Sample records for alpha 1-acid glycoprotein

  1. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S


    glycoprotein glycoforms to their receptors is inhibited by steroids. Testosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoform A to its receptor. Cortisone, aldosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoforms B......A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...... and C to their receptor. A difference in the cellular content of alpha 1-acid glycoprotein glycoforms and alpha 1-acid glycoprotein receptors separates the spermatocytes and the early spermatids from the late spermatids. The difference in receptor composition implies a difference in the effect...

  2. Zonal variation in the distribution of an alpha 1-acid glycoprotein glycoform receptor in human adrenal cortex

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S


    Using a histochemical technique with three different alpha 1-acid glycoprotein glycoform one glycoform specific receptor has been identified in human adrenal cortex. The receptor is associated to alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C. The glycoform specific...... specific receptor. The binding of alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C to the glycoform specific receptor is inhibited by the steroid hormones cortisone, aldosterone, estradiol and progesterone but not by testosterone. The pronounced changes in the distribution....... The binding activity was dependent on the presence of calcium ions and not on thiol reagents. Thus the lectin-like receptor may belong to the C-type lectin family. Using an antibody to alpha 1-acid glycoprotein the presence of alpha 1-acid glycoprotein was observed in the same location as the glycoform...

  3. Alpha 1-acid glycoprotein has immunomodulatory effects in neonatal swine adipose tissue (United States)

    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...

  4. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket. (United States)

    Albani, Jihad R


    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.

  5. Alpha1-acid glycoprotein post-translational modifications: a comparative two dimensional electrophoresis based analysis

    Directory of Open Access Journals (Sweden)

    P. Roncada


    Full Text Available Alpha1-acid glycoprotein (AGP is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. A proteomic approach based on two dimensional electrophoresis, immunoblotting and staining of 2DE gels with dyes specific for post-translational modifications (PTMs such as glycosylation and phosphorylation has been used to evaluate the differential interspecific protein expression of AGP purified from human, bovine and ovine sera. By means of these techniques, several isoforms have been identified in the investigated species: they have been found to change both with regard to the number of isoforms expressed under physiological condition and with regard to the quality of PTMs (i.e. different oligosaccharidic chains, presence/absence of phosphorilations. In particular, it is suggested that bovine serum AGP may have one of the most complex pattern of PTMs among serum proteins of mammals studied so far.

  6. Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene. (United States)

    Merritt, C M; Board, P G


    Human alpha 1-acid glycoprotein (AGP), also known as orosomucoid, is a major acute-phase plasma protein. The amino acid sequence of AGP, which was determined by sequencing from protein isolated from pooled plasma, contained amino acid substitutions in 21 different positions. Genomic and cDNA clones which correspond to one of the possible amino acid sequences have been previously reported. In this paper we present the complete nucleotide sequence of a second gene, AGP2 which is located approx. 3.3 kb downstream from AGP1. The derived amino acid sequence of AGP2 contains 19 of the possible alternative amino acid substitutions as well as two additional differences. It is clear from the results presented here that the AGP in human plasma is the product of two separate gene loci.

  7. Evolution of human alpha 1-acid glycoprotein genes and surrounding Alu repeats. (United States)

    Merritt, C M; Easteal, S; Board, P G


    There is a mosaic pattern of variation between the two tandemly arranged human alpha 1-acid glycoprotein genes. Both the synonymous and the nonsynonymous sites of exons 3 and 4 are more divergent than the rest of the gene, suggesting that they have had a different evolutionary history. Comparisons of the two gene sequences with rat AGP indicate that exons 3 and 4 of AGP2 have been evolving without functional constraint since their divergence from AGP1. It is proposed that the conserved region of the gene has been homogenized recently by gene conversion with the homologous regions of AGP1. The Alu sequences surrounding the genes appear to have been involved in both the gene duplication and the gene conversion events.

