development of atherothrombotic stroke were not significantly related. The effect of aspirin can change by time, dosage and type of preparation used. There are no relationships among glycoprotein IIIa P1A1/A2 polymorphism, aspirin resistance and development of atherothrombotic stroke.

  5. Expression of Glycoproteins in Wild-Type and Vaccine Strains of Varicella Zoster Virus

    DTIC Science & Technology

    1990-06-18

    Type and Vaccine Strains of varicella Zoster Virus" beyond brief excerpts is with the permission of the copyright owner, and will save and hold...Type and Vaccine Strains of Varicella Zoster Virus Paul D. Ling, Doctor of Philosophy, 1990 Dissertation directed by: John Hay, Ph.D., Professor and...Vice-chairman, Department of Microbiology Characterization of varicella zoster virus(VZV) glycoprotein transcripts, polypeptides, and biologic

  6. Influence of spermine on intestinal maturation of the glycoprotein glycosylation process in neonatal rats.

    PubMed Central

    Greco, S; Hugueny, I; George, P; Perrin, P; Louisot, P; Biol, M C

    2000-01-01

    Previous work has shown an inverse evolution of the rat intestinal glycoprotein sialylation that decreases from birth to weaning and of fucosylation that increases markedly after weaning during postnatal development. At weaning time, an increase in the intestinal level of polyamines (and especially that of spermine) was observed, owing partly to the higher level of spermine found in solid food given to rats at this period in comparison with the level found in milk. To study the role of this polyamine as a possible maturation factor of the glycoprotein glycosylation, suckling rats were treated for 4 days with spermine administered orally. This treatment allowed us to mimic the spermine increase that was observed naturally in rat small intestine after weaning because, in intestines of spermine-treated suckling rats, spermine was the only polyamine to be increased and was at a level similar to that of weaned rats. Spermine treatment did not induce appreciable changes in sialyltransferase activity or in sialylation of the brush-border-membrane glycoproteins. On the contrary, this treatment induced a rise in an alpha-1, 2-fucosyltransferase activity that was regulated at the transcriptional level, but not by its inhibitor (fuctinin), and no change in the availability of substrate (GDP-fucose). As a consequence of the increase in alpha-1,2-fucosyltransferase level and of the decrease in alpha-l-fucosidase level after treatment with spermine, several alpha-1,2-fucoproteins, naturally found in brush border membranes after weaning time, appeared precociously in these membranes after the treatment of the immature suckling rats. These results indicate that spermine is a maturation factor for the fucosylation of intestinal brush-border-membrane glycoproteins but not for their sialylation, and that this polyamine might be implicated in the increased fucosylation naturally occurring at weaning time during postnatal development. PMID:10600640

  7. Mosquito cell line glycoproteins: an unsuitable model system for the Plasmodium ookinete-mosquito midgut interaction?

    PubMed Central

    2010-01-01

    Background Mosquito midgut glycoproteins may act as key recognition sites for the invading malarial ookinete. Effective transmission blocking strategies require the identification of novel target molecules. We have partially characterised the surface glycoproteins of two cell lines from two mosquito species; Anopheles stephensi and Anopheles gambiae, and investigated the binding of Plasmodium berghei ookinetes to carbohydrate ligands on the cells. Cell line extracts were run on SDS-PAGE gels and carbohydrate moieties determined by blotting against a range of biotinylated lectins. In addition, specific glycosidases were used to cleave the oligosaccharides. Results An. stephensi 43 and An. gambiae 55 cell line glycoproteins expressed oligosaccharides containing oligomannose and hybrid oligosaccharides, with and without α1-6 core fucosylation; N-linked oligosaccharides with terminal Galβ1-3GalNAc or GalNAcβ1-3Gal; O-linked α/βGalNAc. An. stephensi 43 cell line glycoproteins also expressed N-linked Galβ1-4R and O-linked Galβ1-3GalNAc. Although P. berghei ookinetes bound to both mosquito cell lines, binding could not be inhibited by GlcNAc, GalNAc or Galactose. Conclusions Anopheline cell lines displayed a limited range of oligosaccharides. Differences between the glycosylation patterns of the cell lines and mosquito midgut epithelial cells could be a factor why ookinetes did not bind in a carbohydrate inhibitable manner. Anopheline cell lines are not suitable as a potential model system for carbohydrate-mediated adhesion of Plasmodium ookinetes. PMID:20338056

  8. Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens

    PubMed Central

    da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping

    2017-01-01

    ABSTRACT The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that

  9. Detection of pregnancy specific beta1-glycoprotein in formalin-fixed tissues.

    PubMed Central

    Horne, C H; Towler, C M; Milne, G D

    1977-01-01

    Using an enzyme-bridge immunoperoxidase method, pregnancy specific beta1-glycoprotein (PSbetaG) has been demonstrated in the cytoplasm of the trophoblast in several formalin-fixed tissues, namely, implantation sites of ovum, normal placentae, hydatidiform moles, invasive moles, and choriocarcinomata of uterus and testis. It is suggested that this technique may prove helpful in the detection of choriocarcinomatous elements in malignant tumours. Images PMID:190273

  10. Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus

    USGS Publications Warehouse

    Huang, C.; Chien, M.S.; Landolt, M.L.; Batts, W.; Winton, J.

    1996-01-01

    Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.

  11. Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

    PubMed

    Ji, Xin; Olinger, Gene G; Aris, Sheena; Chen, Ying; Gewurz, Henry; Spear, Gregory T

    2005-09-01

    Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

  12. A hybrid monolithic column based on boronate-functionalized graphene oxide nanosheets for online specific enrichment of glycoproteins.

    PubMed

    Zhou, Chanyuan; Chen, Xiaoman; Du, Zhuo; Li, Gongke; Xiao, Xiaohua; Cai, Zongwei

    2017-05-19

    A hybrid monolithic column based on aminophenylboronic acid (APBA)-functionalized graphene oxide (GO) has been developed and used for selective enrichment of glycoproteins. The APBA/GO composites were homogeneously incorporated into a polymer monolithic column with the help of oligomer matrix and followed by in situ polymerization. The effect of dispersion of APBA/GO composites in the polymerization mixture on the performance of the monolithic column was explored in detail. The presence of graphene oxide not only enlarged the BET surface area from 6.3m 2 /g to 169.4m 2 /g, but also provided abundant boronic acid moieties for glycoprotein extraction, which improved the enrichment selectivity and efficiency for glycoproteins. The APBA/GO hybrid monolithic column was incorporated into a sequential injection system, which facilitated online extraction of proteins. Combining the superior properties of extraordinary surface area of GO and the affinity interaction of APBA to glycoproteins, the APBA/GO hybrid monolithic column showed higher enrichment factors for glycoproteins than other proteins without cis-diol-containing groups. Also, under comparable or even shorter processing time and without the addition of any organic solvent, it showed higher binding capacity toward glycoproteins compared with the conventional boronate affinity monolithic column. The practical applicability of this system was demonstrated by processing of egg white samples for extraction of ovalbumin and ovotransferrin, and satisfactory results were obtained by assay with SDS-PAGE. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Chang, Jung-San

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling ofmore » 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.« less

  14. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket.

    PubMed

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  15. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

    PubMed

    Tang, Xing Hua; Wu, Xun Yi; Xu, Lan; Fang, You Xin; Wang, Ping; Zhu, Guo Xing; Hong, Zhen

    2015-01-01

    We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs). Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR. Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs. eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  16. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

    PubMed Central

    Tang, Xing Hua; Wu, Xun Yi; Xu, Lan; Fang, You Xin; Wang, Ping; Zhu, Guo Xing; Hong, Zhen

    2015-01-01

    Objective We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs). Methods Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR. Results Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs. Conclusions eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent. PMID:25962137

  17. LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.

    PubMed

    Song, Ehwang; Zhu, Rui; Hammoud, Zane T; Mechref, Yehia

    2014-11-07

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC-MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC-ESI-MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts.

  18. Endoplasmic reticulum-associated N-glycan degradation of cold-upregulated glycoproteins in response to chilling stress in Arabidopsis

    PubMed Central

    Ma, Jun; Wang, Dinghe; She, Jessica; Li, Jianming; Zhu, Jian-Kang; She, Yi-Min

    2017-01-01

    Summary N-glycosylation has a great impact on glycoprotein structure, conformation, stability, solubility, immunogenicity and enzyme activity. Structural characterization of N-glycoproteome has been challenging but can provide insights into the extent of protein folding and surface topology. We describe a highly sensitive proteomics method for large-scale identification and quantification of glycoproteins in Arabidopsis through 15N-metabolic labeling, selective enrichment of glycopeptides, data-dependent MS/MS analysis and automated database searching.In-house databases of Arabidopsis glycoproteins and glycopeptides containing Asn-X-Ser/Thr/Cys motifs were constructed by reducing 20% and 90% of the public database size, respectively, to enable a rapid analysis of large datasets for comprehensive identification and quantification of glycoproteins and heterogeneous N-glycans in a complex mixture.Proteome-wide analysis identified c. 100 stress-related N-glycoproteins, of which the endoplasmic reticulum (ER) resident proteins were examined to be up-regulated. Quantitative measurements provided a molecular signature specific to glycoproteins for determining the degree of plant stress at low temperature.Structural N-glycoproteomics following time-course cold treatments revealed the stress-responsive degradation of high-mannose type N-glycans in ER in response to chilling stress, which may aid in elucidating the cellular mechanisms of protein relocation, transport, trafficking, misfolding and degradation under stress conditions. PMID:27558752

  19. Contribution of tumor endothelial cells to drug resistance: anti-angiogenic tyrosine kinase inhibitors act as p-glycoprotein antagonists.

    PubMed

    Bani, MariaRosa; Decio, Alessandra; Giavazzi, Raffaella; Ghilardi, Carmen

    2017-05-01

    Tumor endothelial cells (TEC) differ from the normal counterpart, in both gene expression and functionality. TEC may acquire drug resistance, a characteristic that is maintained in vitro. There is evidence that TEC are more resistant to chemotherapeutic drugs, substrates of ATP-binding cassette (ABC) transporters. TEC express p-glycoprotein (encoded by ABCB1), while no difference in other ABC transporters was revealed compared to normal endothelia. A class of tyrosine kinase inhibitors (TKI), used as angiostatic compounds, interferes with the ATPase activity of p-glycoprotein, thus impairing its functionality. The exposure of ovarian adenocarcinoma TEC to the TKIs sunitinib or sorafenib was found to abrogate resistance (proliferation and motility) to doxorubicin and paclitaxel in vitro, increasing intracellular drug accumulation. A similar effect has been reported by the p-glycoprotein inhibitor verapamil. No beneficial effect was observed in combination with cytotoxic drugs that are not p-glycoprotein substrates. The current paper reviews the mechanisms of TEC chemoresistance and shows the role of p-glycoprotein in mediating such resistance. Inhibition of p-glycoprotein by anti-angiogenic TKI might contribute to the beneficial effect of these small molecules, when combined with chemotherapy, in counteracting acquired drug resistance.