  8. Two lectin-like receptors for alpha 1-acid glycoprotein in mouse testis

    DEFF Research Database (Denmark)

    Andersen, U O; Kirkeby, S; Bøg-Hansen, T C


    Three glycoforms of alpha 1-acid glycoprotein (AGP) were biotinylated to examine their binding in mouse testis by light microscopy. The transition from one stage to another in the spermatogenic cycle is marked with an appearance of a receptor for the Concanavalin A (Con A) non-reactive glycoform...... AGP-A in the cytoplasm of spermatocytes, young spermatids and Sertoli cells. This receptor disappears in the late stages of the spermatids. The Con-A intermediately reactive and the Con-A reactive glycoforms, AGP-B and AGP-C, showed weak reaction in the cytoplasm of spermatocytes, spermatids....... AGP-A was inhibited by testosterone, oestradiol and progesterone, while AGP-B and AGP-C were inhibited by mannose, GlcNAc, cortisone, aldosterone, oestradiol and progesterone. The receptors and the corresponding AGP glycoforms may be adhesion molecules between Sertoli cells and the spermatogenic cells...

  9. Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein. (United States)

    Albani, Jihad R


    We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.

  10. Characterization of genomic rearrangements of the alpha1-acid glycoprotein/orosomucoid gene in Ghanaians. (United States)

    Yuasa, I; Nakamura, H; Henke, L; Henke, J; Nakagawa, M; Irizawa, Y; Umetsu, K


    In this study, the structure of the alpha1-acid glycoprotein (AGP), or orosomucoid (ORM), gene was investigated in a Ghanaian mother and her child, who shared an unusual variant, ORM1 S2(C), found by isoelectric focusing. Three remarkable changes of nucleotide sequence were observed: (1) The two ORM1 alleles, ORMI*S and ORMI*S2(C), had the AGP2 gene-specific sequence at one and three regions, respectively, in exon 5 to intron 5. The variant allele originating from ORMi*S was characterized by a G-to-A transition, resulting in an amino acid change from valine to methionine, which is also detected in ORM1 F2, a form that is common in Europeans. (2) The AGP2 gene of the child, inherited from the father, was duplicated, as revealed by long-range polymerase chain reaction. (3) Three new mutations were observed in two exons of the AGP2 genes of the mother and child. All of these novel genomic rearrangements, which were not observed in Japanese subjects, may have arisen through point mutation, gene conversion, and unequal crossover events. It is likely that the rearrangement of the AGP gene has often occurred in Africans.

  11. Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism. (United States)

    Matsunaga, Hisami; Sadakane, Yutaka; Haginaka, Jun


    Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.

  12. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture. (United States)

    Caperna, T J; Shannon, A E; Stoll, M; Blomberg, L A; Ramsay, T G


    Alpha-1 acid glycoprotein (AGP, orosomucoid, ORM-1) is a highly glycosylated mammalian acute-phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute-phase protein in this species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

  13. Lectin-like receptor for alpha 1-acid glycoprotein in the epithelium of the rat prostate gland and seminal vesicles

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S


    BACKGROUND: A receptor for alpha 1-acid glycoprotein glycoforms AGP-B and AGP-C in the epithelium of the rat prostate gland and seminal vesicles is described. METHODS: The interaction between AGP-glycoforms and their receptor is a lectin-like interaction confirmed by inhibition of the binding...... in rat prostate and seminal vesicles. The localization of the AGP lectin receptor is compared to the localization of glycoprotein AGP, and small differences are found. CONCLUSIONS: It is proposed the AGP receptors in the prostate and seminal vesicles belong to a group of lectins in the control...

  14. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors. (United States)

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László


    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  15. Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies. (United States)

    Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S


    A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

  16. alpha1-acid glycoprotein as a putative biomarker for monitoring the development of the type II reactional stage of leprosy. (United States)

    Gupta, Nishma; Shankernarayan, Nallakandy P; Dharmalingam, Kuppamuthu


    Leprosy, a spectral disease manifested on the basis of host immune responses, is complicated by its reactional stages, namely type I reversal reaction (RR) and type II erythema nodosum leprosum (ENL). These reactional stages are characterized by uncontrolled and aberrant immune responses. Biomarkers for reactional stages would aid in early diagnosis, efficient treatment, prevention of neurological complications and prediction of predisposition to reactional stages. In this study, comparative analysis of the serum proteome of leprosy patients by two-dimensional electrophoresis (2DE) followed by mass spectrometry showed differential expression of acute-phase protein alpha (1)-acid glycoprotein (AGP; also known as orosomucoid). AGP levels in untreated ENL cases were significantly higher than in lepromatous leprosy (LL; a non-reactional disease stage) (P=0.0126), RR (P=0.0176) and healthy controls (P=0.0030). These data were confirmed using ELISA. The levels of AGP decreased to normal levels after treatment with multidrug therapy and thalidomide (P =0.0167). In a follow-up study, AGP levels, which were high in the untreated ENL stage, decreased significantly at 5 days ( P=0.0084) and 21 days (P=0.0027) post-treatment. A stage-dependent increase in AGP in an LL patient who progressed into the ENL stage was also shown. Glycosylation analysis by 2DE showed differential expression of acidic glycoforms of AGP in untreated ENL cases. Changes in AGP concentration and differential expression of isoforms correlated with the inflammatory condition in ENL and also with the treatment regimen. Thus, initial validation of AGP as an ENL-specific biomarker and treatment indicator was shown in this study.

  17. Förster energy-transfer studies between Trp residues of alpha1-acid glycoprotein (orosomucoid) and the glycosylation site of the protein. (United States)

    Albani, Jihad R


    Energy-transfer studies between Trp residues of alpha(1)-acid glycoprotein and the fluorescent probe Calcofluor White were performed. Calcofluor White interacts with carbohydrate residues of the protein, while the three Trp residues are located at the surface (Trp-160) and in hydrophobic domains of the protein (Trp-25 and Trp-122). Binding of Calcofluor to the protein induces a decrease in the fluorescence intensity of the Trp residues accompanied by an increase of that of Calcofluor White. Efficiency (E) of Trp fluorescence quenching was determined to be equal to 45%, and the Förster distance R(o), at which the efficiency of energy transfer is 50%, was calculated to be 18.13 A. This low distance and the value of the efficiency clearly indicate that energy transfer between Trp residues and Calcofluor White is weak.

  18. Exogenous alpha-1-acid glycoprotein protects against renal ischemia-reperfusion injury by inhibition of inflammation and apoptosis

    NARCIS (Netherlands)

    de Vries, B; Walter, SJ; Wolfs, TGAM; Hochepied, T; Rabina, J; Heeringa, P; Parkkinen, J; Libert, C; Buurman, WA


    Background. Although ischemia-reperfusion (I/R) injury represents a major problem in posttransplant organ failure, effective treatment is not available. The acute phase protein a-l-acid glycoprotein (AGP) has been shown to be protective against experimental I/R injury. The effects of AGP are thought

  19. In Vivo Clearance of Alpha-1 Acid Glycoprotein Is Influenced by the Extent of Its N-Linked Glycosylation and by Its Interaction with the Vessel Wall

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    Teresa R. McCurdy


    Full Text Available Alpha-1 acid glycoprotein (AGP is a highly glycosylated plasma protein that exerts vasoprotective effects. We hypothesized that AGP’s N-linked glycans govern its rate of clearance from the circulation, and followed the disappearance of different forms of radiolabeled human AGP from the plasma of rabbits and mice. Enzymatic deglycosylation of human plasma-derived AGP (pdAGP by Peptide: N-Glycosidase F yielded a mixture of differentially deglycosylated forms (PNGase-AGP, while the introduction of five Asn to Gln mutations in recombinant Pichia pastoris-derived AGP (rAGP-N(5Q eliminated N-linked glycosylation. PNGase-AGP was cleared from the rabbit circulation 9-fold, and rAGP-N(5Q, 46-fold more rapidly than pdAGP, primarily via a renal route. Pichia pastoris-derived wild-type rAGP differed from pdAGP in expressing mannose-terminated glycans, and, like neuraminidase-treated pdAGP, was more rapidly removed from the rabbit circulation than rAGP-N(5Q. Systemic hyaluronidase treatment of mice transiently decreased pdAGP clearance. AGP administration to mice reduced vascular binding of hyaluronic acid binding protein in the liver microcirculation and increased its plasma levels. Our results support a critical role of N-linked glycosylation of AGP in regulating its in vivo clearance and an influence of a hyaluronidase-sensitive component of the vessel wall on its transendothelial passage.