  20. IgA antibodies against β2 glycoprotein I in hemodialysis patients are an independent risk factor for mortality.

    PubMed

    Serrano, Antonio; García, Florencio; Serrano, Manuel; Ramírez, Elisa; Alfaro, F Javier; Lora, David; de la Cámara, Agustín Gómez; Paz-Artal, Estela; Praga, Manuel; Morales, Jose M

    2012-06-01

    Cardiovascular complications are the most important cause of death in patients on dialysis with end-stage renal disease. Antibodies reacting with β-glycoprotein I seem to play a pathogenic role in antiphospholipid syndrome and stroke and are involved in the origin of atherosclerosis. Here we evaluated the presence of anticardiolipin and anti-β-glycoprotein I antibodies together with other vascular risk factors and their relationship with mortality and cardiovascular morbidity in a cohort of 124 hemodialysis patients prospectively followed for 2 years. Of these, 41 patients were significantly positive for IgA anti-β-glycoprotein I, and the remaining had normal values. At 24 months, overall and cardiovascular mortality and thrombotic events were all significantly higher in patients with high anti-β-glycoprotein I antibodies. Multivariate analysis using Cox regression modeling found that age, hypoalbuminemia, use of dialysis catheters, and IgA β-glycoprotein I antibodies were independent risk factors for death. Thus, IgA antibodies to β-glycoprotein I are detrimental to the clinical outcome of hemodialysis patients.

  1. A synthetic peptide corresponding to the carboxy terminus of human immunodeficiency virus type 1 transmembrane glycoprotein induces alterations in the ionic permeability of Xenopus laevis oocytes.

    PubMed

    Comardelle, A M; Norris, C H; Plymale, D R; Gatti, P J; Choi, B; Fermin, C D; Haislip, A M; Tencza, S B; Mietzner, T A; Montelaro, R C; Garry, R F

    1997-11-20

    The carboxy-terminal 29 amino acids of the human immunodeficiency virus type 1 transmembrane glycoprotein (HIV-1 TM) are referred to as lentivirus lytic peptide 1 (LLP-1). Synthetic peptides corresponding to LLP-1 have been shown to induce cytolysis and to alter the permeability of cultured cells to various small molecules. To address the mechanisms by which LLP-1 induces cytolysis and membrane permeability changes, various concentrations of LLP-1 were incubated with Xenopus laevis oocytes, and two-electrode, voltage-clamp recording measurements were performed. LLP-1 at concentrations of 75 nM and above induced dramatic alterations in the resting membrane potential and ionic permeability of Xenopus oocytes. These concentrations of LLP-1 appeared to induce a major disruption of plasma membrane electrophysiological integrity. In contrast, concentrations of LLP-1 of 20-50 nM induced changes in membrane ionic permeability that mimic changes induced by compounds, such as the bee venom peptide melittin, that are known to form channel-like structures in biological membranes at sublytic concentrations. An analog of LLP-1 with greatly reduced cytolytic activity failed to alter the electrophysiological properties of Xenopus oocytes. Thus, by altering plasma membrane ionic permeability, the carboxy terminus of TM may contribute to cytolysis of HIV-1-infected CD4+ cells.

  2. Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation

    PubMed Central

    Willensky, Shmuel; Bignon, Eduardo A.; Tischler, Nicole D.; Dessau, Moshe

    2016-01-01

    Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens. PMID:27783673

  3. Glycoprotein CA19.9-specific monoclonal antibodies recognize sialic acid-independent glycotope.

    PubMed

    Chugh, Manoj; Piskarev, Vladimir; Galanina, Oxana; Khasbiullina, Nailya; Kadam, Pallavi; Shilova, Nadezhda; Pazynina, Galina; Dobrochaeva, Kira; Bhanushali, Paresh; Kozlov, Nikolay; Tupitsin, Nikolay; Bovin, Nicolai

    2017-10-01

    A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9. Fine epitope specificity of the antibodies was determined using printed glycan array, enzyme-linked immunosorbent assay, and inhibitory enzyme-linked immunosorbent assay. Unexpectedly, both immunoglobulins did not bind key epitope of CA19.9 glycoprotein, tetrasaccharide SiaLe A , as well as its defucosylated form sialyl Le C (known as CA-50 epitope). The antibodies were found to have different glycan-binding profiles; however, they recognized similar glycotopes with common motif Galβ1-3GlcNAcβ (Le C ), thus resembling specificity of human natural cancer-associated anti-Le C antibodies. We propose that cancer-specific glycopeptide epitope includes Galβ1-3GlcNAcβ fragment of a glycoprotein O-chain in combination with proximal hydrophobic amino acid(s) of the polypeptide chain.

  4. Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

    PubMed

    Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

    2016-07-01

    Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection.

  5. Glycoprotein lysosomal storage disorders: alpha- and beta-mannosidosis, fucosidosis and alpha-N-acetylgalactosaminidase deficiency.

    PubMed

    Michalski, J C; Klein, A

    1999-10-08

    Glycoproteinoses belong to the lysosomal storage disorders group. The common feature of these diseases is the deficiency of a lysosomal protein that is part of glycan catabolism. Most of the lysosomal enzymes involved in the hydrolysis of glycoprotein carbohydrate chains are exo-glycosidases, which stepwise remove terminal monosaccharides. Thus, the deficiency of a single enzyme causes the blockage of the entire pathway and induces a storage of incompletely degraded substances inside the lysosome. Different mutations may be observed in a single disease and in all cases account for the nonexpression of lysosomal glycosidase activity. Different clinical phenotypes generally characterize a specific disorder, which rather must be described as a continuum in severity, suggesting that other biochemical or environmental factors influence the course of the disease. This review provides details on clinical features, genotype-phenotype correlations, enzymology and biochemical storage of four human glycoprotein lysosomal storage disorders, respectively alpha- and beta-mannosidosis, fucosidosis and alpha-N-acetylgalactosaminidase deficiency. Moreover, several animal disorders of glycoprotein metabolism have been found and constitute valuable models for the understanding of their human counterparts.

  6. Carbohydrates and glycoproteins of Bacillus anthracis and related bacilli: targets for biodetection.

    PubMed

    Fox, Alvin; Stewart, George C; Waller, Lashanda N; Fox, Karen F; Harley, William M; Price, Robert L

    2003-08-01

    The spore is the form released in a bioterrorism attack. There is a real need for definition of new targets for Bacillus anthracis that might be incorporated into emerging biodetection technologies. Particularly of interest are macromolecules found in B. anthracis that are (1) spore-specific, (2) readily accessible on the spore surface and (3) distinct from those present in related organisms. One of the few biochemical methods to identify the spores of B. anthracis is based on the presence of rhamnose and 3-O-methyl rhamnose as determined by gas chromatography-mass spectrometry. Related organisms additionally contain 2-O-methyl rhamnose and fucose. Carbohydrates and glycoproteins of the B. cereus group of organisms and the related B. subilis group are reviewed here. It is hypothesized that the spore-specific carbohydrate is a component of the newly described glycoprotein of the exosporium of B. anthracis. Further work to define the protein and carbohydrate components of the glycoprotein of B. anthracis could be highly useful in developing new technologies for rapid biodetection.

  7. The changing fate of a secretory glycoprotein in developing maize endosperm.

    PubMed

    Arcalis, Elsa; Stadlmann, Johannes; Marcel, Sylvain; Drakakaki, Georgia; Winter, Verena; Rodriguez, Julian; Fischer, Rainer; Altmann, Friedrich; Stoger, Eva

    2010-06-01

    Zeins are the major storage proteins in maize (Zea mays) endosperm, and their accumulation in zein bodies derived from the endoplasmic reticulum is well characterized. In contrast, relatively little is known about post-Golgi compartments or the trafficking of vacuolar proteins in maize endosperm, specifically the presence of globulins in structures resembling protein storage vacuoles that appear in early to mid-stage seed development. We investigated this pathway by expressing and analyzing a recombinant reporter glycoprotein during endosperm maturation, using a combination of microscopy and sensitive glycopeptide analysis. Specific N-glycan acceptor sites on the protein were followed through the stages of grain development, revealing a shift from predominantly paucimannosidic vacuolar glycoforms to predominantly trimmed glycan structures lacking fucose. This was accompanied by a change in the main subcellular localization of the protein from large protein storage vacuole-like post-Golgi organelles to the endoplasmic reticulum and zein bodies. The endogenous storage proteins corn alpha-globulin and corn legumin-1 showed a similar spatiotemporal profile both in transgenic plants expressing the reporter glycoprotein and in wild-type plants. This indicates that the shift of the intracellular trafficking route, as observed with our reporter glycoprotein, may be a common strategy in maize seed development.

  8. A glycoprotein hormone expressed in corticotrophs exhibits unique binding properties on thyroid-stimulating hormone receptor.

    PubMed

    Okada, Shannon L; Ellsworth, Jeff L; Durnam, Diane M; Haugen, Harald S; Holloway, James L; Kelley, Merideth L; Lewis, Katherine E; Ren, Hongping; Sheppard, Paul O; Storey, Harold M; Waggie, Kimberly S; Wolf, Anitra C; Yao, Lena Y; Webster, Philippa J

    2006-02-01

    Corticotroph-derived glycoprotein hormone (CGH), also referred to as thyrostimulin, is a noncovalent heterodimer of glycoprotein hormone alpha 2 (GPHA2) and glycoprotein hormone beta 5 (GPHB5). Here, we demonstrate that both subunits of CGH are expressed in the corticotroph cells of the human anterior pituitary, as well as in skin, retina, and testis. CGH activates the TSH receptor (TSHR); (125)I-CGH binding to cells expressing TSHR is saturable, specific, and of high affinity. In competition studies, unlabeled CGH is a potent competitor for (125)I-TSH binding, whereas unlabeled TSH does not compete for (125)I-CGH binding. Binding and competition analyses are consistent with the presence of two binding sites on the TSHR transfected baby hamster kidney cells, one that can interact with either TSH or CGH, and another that binds CGH alone. Transgenic overexpression of GPHB5 in mice produces elevations in serum T(4) levels, reductions in body weight, and proptosis. However, neither transgenic overexpression of GPHA2 nor deletion of GPHB5 produces an overt phenotype in mice. In vivo administration of CGH to mice produces a dose-dependent hyperthyroid phenotype including elevation of T(4) and hypertrophy of cells within the inner adrenal cortex. However, the distinctive expression patterns and binding characteristics of CGH suggest that it has endogenous biological roles that are discrete from those of TSH.

  9. A lectin-based gold nanoparticle assay for probing glycosylation of glycoproteins.

    PubMed

    Sánchez-Pomales, Germarie; Morris, Todd A; Falabella, James B; Tarlov, Michael J; Zangmeister, Rebecca A

    2012-09-01

    We report a glycoanalysis method in which lectins are used to probe the glycans of therapeutic glycoproteins that are adsorbed on gold nanoparticles. A model mannose-presenting glycoprotein, ribonuclease B (RNase B), and the therapeutic monoclonal antibody (mAb) rituximab, were found to adsorb spontaneously and non-specifically to bare gold nanoparticles such that glycans were accessible for lectin binding. Addition of a multivalent binding lectin, such as concanavalin A (Con A), to a solution of the modified gold nanoparticles resulted in cross-linking of the nanoparticles. This phenomenon was evidenced within 1 min by a change in the hydrodynamic diameter, D(H), measured by dynamic light scattering (DLS) and a shift and increase in absorbance of the plasmon resonance band of the gold nanoparticles. By combining the sugar-binding specificity and the cross-linking capabilities of lectins, the non-specific adsorption of glycoproteins to gold surfaces, and the unique optical reporting properties of gold nanoparticles, a glycosylation pattern of rituximab could be generated. This assay provides advantages over currently used glycoanalysis methods in terms of short analysis time, simplicity of the conjugation method, convenience of simple spectroscopic detection, and feasibility of providing glycan characterization of the protein drug product by using a variety of binding lectins. Copyright © 2012 Wiley Periodicals, Inc.

  10. A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins.

    PubMed

    Pfeiffer, Anett; Stephanowitz, Heike; Krause, Eberhard; Volkwein, Corinna; Hirsch, Christian; Jarosch, Ernst; Sommer, Thomas

    2016-06-03

    A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. SERINC5 protein inhibits HIV-1 fusion pore formation by promoting functional inactivation of envelope glycoproteins

    PubMed Central

    Sood, Chetan; Marin, Mariana; Chande, Ajit; Pizzato, Massimo; Melikyan, Gregory B.

    2017-01-01

    The host proteins, SERINC3 and SERINC5, have been recently shown to incorporate into HIV-1 particles and compromise their ability to fuse with target cells, an effect that is antagonized by the viral Nef protein. Envelope (Env) glycoproteins from different HIV-1 isolates exhibit a broad range of sensitivity to SERINC-mediated restriction, and the mechanism by which SERINCs interfere with HIV-1 fusion remains unclear. Here, we show that incorporation of SERINC5 into virions in the absence of Nef inhibits the formation of small fusion pores between viruses and cells. Strikingly, we found that SERINC5 promotes spontaneous functional inactivation of sensitive but not resistant Env glycoproteins. Although SERINC5-Env interaction was not detected by co-immunoprecipitation, incorporation of this protein enhanced the exposure of the conserved gp41 domains and sensitized the virus to neutralizing antibodies and gp41-derived inhibitory peptides. These results imply that SERINC5 restricts HIV-1 fusion at a step prior to small pore formation by selectively inactivating sensitive Env glycoproteins, likely through altering their conformation. The increased HIV-1 sensitivity to anti-gp41 antibodies and peptides suggests that SER5 also delays refolding of the remaining fusion-competent Env trimers. PMID:28179429

  12. Signaling to P-glycoprotein-A new therapeutic target to treat drug-resistant epilepsy?

    PubMed

    Hartz, Anika M S; Notenboom, Sylvia; Bauer, Bjorn

    2009-09-01

    Epilepsy affects more than 60 million people worldwide. While most patients can be treated with antiepileptic drugs, up to 40% of patients respond poorly to pharmacotherapy. This drug resistance is not well understood and presents a major clinical problem. In this short review we provide background information on one potential cause of antiepileptic drug resistance, namely, upregulation of the drug efflux transporter P-glycoprotein at the blood-brain barrier. We summarize recent findings that connect antiepileptic drug resistance with P-glycoprotein upregulation and show a mechanistic link between seizures and upregulation of this transporter. We provide an overview of results demonstrating that glutamate released during seizures signals through N-methyl-Daspartate (NMDA) receptor and cyclooxygenase-2 (COX-2) to increase P-glycoprotein. In this context we discuss the NMDA receptor and COX-2 as potential therapeutic targets and provide information on current clinical trials on drugresistant epilepsy involving blood-brain barrier efflux transporters. Finally, we provide a perspective on future research that could help improve the treatment of drug-resistant epilepsy. Copyright 2009 Prous Science, S.A.U. or its licensors. All rights reserved.

  13. Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

    SciTech Connect

    Mahmoud, Nora F.; Faculty of Pharmacy, Suez Canal University, Ismailia; Jasirwan, Chyntia

    2016-03-15

    Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highlymore » conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.« less

  14. A Cell-Cell Fusion Assay to Assess Arenavirus Envelope Glycoprotein Membrane-Fusion Activity.

    PubMed

    York, Joanne; Nunberg, Jack H

    2018-01-01

    For many viruses that enter their target cells through pH-dependent fusion of the viral and endosomal membranes, cell-cell fusion assays can provide an experimental platform for investigating the structure-function relationships that promote envelope glycoprotein membrane-fusion activity. Typically, these assays employ effector cells expressing the recombinant envelope glycoprotein on the cell surface and target cells engineered to quantitatively report fusion with the effector cell. In the protocol described here, Vero cells are transfected with a plasmid encoding the arenavirus envelope glycoprotein complex GPC and infected with the vTF7-3 vaccinia virus expressing the bacteriophage T7 RNA polymerase. These effector cells are mixed with target cells infected with the vCB21R-lacZ vaccinia virus encoding a β-galactosidase reporter under the control of the T7 promoter. Cell-cell fusion is induced upon exposure to low-pH medium (pH 5.0), and the resultant expression of the β-galactosidase reporter is quantitated using a chemiluminescent substrate. We have utilized this robust microplate cell-cell fusion assay extensively to study arenavirus entry and its inhibition by small-molecule fusion inhibitors.

  15. Biosynthesis of an unglycosylated envelope glycoprotein of Rous sarcoma virus in the presence of tunicamycin.

    PubMed Central

    Diggelmann, H

    1979-01-01

    Cells stably infected with Rous sarcoma virus were treated with tunicamycin to prevent the glycosylation of the precursor (pr92gp) to the two viral envelope glycoproteins gp85 and gp35. Pretreatment of the cells for 4 h with the antibiotic resulted in a 90% reduction in [3H]mannose incorporation into total cellular glycoproteins, intracellular viral glycoproteins, and released virus particles. Protein synthesis and virus particle formation were not significantly affected by the treatment. A new polypeptide made in the presence of the drug was identified by immunoprecipitation of pulse-labeled cell lysates with monospecific anti-gp85 and anti-gp35 sera. This polypeptide, migrating on sodium dodecyl sulfate-polyacrylamide gels as a molecule of 62,000 daltons (pr62), contained no [3H]mannose, was labeled with [S35]methionine and [3H]arginine, could not be chased into the higher-molecular-weight glycosylated form, and contained the same [3H]arginine tryptic peptides as pr92gp. The unglycosylated pr62 was still detectable 2 h after the pulse labeling of the cells. The lack of glycosylation of pr62 did not seem to reduce its stability. No clear evidence for the incorporation of this molecule or its cleavage products into viral particles could be obtained. To code for an envelope polypeptide of 62,000 daltons, only about 1,500 nucleotides or 15% of the total coding capacity of the virus are needed. Images PMID:90167

  16. Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

    PubMed Central

    Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

    2015-01-01

    Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

  17. Immune responses of cattle and mice to the G glycoprotein of vesicular stomatitis virus.

    PubMed

    Yilma, T; Breeze, R G; Ristow, S; Gorham, J R; Leib, S R

    1985-01-01

    A subunit vaccine for vesicular stomatitis was developed from a purified vesicular stomatitis virus preparation by selectively removing the immunogenic G glycoprotein of the virus with the dialyzable, nonionic detergent, beta-D-octylglucoside. Cattle immunized intramuscularly with a single dose of 112 micrograms of G glycoprotein preparation in complete Freund's adjuvant did not develop vesicular disease following challenge by intralingual inoculation of 400 times the infectious dose of the virus. Similarly, mice vaccinated subcutaneously with a single dose of 10 micrograms of G glycoprotein preparation, with or without complete Freund's adjuvant, were protected from lethal encephalitis caused by vesicular stomatitis virus. A subunit vaccine for vesicular stomatitis of cattle, horses, and swine avoids the hazards associated with attenuated and inactivated vaccines, such as vaccine breaks, reversion to virulence, or introduction of virus into potential wild reservoirs or arthropod hosts. Further, it is possible to distinguish serologically animals vaccinated with the subunit preparation from those that have had the clinical disease or that have been vaccinated with whole virus. This is an essential consideration both for epidemiological studies and for disease control or establishment of quarantine programs.

  18. Herpes Simplex Virus Glycoproteins H/L Bind to Cells Independently of αVβ3 Integrin and Inhibit Virus Entry, and Their Constitutive Expression Restricts Infection ▿

    PubMed Central

    Gianni, Tatiana; Cerretani, Arianna; DuBois, Rebecca; Salvioli, Stefano; Blystone, Scott S.; Rey, Felix; Campadelli-Fiume, Gabriella

    2010-01-01

    Herpes simplex virus (HSV) fusion with cells requires the gD, gB, and gH/gL glycoprotein quartet. gD serves as a receptor binding glycoprotein. gB and gH/gL execute fusion in an as-yet-unclear manner. To better understand the role of gH/gL in HSV entry, we produced a soluble version of gH/gL carrying a One-STrEP tag (gHt.st/gL). Previous findings implicated integrins as possible ligands to gH/gL (C. Parry et al., J. Gen. Virol. 86:7-10, 2005). We report that (i) gHt.st/gL bound a number of cells in a dose-dependent manner at concentrations similar to those required for the binding of soluble gB or gD. (ii) gHt.st/gL inhibited HSV entry at the same concentrations required for binding. It also inhibited cell-cell fusion in transfected cells. (iii) The absence of β3 integrin did not prevent the binding of gHt.st/gL to CHO cells and infection inhibition. Conversely, integrin-negative K562 cells did not acquire the ability to bind gHt.st/gL when hyperexpressing αVβ3 integrin. (iv) Constitutive expression of wild-type gH/gL (wt-gH/gL) restricted infection in all of the cell lines tested, a behavior typical of glycoproteins which bind cellular receptors. The extent of restriction broadly paralleled the efficiency of gH/gL transfection. RGD motif mutant gH/gL could not be differentiated from wt-gH with respect to restriction of infection. Cumulatively, the present results provide several lines of evidence that HSV gH/gL interacts with a cell surface cognate protein(s), that this protein is not necessarily an αVβ3 integrin, and that this interaction is required for the process of virus entry/fusion. PMID:20147400

  19. 55,000-dalton, retrovirus-associated, cell membrane glycoprotein: purification and quantitative measurements of expression in viruses, cells, and tissues.