  20. The rearrangement of the human alpha(1)-acid glycoprotein/orosomucoid gene: evidence for tandemly triplicated genes consisting of two AGP1 and one AGP2. (United States)

    Nakamura, H; Yuasa, I; Umetsu, K; Nakagawa, M; Nanba, E; Kimura, K


    The human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is controlled by the two tandemly arranged genes, AGP1 and AGP2. The further duplication of the AGP1 gene has been suggested by a few duplicated ORM1 locus haplotypes including ORM1*F1. S and ORM1*B9. S, detected by isoelectric focusing. To clarify the triplication of the AGP gene, 39 DNA samples from Japanese subjects were studied by the long-range PCR of intergenic regions. The analysis of PCR products showed that the tandemly triplicated genes, AGP1A-AGP1B-AGP2, occurred on about 20% of chromosomes. These composites were divided into ORM1A*F1-ORM1B*S-ORM2*M and ORM1A*B9-ORM1B*S-ORM2*M by allelic variations. Furthermore, the former was classified into a few haplotypes by three synonymous sequence variations, which might have arisen through gene conversion-like events. The recombination breakpoints existed between the 5' flanking region and intron 2 of the AGP1B gene. Thus, it is likely that the rearrangement of the AGP gene has often occurred.

  1. Retention behaviour of proteins on poly(vinylimidazole)-copper(II) complexes supported on silica: application to the fractionation of desialylated human alpha 1-acid glycoprotein variants. (United States)

    Millot, M C; Hervé, F; Sébille, B


    The retention behaviour of various amino acids, peptides and proteins on poly(vinylimidazole)-Cu(II) complexes supported on silica was investigated. Free amino acids and peptides containing one histidine and in some instances one additional tryptophan residue in their primary structure were found to elute from the supports only after addition of a competing complexing agent to the mobile phase. However, the results obtained the proteins containing metal binding groups suggested that, in addition to the presence of donor-acceptor interactions between the macromolecules and the immobilized metal, other additional (essentially ionic and/or hydrophobic) interactions took place between the proteins and the surrounding of the metal. When donor-acceptor interactions were predominant, proteins were strongly adsorbed on the stationary phase and their elution required the addition of a competing complexing agent in the mobile phase. However, when the binding between the proteins and the supports via donor-acceptor interactions was less favourable, proteins were eluted from the columns without the addition of a competing agent in the mobile phase. With respect to the binding of these proteins, ionic and/or hydrophobic interactions were no longer negligible during the chromatographic process and the retention of the macromolecules by the stationary phase depended on the elution conditions (ionic strength, pH, etc.). These supports were used in the fractionation of the three main genetic variants of desialylated alpha 1-acid glycoprotein.

  2. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography. (United States)

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S


    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  3. Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues. (United States)

    Albani, Jihad René


    We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.

  4. Critical assessment of the diagnostic value of feline alpha1-acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach. (United States)

    Paltrinieri, Saverio; Saverio, Paltrinieri; Giordano, Alessia; Alessia, Giordano; Tranquillo, Vito; Vito, Tranquillo; Guazzetti, Stefano; Stefano, Guazzetti