    PubMed Central

    Scheinberg, D A; Strand, M

    1981-01-01

    We have purified to homogeneity and characterized a 55,000-dalton rat cell membrane glycoprotein, gp55. This protein was originally identified in preparations of a defective pseudotype of the Kirsten sarcoma virus and shown to be present in several rodent retrovirus particles. The gp55 was purified from this defective virus by concanavalin A and heparin affinity chromatography, as well as by preparative sodium dodecyl sulfate-gel electrophoresis. Both preparations displayed similar purity and antigenic characteristics. The 125I-labeled gp55 was precipitated by antisera against rodent retroviruses, but not by monospecific antisera against purified type C virus structural proteins, thus indicating that gp55 was retrovirus associated, but unrelated to known retrovirus structural proteins. Competition radioimmunoassay with an anti-rat virus serum which recognized rodent group-specific antigens on gp55 indicated: the presence of gp55 antigens in 15 rodent cell lines, but not 10 nonrodent cell lines; no effect of viral infection or cell transformation on the amount of gp55 expressed; up to 100-fold increases in the concentration of the gp55 antigens in nine rodent retroviruses, but not in five nonrodent viruses, as compared to cells; the presence of gp55 in rodent sera, especially of the NZB mouse, where anti-gp55 antibody was also detected; a lymphoid and epithelial tissue distribution of gp55 in rats and mice. Additional competition radioimmunoassays with a broad-reacting antivirus serum also detected the presence of gp55 in nonrodent, mink, and human cells and thus distinguished rat type, rodent group, and interspecies antigenic determinants on gp55. In conclusion, gp55 is a cell membrane glycoprotein associated in high concentration with retroviruses. Images PMID:6965096

  20. Polymethoxylated flavones and other phenolic derivates from citrus in their inhibitory effects on P-glycoprotein-mediated transport of talinolol in Caco-2 cells.

    PubMed

    Mertens-Talcott, Susanne U; De Castro, Whocely Victor; Manthey, John A; Derendorf, Hartmut; Butterweck, Veronika

    2007-04-04

    Many studies investigating drug interactions with citrus compounds focus on the major grapefruit furanocoumarins bergamottin, dihydroxybergamottin, and the flavonoid naringenin. This study evaluated the influence of polymethoxylated flavones (PMFs), tangeretin, nobiletin, 3,5,6,7,8,3,4'-heptamethoxyflavone, and sinensetin, as well as other minor occurring citrus phenols, hesperetin, limettin, 7-OH-coumarin, 7-geranyloxycoumarin, and eriodictyol, on P-glycoprotein-mediated transport of the beta-blocker talinolol using the Caco-2 cell monolayer model and was used to determine the structure-function aspects of the interaction. The transport of talinolol across Caco-2 cells monolayers was determined in the absence and presence of distinct concentrations of the calcium-channel blocker verapamil (a known inhibitor of P-glycoprotein) and citrus compounds. A sigmoid dose-response model was used to fit the data and to estimate the IC50 values of the potential inhibitors. Results from this study show that PMFs significantly decreased talinolol transport from the basolateral to apical side, where tangeretin had the lowest IC50 of 3.2 micromol/L, followed by nobiletin, heptamethoxyflavone, and sinensetin with IC50 values of 3.5, 3.8, and 3.9 micromol/L, respectively. However, the efficacy of the compounds did not appear to be dependent on the number of methoxy groups. Other citrus compounds did not have any significant effect on the transport of talinolol. This study suggests that PMFs have a high potential in the interaction with P-gp-mediated talinolol transport in Caco-2 cells. Based on their relatively low concentrations (< or =3 microg/mL) in citrus, the clinical relevance of these interactions needs to be further elucidated in in vivo studies.

  1. In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture

    PubMed Central

    Follit, Courtney A; Brewer, Frances K; Wise, John G; Vogel, Pia D

    2015-01-01

    Failure of cancer chemotherapies is often linked to the over expression of ABC efflux transporters like the multidrug resistance P-glycoprotein (P-gp). P-gp expression in cells leads to the elimination of a variety of chemically unrelated, mostly cytotoxic compounds. Administration of chemotherapeutics during therapy frequently selects for cells that over express P-gp and are therefore capable of robustly exporting diverse compounds, including chemotherapeutics, from the cells. P-gp thus confers multidrug resistance to a majority of drugs currently available for the treatment of cancers and diseases like HIV/AIDS. The search for P-gp inhibitors for use as co-therapeutics to combat multidrug resistances has had little success to date. In a previous study (Brewer et al., Mol Pharmacol 86: 716–726, 2014), we described how ultrahigh throughput computational searches led to the identification of four drug-like molecules that specifically interfere with the energy harvesting steps of substrate transport and inhibit P-gp catalyzed ATP hydrolysis in vitro. In the present study, we demonstrate that three of these compounds reversed P-gp-mediated multidrug resistance of cultured prostate cancer cells to restore sensitivity comparable to naïve prostate cancer cells to the chemotherapeutic drug, paclitaxel. Potentiation concentrations of the inhibitors were <3 μmol/L. The inhibitors did not exhibit significant toxicity to noncancerous cells at concentrations where they reversed multidrug resistance in cancerous cells. Our results indicate that these compounds with novel mechanisms of P-gp inhibition are excellent leads for the development of co-therapeutics for the treatment of multidrug resistances. PMID:26516582

  2. In silico identified targeted inhibitors of P-glycoprotein overcome multidrug resistance in human cancer cells in culture.

    PubMed

    Follit, Courtney A; Brewer, Frances K; Wise, John G; Vogel, Pia D

    2015-10-01

    Failure of cancer chemotherapies is often linked to the over expression of ABC efflux transporters like the multidrug resistance P-glycoprotein (P-gp). P-gp expression in cells leads to the elimination of a variety of chemically unrelated, mostly cytotoxic compounds. Administration of chemotherapeutics during therapy frequently selects for cells that over express P-gp and are therefore capable of robustly exporting diverse compounds, including chemotherapeutics, from the cells. P-gp thus confers multidrug resistance to a majority of drugs currently available for the treatment of cancers and diseases like HIV/AIDS. The search for P-gp inhibitors for use as co-therapeutics to combat multidrug resistances has had little success to date. In a previous study (Brewer et al., Mol Pharmacol 86: 716-726, 2014), we described how ultrahigh throughput computational searches led to the identification of four drug-like molecules that specifically interfere with the energy harvesting steps of substrate transport and inhibit P-gp catalyzed ATP hydrolysis in vitro. In the present study, we demonstrate that three of these compounds reversed P-gp-mediated multidrug resistance of cultured prostate cancer cells to restore sensitivity comparable to naïve prostate cancer cells to the chemotherapeutic drug, paclitaxel. Potentiation concentrations of the inhibitors were <3 μmol/L. The inhibitors did not exhibit significant toxicity to noncancerous cells at concentrations where they reversed multidrug resistance in cancerous cells. Our results indicate that these compounds with novel mechanisms of P-gp inhibition are excellent leads for the development of co-therapeutics for the treatment of multidrug resistances.

  3. Lactation Stage-Related Expression of Sialylated and Fucosylated Glycotopes of Human Milk α-1-Acid Glycoprotein

    PubMed Central

    Hirnle, Lidia; Berghausen-Mazur, Marta; Kątnik-Prastowska, Iwona M.

    2014-01-01

    Abstract Background: Because terminal sugars of α-1-acid glycoprotein (AGP) are reported to be involved in anti-inflammatory and immunomodulatory processes, their expressions might have an influence on the proper function of immune system of newborns. Here, relative amounts of sialylated and fucosylated glycotopes on human milk AGP over normal lactation were investigated. Materials and Methods: AGP concentration and relative amounts of its sialylated and fucosylated glycovariants were analyzed in early colostrum, colostrum, and transitional and mature milk samples of 127 healthy mothers by lectin–AGP enzyme-linked immunosorbent assay using α2,3- and α2,6-sialic acid and α1,2-, α1,3-, and α1,6-fucose specific biotinylated Maackia amurensis, Sambucus nigra, Ulex europaeus, Tetragonolobus purpureus, and Lens culinaris lectins, respectively. Results: AGP concentration in human milk was about 30 times lower than in plasma of lactating mothers and decreased gradually over lactation. Milk AGP showed significantly higher expression of sialylated and fucosylated glycotopes in comparison with those of plasma AGP. Milk AGP glycovariants containing α2,6-sialylated and α1,6- and α1,2-fucosylated glycotopes showed the highest relative amounts in early colostrums. With progression of lactation, the expressions of glycotopes α1,2-fucosylated decreased starting from Day 4 and those of α2,6-sialylated and α1,6-fucosylated from Day 8 of lactation, whereas the level of α2,3-sialyl-glycotope was almost constant over 45 days of lactation. In contrast, the expression of α1,3-linked fucose on AGP was low in colostrums and significantly higher in transitional and mature milk. Conclusions: The relative amounts of sialylated and fucosylated glycovariants of human hindmilk AGP significantly varied between Days 2 and 45 of normal lactation. PMID:24892765

  4. Effects of Phytochemical P-Glycoprotein Modulators on the Pharmacokinetics and Tissue Distribution of Doxorubicin in Mice.

    PubMed

    Kim, Tae Hwan; Shin, Soyoung; Yoo, Sun Dong; Shin, Beom Soo

    2018-02-07

    Pungent spice constituents such as piperine, capsaicin and [6]-gingerol consumed via daily diet or traditional Chinese medicine, have been reported to possess various pharmacological activities. These dietary phytochemicals have also been reported to inhibit P-glycoprotein (P-gp) in vitro and act as an alternative to synthetic P-gp modulators. However, the in vivo effects on P-gp inhibition are currently unknown. This study aimed to test the hypothesis that phytochemical P-gp inhibitors, i.e., piperine, capsaicin and [6]-gingerol, modulate the in vivo tissue distribution of doxorubicin, a representative P-gp substrate. Mice were divided into four groups and each group was pretreated with intraperitoneal injections of control vehicle, piperine, capsaicin, or [6]-gingerol and doxorubicin (1 mg/kg) was administered via the penile vein. The concentrations of the phytochemicals and doxorubicin in the plasma and tissues were determined by LC-MS/MS. The overall plasma concentration-time profiles of doxorubicin were not significantly affected by piperine, capsaicin, or [6]-gingerol. In contrast, doxorubicin accumulation was observed in tissues pretreated with piperine or capsaicin. The tissue to plasma partition coefficients, K p , for the liver and kidney were higher in the piperine-pretreated group, while the K p for kidney, brain and liver were higher in the capsaicin-pretreated group. [6]-Gingerol did not affect doxorubicin tissue distribution. The data demonstrated that the phytochemicals modulated doxorubicin tissue distribution, which suggested their potential to induce food-drug interactions and act as a strategy for the delivery of P-gp substrate drugs to target tissues and tumors.

  5. The Two Caenorhabditis elegans UDP-Glucose:Glycoprotein Glucosyltransferase Homologues Have Distinct Biological Functions

    PubMed Central

    Buzzi, Lucila I.; Simonetta, Sergio H.; Parodi, Armando J.; Castro, Olga A.

    2011-01-01

    The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans

  6. Myelin oligodendrocyte glycoprotein and aquaporin-4 antibodies are highly specific in children with acquired demyelinating syndromes.

    PubMed

    Duignan, Sophie; Wright, Sukhvir; Rossor, Tom; Cazabon, John; Gilmour, Kimberly; Ciccarelli, Olga; Wassmer, Evangeline; Lim, Ming; Hemingway, Cheryl; Hacohen, Yael

    2018-02-22

    Our objectives were to evaluate the utility of measuring myelin oligodendrocyte glycoprotein (MOG) and aquaporin-4 (AQP4) antibodies (Ab) in clinical practice and describe their associated neurological phenotypes in children. Between 2012 and 2017, 371 children with suspected acquired demyelinating syndromes (ADS) seen in three tertiary centres were tested for MOG-Ab and AQP4-Ab. Medical notes were retrospectively reviewed, and clinical and demographic data compiled. Clinical phenotyping was performed blinded to the antibody results. After review, 237 of the 371 were diagnosed with ADS. Of these, 76 out of 237 (32.1%) were MOG-Ab positive and 14 out of 237 (5.9%) were AQP4-Ab positive. None were positive for both autoantibodies. All 134 patients with non-ADS were negative for MOG-Ab. MOG-Ab were identified in 45 out of 70 (64.3%) patients presenting with acute disseminated encephalomyelitis (ADEM) and in 24 out of 25 patients with relapsing ADEM. Thirty-six out of 75 (48%) MOG-Ab positive patients relapsed. Of the 33 children with neuromyelitis optic spectrum disorder, 14 were AQP4-Ab positive, 13 were MOG-Ab positive, and 6 were seronegative. Of the children with longitudinal samples, 8 out of 13 AQP4-Ab remained positive during the disease course compared to 35 out of 43 MOG-Ab (13/16 monophasic and 22/27 relapsing). Myelin oligodendrocyte glycoprotein antibodies were identified in a third of children with ADS. Almost half of the MOG-Ab positive children relapsed and the majority of them remained antibody positive over 4-years follow-up. Myelin oligodendrocyte glycoprotein antibodies (MOG-Ab) are highly specific for acquired demyelinating syndromes (ADS). Myelin oligodendrocyte glycoprotein antibodies are not identified in children with peripheral demyelination or genetic leukodystrophies/hypomyelination. Up to 48% of MOG-Ab ADS paediatric patients relapse, higher than previously thought. Seroconversion to MOG-Ab negative status is infrequent; patients may test

  7. Construction of P-glycoprotein incorporated tethered lipid bilayer membranes.

    PubMed

    Inci, Fatih; Celik, Umit; Turken, Basak; Özer, Hakan Özgür; Kok, Fatma Nese

    2015-07-01

    To investigate drug-membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid ( i.e. , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03 mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro . Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance-Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.

  8. Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance.

    PubMed Central

    Gekeler, V.; Boer, R.; Uberall, F.; Ise, W.; Schubert, C.; Utz, I.; Hofmann, J.; Sanders, K. H.; Schächtele, C.; Klemm, K.; Grunicke, H.

    1996-01-01

    Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H

  9. Gastric carcinoma: expression of c-erbB-2/neu oncoprotein, epidermal growth factor receptor, cathepsin D, progesterone receptor and tumor associated glycoprotein-72 in different histological types.

    PubMed

    Peláez Buján, M del C; Ruibal Morell, A; Aza González, J

    1999-12-01

    Gastric carcinoma can be divided into two main histological and clinical types: diffuse and intestinal. The aim of this study was to investigate the expression of neu/c-erbB-2 oncoprotein, epidermal growth factor receptor (EGFR), cathepsin D (catD), progesterone receptor (PR) and tumor-associated glycoprotein-72 (TAG-72) in gastric carcinoma of these histological types. In this randomized, prospective study we analyzed 85 biopsy samples from patients with gastric adenocarcinoma. The control group consisted of 40 specimens from normal gastric mucosa. Neu oncoprotein and PR were determined by ELISA. CatD and TAG-72 were quantified with immunoradiometric (IRMA) methods, and EGFR were studied by radioimmunoassay (RIA). Neu, EGFR, catD and TAG-72 concentrations were higher in the tumoral group (p = 0.02, p = 0.00001, p = 0.002 and p = 0.007, respectively). In diffuse adenocarcinomas, catD and PR expression was increased (p = 0.01 and p = 0.04 respectively), whereas TAG-72 concentration, which correlated with neu (r = 0.57), was higher in the intestinal type (p = 0.04). No significant differences in EGFR and neu concentrations were seen between the two histological types. The higher PR and catD concentrations in diffuse adenocarcinomas, and the overexpression of TAG-72 in the intestinal type, support the existence of two modes of gastric carcinogenesis.

  10. Histochemical analysis of glycoproteins in the secretory cells in the epidermis of the head skin of Indian Major Carp, Labeo rohita.

    PubMed

    Rai, A K; Srivastava, N; Kumari, U; Mittal, S; Mittal, A K

    2012-12-01

    A series of histochemical procedures were employed to localise and characterise glycoprotein (GP) classes produced by the epithelial cells, the type A and the type B mucous goblet cells (MGCs) and the club cells in the epidermis of Labeo rohita. The epithelial cells secreted GPs with oxidizable vicinal diols and GPs with sialic acid residues without O-acyl substitution in low concentrations. The type A MGCs and the type B MGCs, in contrast, produced these GPs in high concentrations. Further, these MGCs produced GPs with O-sulphate esters as well. GPs with O-sulphate esters were produced in high concentration by the type A MGCs and in low concentration by the type B MGCs. The club cells produced GPs with oxidizable vicinal diols in trace amounts. Production of more than one type of GPs suggested a basis for functional discrimination in their role in the mucous secretions at the skin surface. This is considered an adaptation to environment inhabited by the fish and is discussed in relation to their role in lubrication, protection and inhibition of the invasion and proliferation of pathogenic micro-organisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Tailored Multivalent Neo-Glycoproteins: Synthesis, Evaluation, and Application of a Library of Galectin-3-Binding Glycan Ligands.

    PubMed

    Laaf, Dominic; Bojarová, Pavla; Pelantová, Helena; Křen, Vladimír; Elling, Lothar

    2017-11-15

    Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.

  12. Association between MDR1 gene of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs.

    PubMed

    Su, Jian-Li; Wang, Cheng-Hong; Kang, Hong-Gang; Zhang, Jing; Wang, Bao-Zhong; Liu, Mei-Rong; Zhao, Jun; Liu, Lin

    2017-09-01

    The aim of the present study was to examine and discuss the association between multidrug resistance 1 gene ( MDR1 ) of gastrointestinal tumors, the expression of P-glycoprotein and resistance to chemotherapeutic drugs. In this study, 126 cases of patients with gastrointestinal tumors admitted to hospital from February 2013 to February 2015 were selected. The expression levels of MDR1 gene were obsreved in the control population and patients before and after treatment by fluoresecent quantitative PCR. The protein expression level of P-glycoprotein was determined using western blotting and enzyme-linked immunosorbent assay. In addition, drug resistance was assessed by ATP-TCA chemosensitivity experiments. The results showed that before treatment, the expression of mRNA in MDR1 of tissues of gastrointestinal tract of the 126 cases was 108-fold larger than that of the gastrointestinal tract of the controls (p<0.05), P-glycoprotein was 87-fold larger than the expression level of the controls (p<0.05). The sensitivity of 126 tumor tissues to different chemotherapeutic drugs was determined, and the results showed that most of the tumor tissues were sensitive to chemotherapeutic drugs, and the sensitivity rate reached 96.4%. Following chemotherapy, the expression of mRNA in MDR1 of tumor tissues and the expression of P-glycoprotein decreased (p<0.05). In conclusion, the MDR1 gene and P-glycoprotein have a positive correlation with the occurrence of gastrointestinal tumors, and a negative correlation between the MDR1 gene and P-glycoprotein with resistance of chemotherapeutic drugs. Therefore, the MDR1 gene and P-glycoprotein can be used as references in the identification and diagnosis of gastrointestinal tumors.

  13. Decreased levels of alpha-1-acid glycoprotein are related to the mortality of septic patients in the emergency department

    PubMed Central

    Barroso-Sousa, Romualdo; Lobo, Romulo R.; Mendonça, Patricia R.; Memória, Renan R.; Spiller, Fernando; Cunha, Fernando Q.; Pazin-Filho, Antonio

    2013-01-01

    OBJECTIVE: To determine the validity of alpha-1-acid glycoprotein as a novel biomarker for mortality in patients with severe sepsis. METHODS: We prospectively included patients with severe sepsis or septic shock at the emergency department at a single tertiary referral teaching hospital. All of the patients were enrolled within the first 24 hours of emergency department admission, and clinical data and blood samples were obtained. As the primary outcome, we investigated the association of serum levels of alpha-1-acid glycoprotein and 96-hour mortality with logistic regression analysis and generalized estimating equations adjusted for age, sex, shock status and Acute Physiology and Chronic Health Evaluation II score. RESULTS: Patients with septic shock had lower alpha-1-acid glycoprotein levels at the time of emergency department admission compared to patients without shock (respectively, 149.1±42.7 vs. 189.8±68.6; p = 0.005). Similarly, non-survivors in the first 96 hours were also characterized by lower levels of alpha-1-acid glycoprotein at the time of emergency department admission compared to survivors (respectively, 132.18±50.2 vs. 179.8±61.4; p = 0.01). In an adjusted analysis, alpha-1-acid glycoprotein levels ≤120 mg/dL were significantly associated with 96-hour mortality (odds ratio = 14.37; 95% confidence interval = 1.58 to 130.21). CONCLUSION: Septic shock patients exhibited lower circulating alpha-1-acid glycoprotein levels than patients without shock. Alpha-1-acid glycoprotein levels were independently associated with 96-hour mortality in individuals with severe sepsis. PMID:24037010

  14. Decreased levels of alpha-1-acid glycoprotein are related to the mortality of septic patients in the emergency department.

    PubMed

    Barroso-Sousa, Romualdo; Lobo, Romulo R; Mendonça, Patricia R; Memória, Renan R; Spiller, Fernando; Cunha, Fernando Q; Pazin-Filho, Antonio

    2013-01-01

    To determine the validity of alpha-1-acid glycoprotein as a novel biomarker for mortality in patients with severe sepsis. We prospectively included patients with severe sepsis or septic shock at the emergency department at a single tertiary referral teaching hospital. All of the patients were enrolled within the first 24 hours of emergency department admission, and clinical data and blood samples were obtained. As the primary outcome, we investigated the association of serum levels of alpha-1-acid glycoprotein and 96-hour mortality with logistic regression analysis and generalized estimating equations adjusted for age, sex, shock status and Acute Physiology and Chronic Health Evaluation II score. Patients with septic shock had lower alpha-1-acid glycoprotein levels at the time of emergency department admission compared to patients without shock (respectively, 149.1 ±42.7 vs. 189.8 ±68.6; p = 0.005). Similarly, non-survivors in the first 96 hours were also characterized by lower levels of alpha-1-acid glycoprotein at the time of emergency department admission compared to survivors (respectively, 132.18 ±50.2 vs. 179.8 ±61.4; p = 0.01). In an adjusted analysis, alpha-1-acid glycoprotein levels ≤120 mg/dL were significantly associated with 96-hour mortality (odds ratio = 14.37; 95% confidence interval = 1.58 to 130.21). Septic shock patients exhibited lower circulating alpha-1-acid glycoprotein levels than patients without shock. Alpha-1-acid glycoprotein levels were independently associated with 96-hour mortality in individuals with severe sepsis.

  15. Juxtamembrane basic residues in glycoprotein Ibβ cytoplasmic domain are required for assembly and surface expression of glycoprotein Ib-IX complex

    PubMed Central

    Mo, Xi; Luo, Shi-Zhong; López, José A.; Li, Renhao

    2008-01-01

    Platelet glycoprotein (GP) Ib-IX complex requires all its three subunits for efficient expression on the cell surface, but the underlying molecular basis is not fully clear. Using transfected Chinese hamster ovary cells as the model system, we demonstrate that juxtamembrane residues 149–154 in the cytoplasmic domain of the GPIbβ subunit is required for assembly and surface expression of the GPIb-IX complex. The complex, or GPIbβ by itself, lacking these residues is retained in the endoplasmic reticulum. Our results thus have illustrated an important role of the GPIbβ cytoplasmic domain in biosynthesis of the GPIb-IX complex. Structured summary MINT-6742751, MINT-6742907: GPIX (uniprotkb:P14770), GPIbalpha (uniprotkb:P07359) and GPIbbeta (uniprotkb:P13224) physically interact (MI:0218) by fluorescence-activated cell sorting (MI:0054) PMID:18789323

  16. In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon

    PubMed Central

    1981-01-01

    Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive, both erythrocyte-rosetting and non-erythrocyte- rosetting, as assessed by both positive and negative selection experiments. The induction of nonspecific CMC by viral glycoproteins either in the soluble state or inserted into artificial membranes could be segregated from the CMC associated with whole virions. First, on kinetics studies, purified viral glycoproteins induced CMC more rapidly than did whole virus. Second, viral glycoprotein-produced response occurred in the absence of detectable release of interferon into the culture medium, whereas CMC activity due to whole virions was associated with interferon release. The fact that purified measles virus glycoproteins integrated into artificial membrane bilayers were as efficient as their soluble counterparts in inducing CMC suggests that the hydrophobic portion of the glycoproteins was not involved in the induction and expression of the lytic activity. Purified glycoproteins from lymphocytic choriomeningitis virus behave similarly, although this virus is unrelated to measles virus. It is inferred that interferon-independent CMC induced by viral glycoproteins might account for some of the biological reactions occurring early in the control of a viral infection. PMID:7276828

  17. In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon.

    PubMed

    Casali, P; Sissons, J G; Buchmeier, M J; Oldstone, M B

    1981-09-01

    Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive, both erythrocyte-rosetting and non-erythrocyte-rosetting, as assessed by both positive and negative selection experiments. The induction of nonspecific CMC by viral glycoproteins either in the soluble state or inserted into artificial membranes could be segregated from the CMC associated with whole virions. First, on kinetics studies, purified viral glycoproteins induced CMC more rapidly than did whole virus. Second, viral glycoprotein-produced response occurred in the absence of detectable release of interferon into the culture medium, whereas CMC activity due to whole virions was associated with interferon release. The fact that purified measles virus glycoproteins integrated into artificial membrane bilayers were as efficient as their soluble counterparts in inducing CMC suggests that the hydrophobic portion of the glycoproteins was not involved in the induction and expression of the lytic activity. Purified glycoproteins from lymphocytic choriomeningitis virus behave similarly, although this virus is unrelated to measles virus. It is inferred that interferon-independent CMC induced by viral glycoproteins might account for some of the biological reactions occurring early in the control of a viral infection.

  18. Effect of collecting duct-specific deletion of both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on renal response to metabolic acidosis

    PubMed Central

    Lee, Hyun-Wook; Verlander, Jill W.; Handlogten, Mary E.; Han, Ki-Hwan

    2013-01-01

    The Rhesus (Rh) glycoproteins, Rh B and Rh C Glycoprotein (Rhbg and Rhcg, respectively), are ammonia-specific transporters expressed in renal distal nephron and collecting duct sites that are necessary for normal rates of ammonia excretion. The purpose of the current studies was to determine the effect of their combined deletion from the renal collecting duct (CD-Rhbg/Rhcg-KO) on basal and acidosis-stimulated acid-base homeostasis. Under basal conditions, urine pH and ammonia excretion and serum HCO3− were similar in control (C) and CD-Rhbg/Rhcg-KO mice. After acid-loading for 7 days, CD-Rhbg/Rhcg-KO mice developed significantly more severe metabolic acidosis than did C mice. Acid loading increased ammonia excretion, but ammonia excretion increased more slowly in CD-Rhbg/Rhcg-KO and it was significantly less than in C mice on days 1–5. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice on days 1, 3, and 5 of acid loading. Metabolic acidosis increased phosphenolpyruvate carboxykinase (PEPCK) and Na+/H+ exchanger NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that 1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis; 2) the significantly greater changes in PEPCK, NHE-3, and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared with acid-loaded C mice cause the role of Rhbg and Rhcg to be underestimated quantitatively; and 3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3, and GS expression despite the presence of persistent metabolic acidosis. PMID:24338819

  19. The H2 receptor antagonist nizatidine is a P-glycoprotein substrate: characterization of its intestinal epithelial cell efflux transport.

    PubMed

    Dahan, Arik; Sabit, Hairat; Amidon, Gordon L

    2009-06-01

    The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H(2) receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05-10 mM in both apical-basolateral (AP-BL) and BL-AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC(50) of verapamil on nizatidine P-gp secretion was 1.2 x 10(-2) mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J(max) = 5.7 x 10(-3) nmol cm(-2) s(-1) and K(m) = 2.2 mM) and one nonsaturable component (K(d) = 7 x 10(-4) microL cm(-2) s(-1)). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V(max) and K(m) estimated for nizatidine P-gp-mediated secretion were 4 x 10(-3) nmol cm(-2) s(-1) and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug-drug interactions.

  20. Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects.

    PubMed

    Branco, Luis M; Grove, Jessica N; Moses, Lina M; Goba, Augustine; Fullah, Mohammed; Momoh, Mambu; Schoepp, Randal J; Bausch, Daniel G; Garry, Robert F

    2010-11-09

    Lassa hemorrhagic fever (LHF) is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. With a high rate of infection that may lead to morbidity and mortality, understanding how the virus interacts with the host's immune system is of great importance for generating vaccines and therapeutics. Previous work by our group identified a soluble isoform of the Lassa virus (LASV) GP1 (sGP1) in vitro resulting from the expression of the glycoprotein complex (GPC) gene [1, 2]. Though no work has directly been done to demonstrate the function of this soluble isoform in arenaviral infections, evidence points to immunomodulatory effects against the host's immune system mediated by a secreted glycoprotein component in filoviruses, another class of hemorrhagic fever-causing viruses. A significant fraction of shed glycoprotein isoforms during viral infection and biogenesis may attenuate the host's inflammatory response, thereby enhancing viral replication and tissue damage. Such shed glycoprotein mediated effects were previously reported for Ebola virus (EBOV), a filovirus that also causes hemorrhagic fever with nearly 90 percent fatality rates [3 - 5]. The identification of an analogous phenomenon in vivo could establish a new correlate of LHF infection leading to the development of sensitive diagnostics targeting the earliest molecular events of the disease. Additionally, the reversal of potentially untoward immunomodulatory functions mediated by sGP1 could potentiate the development of novel therapeutic intervention. To this end, we investigated the presence of sGP1 in the serum of suspected LASV patients admitted to the Kenema Government Hospital (KGH) Lassa Fever Ward (LFW), in Kenema, Sierra Leone that tested positive for viral antigen or displayed classical signs of Lassa fever. It is reasonable to expect that a narrow window exists for detection of sGP1 as the sole

  1. Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins

    PubMed Central

    Stone, Jacquelyn A.; Vemulapati, Bhadra M.; Bradel-Tretheway, Birgit

    2016-01-01

    ABSTRACT The paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions. IMPORTANCE Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G

  2. Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects

    PubMed Central

    2010-01-01

    Background Lassa hemorrhagic fever (LHF) is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. With a high rate of infection that may lead to morbidity and mortality, understanding how the virus interacts with the host's immune system is of great importance for generating vaccines and therapeutics. Previous work by our group identified a soluble isoform of the Lassa virus (LASV) GP1 (sGP1) in vitro resulting from the expression of the glycoprotein complex (GPC) gene [1,2]. Though no work has directly been done to demonstrate the function of this soluble isoform in arenaviral infections, evidence points to immunomodulatory effects against the host's immune system mediated by a secreted glycoprotein component in filoviruses, another class of hemorrhagic fever-causing viruses. A significant fraction of shed glycoprotein isoforms during viral infection and biogenesis may attenuate the host's inflammatory response, thereby enhancing viral replication and tissue damage. Such shed glycoprotein mediated effects were previously reported for Ebola virus (EBOV), a filovirus that also causes hemorrhagic fever with nearly 90% fatality rates [3-5]. The identification of an analogous phenomenon in vivo could establish a new correlate of LHF infection leading to the development of sensitive diagnostics targeting the earliest molecular events of the disease. Additionally, the reversal of potentially untoward immunomodulatory functions mediated by sGP1 could potentiate the development of novel therapeutic intervention. To this end, we investigated the presence of sGP1 in the serum of suspected LASV patients admitted to the Kenema Government Hospital (KGH) Lassa Fever Ward (LFW), in Kenema, Sierra Leone that tested positive for viral antigen or displayed classical signs of Lassa fever. Results It is reasonable to expect that a narrow window exists for detection of sGP1 as the

  3. Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

    PubMed

    Asfor, A S; Wakeley, P R; Drew, T W; Paton, D J

    2014-08-30

    Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Induction of apoptosis and reversal of permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh2

    PubMed Central

    Zhang, Hui; Gong, Jian; Zhang, Huilai; Kong, Di

    2015-01-01

    Multidrug resistance is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs, and permeability glycoprotein derived from the overexpression of multidrug resistance gene 1 has been taken as the most significant cause of multidrug resistance. In the present study, ginsenoside Rh2 was used to reverse permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM cell line. Effects of ginsenoside Rh2 on the apoptotic process and caspase-3 activity of MCF-7 and MCF-7/ADM cell lines were determined using flow cytometry and microplate reader. Methyl thiazolyl tetrazolium test was conducted to assess the IC50 values of ginsenoside Rh2 and adriamycin on MCF-7 and MCF-7/ADM cultures; Rhodamin 123 assay was used to assess the retention of permeability glycoprotein after ginsenoside Rh2 treatment; flow cytometry and real time polymerase chain reaction were used to determine the expression levels of permeability glycoprotein and multidrug resistance gene 1 in drug-resistant cells and their parental cells after exposure to ginsenoside Rh2. The results showed that ginsenoside Rh2, except for inducing apoptosis, had the ability to reverse multidrug resistance in MCF-7/ADM cell line without changing the expression levels of permeability glycoprotein and multidrug resistance gene 1. Our findings provided some valuable information for the application of ginsenoside Rh2 in cancer therapy, especially for multidrug resistance reversal in clinic. PMID:26191135

  5. Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

    PubMed Central

    Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z. Hong; Broder, Christopher C.; Aguilar, Hector C.; Nikolov, Dimitar B.

    2015-01-01

    Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein. PMID:26646856

  6. Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly.

    PubMed

    Xu, Kai; Chan, Yee-Peng; Bradel-Tretheway, Birgit; Akyol-Ataman, Zeynep; Zhu, Yongqun; Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z Hong; Broder, Christopher C; Aguilar, Hector C; Nikolov, Dimitar B

    2015-12-01

    Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.

  7. A Study of Anti Beta-2 Glycoprotein I and Anti-Prothrombin Antibodies in Patients with Unexplained Recurrent Pregnancy Losses.

    PubMed

    Singh, Angad; Nangia, Anita; Sharma, Sunita; Puri, Manju

    2016-06-01

    To compare the levels of IgG and IgM anti beta-2 glycoprotein I antibodies and IgG and IgM anti prothrombin antibodies among women with unexplained recurrent pregnancy losses and women with at least 2 live issues. To compare the prevalence of newer anti beta-2 glycoprotein I & anti prothrombin antibodies with conventional Lupus anticoagulant & anticardiolipin antibodies. 50 women with recurrent pregnancy losses & 50 matched controls were evaluated for the presence of: Lupus anticoagulant-screened by LA sensitive aPTT& DRVV and confirmatory Staclot Assay. ELISA kits were used for detecting IgG & IgM anticardiolipin, anti beta-2 glycoprotein I & anti prothrombin antibodies. 11/50 (22 %) women in study group and none in control group had circulating antiphospholipid antibodies. 2 cases (4 %) had lupus anticoagulant. 1 case (2 %) had anticardiolipin antibody & 6 cases (12 %) were positive for anti beta-2 Glycoprotein I antibody (p value = 0.027). 3 cases (6 %) had anti prothrombin antibody. All were mutually exclusive except for one. Women with recurrent pregnancy losses should be tested for anti beta-2 Glycoprotein I antibodies & anti prothrombin antibodies in addition to conventional lupus anticoagulant and anticardiolipin antibodies. This approach can decrease the incidence of SNAP (seronegative antiphospholipid syndrome) cases while establishing the true prevalence of antiphospholipid syndrome.

  8. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

    PubMed

    Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

    2016-07-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.

  9. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica.

    PubMed

    Roehrig, J T; Bolin, R A; Kelly, R G

    1998-07-05

    Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.

  10. Characterization of the Bas-Congo virus glycoprotein and its function in pseudotyped viruses.

    PubMed

    Steffen, Imke; Liss, Nathan M; Schneider, Bradley S; Fair, Joseph N; Chiu, Charles Y; Simmons, Graham

    2013-09-01

    Bas-Congo virus (BASV) is a novel rhabdovirus recently identified from a patient with acute hemorrhagic fever in the Bas-Congo province of the Democratic Republic of Congo (DRC). Here we show that the BASV glycoprotein (BASV-G) can be successfully used to pseudotype glycoprotein-deficient vesicular stomatitis virus (VSV), allowing studies of BASV-G-driven membrane fusion and viral entry into target cells without replication-competent virus. BASV-G displayed broad tissue and species tropism in vitro, and BASV-G-mediated membrane fusion was pH dependent. The conformational changes induced in BASV-G by acidification were fully reversible and did not lead to inactivation of the viral fusion protein. Our data combined with comparative sequence similarity analyses suggest that BASV-G shares structural and functional features with other rhabdovirus glycoproteins and falls into the group of class III viral fusion proteins. However, activation of BASV-G-driven fusion required a lower pH and higher temperatures than did VSV-G-mediated fusion. Moreover, in contrast to VSV-G, mature BASV-G in VSV pseudotypes consists of a mixture of high-mannose and complex glycans that enables it to bind to certain C-type lectins, thereby enhancing its attachment to target cells. Taken together, the results presented in this study will facilitate future investigations of BASV-G-mediated cell entry and its inhibition in the absence of an infectious cell culture assay for BASV and at lower biosafety levels. Moreover, serology testing based on BASV-G pseudotype neutralization can be used to uncover the prevalence and importance of BASV as a potential novel human pathogen in the DRC and throughout Central Africa.

  11. Structure of a core fragment of glycoprotein H from pseudorabies virus in complex with antibody

    PubMed Central

    Backovic, Marija; DuBois, Rebecca M.; Cockburn, Joseph J.; Sharff, Andrew J.; Vaney, Marie-Christine; Granzow, Harald; Klupp, Barbara G.; Bricogne, Gerard; Mettenleiter, Thomas C.; Rey, Felix A.

    2010-01-01

    Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N-terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a PrV gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially functional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended “flap” masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane. The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a receptor-triggered conformational change of gH, exposing a hydrophobic surface to interact with the viral membrane during the fusion process. PMID:21149698

  12. Beneficial effects of tyrosol on altered glycoprotein components in streptozotocin-induced diabetic rats.

    PubMed

    Chandramohan, Ramasamy; Saravanan, Settu; Pari, Leelavinothan

    2017-12-01

    Olive oil is the major source of tyrosol which is a natural phenolic antioxidant. Olive oil constitutes a major component of the Mediterranean diet that is linked to a reduced incidence of chronic diseases. This study evaluates the effects of tyrosol on altered glycoprotein components in streptozotocin-induced diabetic rats. Diabetes mellitus was induced in male Wistar rats by streptozotocin (40 mg/kg body weight). These rats were administered tyrosol (20 mg/kg body weight) and glibenclamide (600 μg/kg body weight) orally daily for 45 days. Plasma glucose, plasma insulin, glycoprotein components such as hexose, hexosamine, sialic acid and fucose in the plasma, liver and kidney, and histopathogy of tissues were analyzed. Diabetic rats revealed significant (p < 0.05) increase in the levels of glucose, hexose, hexosamine, sialic acid and fucose (277.17, 152.45, 100.43, 79.69 and 49.29 mg/dL) in the plasma; decrease in the levels of palsma insulin (6.12 μU/mL) and sialic acid (4.36 and 5.03 mg/g) in the liver and kidney; significant (p < 0.05) increase in hexose (49.33 and 46.82 mg/g), hexosamine (22.68 and 33.20 mg/g) and fucose (31.63 and 32.44 mg/g) in the liver and kidney. Further, periodic acid-Schiff staining of tissues revealed positive-stain accumulation in diabetic rats. Tyrosol treatment showed significant (p < 0.05) effects on all the biochemical parameters and histopathology studied in streptozotocin- nduced diabetic rats. Also, the in vitro study revealed the antioxidant effect of tyrosol. Thus, tyrosol protects streptozotocin-induced diabetic rats from the altered glycoprotein components. Further, this study can be extrapolated to humans.

  13. Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

    PubMed Central

    Lech, Patrycja J.; Tobin, Gregory J.; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D.; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P.; Russell, Stephen J.; Nara, Peter L.

    2013-01-01

    The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

  14. Hepatitis C Virus Envelope Glycoprotein Fitness Defines Virus Population Composition following Transmission to a New Host

    PubMed Central

    Brown, Richard J. P.; Hudson, Natalia; Wilson, Garrick; Rehman, Shafiq Ur; Jabbari, Sara; Hu, Ke; Tarr, Alexander W.; Borrow, Persephone; Joyce, Michael; Lewis, Jamie; Zhu, Lin Fu; Law, Mansun; Kneteman, Norman; Tyrrell, D. Lorne; McKeating, Jane A.

    2012-01-01

    Genetic variability is a hallmark of RNA virus populations. However, transmission to a new host often results in a marked decrease in population diversity. This genetic bottlenecking is observed during hepatitis C virus (HCV) transmission and can arise via a selective sweep or through the founder effect. To model HCV transmission, we utilized chimeric SCID/Alb-uPA mice with transplanted human hepatocytes and infected them with a human serum HCV inoculum. E1E2 glycoprotein gene sequences in the donor inoculum and recipient mice were determined following single-genome amplification (SGA). In independent experiments, using mice with liver cells grafted from different sources, an E1E2 variant undetectable in the source inoculum was selected for during transmission. Bayesian coalescent analyses indicated that this variant arose in the inoculum pretransmission. Transmitted variants that established initial infection harbored key substitutions in E1E2 outside HVR1. Notably, all posttransmission E1E2s had lost a potential N-linked glycosylation site (PNGS) in E2. In lentiviral pseudoparticle assays, the major posttransmission E1E2 variant conferred an increased capacity for entry compared to the major variant present in the inoculum. Together, these data demonstrate that increased envelope glycoprotein fitness can drive selective outgrowth of minor variants posttransmission and that loss of a PNGS is integral to this improved phenotype. Mathematical modeling of the dynamics of competing HCV variants indicated that relatively modest differences in glycoprotein fitness can result in marked shifts in virus population composition. Overall, these data provide important insights into the dynamics and selection of HCV populations during transmission. PMID:22855498

  15. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc

    PubMed Central

    Barriga, Gonzalo P.; Villalón-Letelier, Fernando; Márquez, Chantal L.; Bignon, Eduardo A.; Acuña, Rodrigo; Ross, Breyan H.; Monasterio, Octavio; Mardones, Gonzalo A.; Vidal, Simon E.; Tischler, Nicole D.

    2016-01-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses

  16. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host.

    PubMed

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q; Kilcoyne, Michelle; Hokke, Cornelis H; Dalton, John P; O'Neill, Sandra M

    2016-10-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Proteomic analysis of platelet N-glycoproteins in PMM2-CDG patients.

    PubMed

    de la Morena-Barrio, M E; Di Michele, M; Lozano, M L; Rivera, J; Pérez-Dueñas, B; Altisent, C; Sevivas, T; Vicente, V; Jaeken, J; Freson, K; Corral, J

    2014-03-01

    PMM2-CDG, the most frequent congenital disorder of N-glycosylation, is an autosomal recessive disease with a multisystem presentation. PMM2-CDG patients show an increased risk for thrombosis, which might be in part due to spontaneous platelet aggregations as previously described. A potential hypoglycosylation of platelet proteins in these patients might explain this increased reactivity, as removal of sialic acid from platelets, particularly of GPIbα, leads to enhance platelet aggregation and clearance from the circulation. This study is the first one that has evaluated the glycosylation status of platelet proteins in 6 PMM2-CDG patients using different approaches including immunoblot, RCA120 lectin binding to platelets and expression of different membrane platelet N-glycoproteins by flow cytometry, as well as by platelet N-glycoproteome analysis. RCA120 lectin binding to the platelet membrane of PMM2-CDG patients showed evidence for decreased sialic acid content. However, immunoblot and flow cytometric analysis of different platelet N-glycoproteins, together with the more sensitive 2D-DIGE analysis, suggest that platelet N-glycoproteins, including GPIbα, seem to be neither quantitatively nor qualitatively significantly affected. The increased binding of RCA120 lectin could be explained by the abnormal glycosylation of hepatic proteins being attached to the platelets. This is the first study that has evaluated the platelet N-glycoproteome. Our findings suggest that platelet proteins are not significantly affected in PMM2-CDG patients. Further studies are still warranted to unravel the mechanism(s) that increase(s) the risk of thrombosis in these patients. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host*

    PubMed Central

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M.; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q.; Kilcoyne, Michelle; Hokke, Cornelis H.; Dalton, John P.; O'Neill, Sandra M.

    2016-01-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. PMID:27466253

  19. Serological diagnosis and prognosis of severe acute pancreatitis by analysis of serum glycoprotein 2.

    PubMed

    Roggenbuck, Dirk; Goihl, Alexander; Hanack, Katja; Holzlöhner, Pamela; Hentschel, Christian; Veiczi, Miklos; Schierack, Peter; Reinhold, Dirk; Schulz, Hans-Ulrich

    2017-05-01

    Glycoprotein 2 (GP2), the pancreatic major zymogen granule membrane glycoprotein, was reported to be elevated in acute pancreatitis in animal models. Enzyme-linked immunosorbent assays (ELISAs) were developed to evaluate human glycoprotein 2 isoform alpha (GP2a) and total GP2 (GP2t) as specific markers for acute pancreatitis in sera of 153 patients with acute pancreatitis, 26 with chronic pancreatitis, 125 with pancreatic neoplasms, 324 with non-pancreatic neoplasms, 109 patients with liver/biliary disease, 67 with gastrointestinal disease, and 101 healthy subjects. GP2a and GP2t levels were correlated with procalcitonin and C-reactive protein in 152 and 146 follow-up samples of acute pancreatitis patients, respectively. The GP2a ELISA revealed a significantly higher assay accuracy in contrast to the GP2t assay (sensitivity ≤3 disease days: 91.7%, specificity: 96.7%, positive likelihood ratio [LR+]: 24.6, LR-: 0.09). GP2a and GP2t levels as well as prevalences were significantly elevated in early acute pancreatitis (≤3 disease days) compared to all control cohorts (p<0.05, respectively). GP2a and GP2t levels were significantly higher in patients with severe acute pancreatitis at admission compared with mild cases (p<0.05, respectively). Odds ratio for GP2a regarding mild vs. severe acute pancreatitis with lethal outcome was 7.8 on admission (p=0.0222). GP2a and GP2t levels were significantly correlated with procalcitonin [Spearman's rank coefficient of correlation (ρ)=0.21, 0.26; p=0.0110, 0.0012; respectively] and C-reactive protein (ρ=0.37, 0.40; p<0.0001; respectively). Serum GP2a is a specific marker of acute pancreatitis and analysis of GP2a can aid in the differential diagnosis of acute upper abdominal pain and prognosis of severe acute pancreatitis.

  20. Primary structure analysis of an integral membrane glycoprotein of the nuclear pore

    PubMed Central

    1989-01-01

    The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the cDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by approximately 9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes. PMID:2738089

  1. Small Integrin-Binding LIgand N-linked Glycoproteins (SIBLINGs): Multifunctional proteins in cancer

    PubMed Central

    Bellahcène, Akeila; Castronovo, Vincent; Ogbureke, Kalu U. E.; Fisher, Larry W.; Fedarko, Neal S.

    2008-01-01

    Numerous components and pathways are involved in the complex interplay between cancer cells and their environment. The family of glycophosphoproteins comprising osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein — small integrin-binding ligand N-linked glycoproteins (SIBLINGs) — are emerging as important players in many stages of cancer progression. From their detection in various human cancers to the demonstration of their key functional roles during malignant transformation, invasion and metastasis, the SIBLINGs are proteins with potential as diagnostic and prognostic tools, as well as new therapeutic targets. PMID:18292776

  2. Structural analysis of glycoproteins: building N-linked glycans with Coot.

    PubMed

    Emsley, Paul; Crispin, Max

    2018-04-01

    Coot is a graphics application that is used to build or manipulate macromolecular models; its particular forte is manipulation of the model at the residue level. The model-building tools of Coot have been combined and extended to assist or automate the building of N-linked glycans. The model is built by the addition of monosaccharides, placed by variation of internal coordinates. The subsequent model is refined by real-space refinement, which is stabilized with modified and additional restraints. It is hoped that these enhanced building tools will help to reduce building errors of N-linked glycans and improve our knowledge of the structures of glycoproteins.

  3. Bactericidal action of a glycoprotein from the body surface mucus of giant African snail.

    PubMed

    Otsuka-Fuchino, H; Watanabe, Y; Hirakawa, C; Tamiya, T; Matsumoto, J J; Tsuchiya, T

    1992-04-01

    1. Bactericidal action of a glycoprotein, Achacin, purified from the giant African snail, Achatina fulica Férussac, has been studied. 2. Achacin kills both gram-positive and gram-negative bacteria, but only in their growing states. 3. Achacin does not have any bacteriolytic activity. 4. The strain which has no cell wall is a little more sensitive than the native strain and the cell membrane-damaged strain. 5. Achacin was observed on the cytoplasmic membrane and on the cell wall of treated Escherichia coli by immunoelectron microscopy. 6. Achacin attacks the cytoplasmic membrane of the cell.

  4. Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

    PubMed

    da Fontoura Budaszewski, Renata; Hudacek, Andrew; Sawatsky, Bevan; Krämer, Beate; Yin, Xiangping; Schnell, Matthias J; von Messling, Veronika

    2017-04-15

    The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains. IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received

  5. Anterograde glycoprotein-dependent transport of newly generated rabies virus in dorsal root ganglion neurons.

    PubMed

    Bauer, Anja; Nolden, Tobias; Schröter, Josephine; Römer-Oberdörfer, Angela; Gluska, Shani; Perlson, Eran; Finke, Stefan

    2014-12-01

    Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of

  6. HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

    Cancer.gov

    The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that Env is the only viral protein expressed on HIV’s surface, knowing its detailed structure—especially in the unbound state—may be critical for designing antibodies and vaccines against HIV.

  7. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    USGS Publications Warehouse

    Kim, W.-S.; Oh, M.-J.; Nishizawa, T.; Park, J.-W.; Kurath, G.; Yoshimizu, M.

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan. ?? 2007 Springer-Verlag.

  8. Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Emmenegger, Eveline J.; Huang, C.; LaPatra, S.; Winton, James R.

    1995-01-01

    Summary ― Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

  9. Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons

    PubMed Central

    Bauer, Anja; Nolden, Tobias; Schröter, Josephine; Römer-Oberdörfer, Angela; Gluska, Shani; Perlson, Eran

    2014-01-01

    ABSTRACT Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCE Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and

  10. Characterization of glycoprotein biopharmaceutical products by Caliper LC90 CE-SDS gel technology.

    PubMed

    Chen, Grace; Ha, Sha; Rustandi, Richard R

    2013-01-01

    Over the last decade, science has greatly improved in the area of protein sizing and characterization. Efficient high-throughput methods are now available to substitute for the traditional labor-intensive SDS-PAGE methods, which alternatively take days to analyze a very limited number of samples. Currently, PerkinElmer(®) (Caliper) has designed an automated chip-based fluorescence detection method capable of analyzing proteins in minutes with sensitivity similar to standard SDS-PAGE. Here, we describe the use and implementation of this technology to characterize and screen a large number of formulations of target glycoproteins in the 14-200 kDa molecular weight range.

  11. Myelin oligodendrocyte glycoprotein has a dual role in T cell autoimmunity against central nervous system myelin.

    PubMed

    't Hart, Bert A; Weissert, Robert

    2016-01-01

    Myelin oligodendrocyte glycoprotein (MOG) is a candidate primary target of the autoimmune attack on the central nervous system (CNS) in multiple sclerosis (MS). However, the physiological function of MOG has been unclear for a long time. We propose that MOG has a central role in the regulation of tolerance and autoimmunity. The interaction of MOG with DC-SIGN, an innate antigen receptor of myeloid antigen-presenting cells (m-APCs), present inside the CNS (microglia) or in draining lymph nodes (dendritic cells; DCs), keeps these cells in an immature/tolerogenic state. We postulate that this tolerogenic mechanism may be disturbed in MS by unknown factors.

  12. Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid glycoprotein. Spectroscopic and molecular docking approach.

    PubMed

    Ajmal, Mohammad Rehan; Nusrat, Saima; Alam, Parvez; Zaidi, Nida; Khan, Mohsin Vahid; Zaman, Masihuz; Shahein, Yasser E; Mahmoud, Mohamed H; Badr, Gamal; Khan, Rizwan Hasan

    2017-02-20

    The binding interaction between clofarabine, an important anticancer drug and two important carrier proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein (AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is quenched by clofarabine and the static mode of fluorescence quenching is operative. UV-vis spectroscopy deciphered the formation of ground state complex between anticancer drug and the two studied proteins. Clofarabine was found to bind at 298K with both AAG and HSA with the binding constant of 8.128×10 3 and 4.120×10 3 for AAG and HSA, respectively. There is stronger interaction of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Arsenite Regulates Prolongation of Glycan Residues of Membrane Glycoprotein: A Pivotal Study via Wax Physisorption Kinetics and FTIR Imaging

    PubMed Central

    Lee, Chih-Hung; Hsu, Chia-Yen; Huang, Pei-Yu; Chen, Ching-Iue; Lee, Yao-Chang; Yu, Hsin-Su

    2016-01-01

    Arsenic exposure results in several human cancers, including those of the skin, lung, and bladder. As skin cancers are the most common form, epidermal keratinocytes (KC) are the main target of arsenic exposure. The mechanisms by which arsenic induces carcinogenesis remains unclear, but aberrant cell proliferation and dysregulated energy homeostasis play a significant role. Protein glycosylation is involved in many key physiological processes, including cell proliferation and differentiation. To evaluate whether arsenite exposure affected protein glycosylation, the alteration of chain length of glycan residues in arsenite treated skin cells was estimated. Herein we demonstrated that the protein glycosylation was adenosine triphosphate (ATP)-dependent and regulated by arsenite exposure by using Fourier transform infrared (FTIR) reflectance spectroscopy, synchrotron-radiation-based FTIR (SR-FTIR) microspectroscopy, and wax physisorption kinetics coupled with focal-plane-array-based FTIR (WPK-FPA-FTIR) imaging. We were able to estimate the relative length of surface protein-linked glycan residues on arsenite-treated skin cells, including primary KC and two skin cancer cell lines, HSC-1 and HaCaT cells. Differential physisorption of wax adsorbents adhered to long-chain (elongated type) and short-chain (regular type) glycan residues of glycoprotein of skin cell samples treated with various concentration of arsenite was measured. The physisorption ratio of beeswax remain/n-pentacosane remain for KC cells was increased during arsenite exposure. Interestingly, this increase was reversed after oligomycin (an ATP synthase inhibitor) pretreatment, suggesting the chain length of protein-linked glycan residues is likely ATP-dependent. This is the first study to demonstrate the elongation and termination of surface protein-linked glycan residues using WPK-FPA-FTIR imaging in eukaryotes. Herein the result may provide a scientific basis to target surface protein-linked glycan

  14. Long-term disability and prognostic factors in polyneuropathy associated with anti-myelin-associated glycoprotein (MAG) antibodies.

    PubMed

    Galassi, Giuliana; Tondelli, Manuela; Ariatti, Alessandra; Benuzzi, Francesca; Nichelli, Paolo; Valzania, Franco

    2017-05-01

    Neuropathy associated with IgM monoclonal gammopathy (MGUS) represents distinctive clinical syndrome, characterized by male predominance, late age of onset, slow progression, predominantly sensory symptoms, deep sensory loss, ataxia, minor motor impairment. More than 50% of patients with neuropathy-associated MGUS possess antibodies against myelin-associated glycoprotein (MAG). Purpose of our study was to assess effects on disease progression of demographic, clinical and neurophysiological variables in our large cohort of patients. Forty-three Caucasians patients were followed every eight months for median duration time of