    Alpha-1-acid glycoprotein (AGP) increases in the blood of cats with feline infectious peritonitis (FIP), a lethal disease caused by feline coronavirus (FCoV). However, the diagnostic potential of AGP might be limited because AGP also increases in pathophysiological conditions other than FIP. In this retrospective study, the diagnostic potential of serum AGP concentration was evaluated on the basis of the pretest probability of disease, according to the Bayesian approach. Serum AGP levels from cats with FIP (group 1; n = 58) and without FIP (group 2; n = 104) were evaluated. Non-FIP cats were further subgrouped as follows: 2a) inflammation (n = 26), 2b) asymptomatic FCoV infection (n = 49), 2c) injection-site sarcoma (n = 19), 2d) postvaccination (n = 7), and 2e) specific pathogen free (n = 3). Standard descriptive analyses by group and empirical receiver-operating characteristic (ROC) curve estimation were performed. Ordinary logistic regression analysis was performed to derive an estimate of the continuous likelihood ratio to produce the posttest probability of disease for any combination of pretest probability and serum AGP value. The comparison of serum AGP levels in the different groups and the analysis of the ROC curve confirmed that serum AGP is a powerful discriminating marker for FIP. The Bayesian approach demonstrated that when the pretest probability of FIP is high, based on history and clinical signs (groups 1 or 2a), moderate serum AGP levels (1.5-2 mg/ml) can discriminate cats with FIP from others, while only high serum AGP levels (>3 mg/ml) can support a diagnosis of FIP in cats with a low pretest probability of disease (groups 2b to 2e).

  5. The immune system modulator a1-acid glycoprotein inhibits insulin and IGF1 induced protein synthesis in C2C12 myotubes (United States)

    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...

  6. Reverse lectin ELISA for detecting fucosylated forms of α1-acid glycoprotein associated with hepatocellular carcinoma (United States)

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


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


    Institute of Scientific and Technical Information of China (English)

    武庆平; 姚尚龙


    目的:通过测定脐血中α1-酸性糖蛋白(alpha-1-acid glycoprotein,AAG)浓度,来比较罗哌卡因和布比卡因分娩镇痛中对胎儿的毒性作用.方法:选择42例产前检查估计能从阴道自然分娩的头位、单胎足月初产妇.随机分为三组,A组为罗哌卡因-芬太尼组;B组为布比卡因-芬太尼组;C组为未行分娩镇痛对照组,每组各14例.分娩镇痛采用病人自控硬膜外镇痛(patient-controlledepidural analgesia,PCEA),PCEA剂量为基础注药速率为6ml/h,冲击量均为2ml,锁定时间均为10min.采用"速率散射比浊法"测定脐血中α1-酸性糖蛋白浓度,进行脐血血气分析,进行胎儿的Apgar评分和SpO2检测,视觉模拟镇痛评分法(VAS)评定产妇疼痛程度,用下肢运动神经阻滞评分法(MBS)评定产妇运动神经阻滞程度.结果:两组产妇的镇痛效果无显著差异.罗哌卡因组脐血α1-酸性糖蛋白浓度和pH值明显高于布比卡因组(P<0.05).罗哌卡因组1分钟胎儿Apgar评分和胎儿血氧饱和度明显高于布比卡因组(P<0.05).布比卡因组对运动神经阻滞大于罗哌卡因组.结论:在分娩镇痛时,罗哌卡因对胎儿毒性低于布比卡因.

  8. Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS. (United States)

    Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Gun Wook; Hwang, Heeyoun; Jeong, Hoi Keun; Yun, Ki Na; Ji, Eun Sun; Kim, Kwang Hoe; Kim, Jun Seok; Kim, Jong Won; Yun, Sung Ho; Choi, Chi-Won; Kim, Seung Il; Lim, Jong-Sun; Jeong, Seul-Ki; Paik, Young-Ki; Lee, Soo-Youn; Park, Jisook; Kim, Su Yeon; Choi, Young-Jin; Kim, Yong-In; Seo, Jawon; Cho, Je-Yoel; Oh, Myoung Jin; Seo, Nari; An, Hyun Joo; Kim, Jin Young; Yoo, Jong Shin


    Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was N-glycopeptide isoforms of AGP from control and disease plasma sample.

  9. Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein. (United States)

    Yeggoni, Daniel Pushparaju; Rachamallu, Aparna; Kallubai, Monika; Subramanyam, Rajagopal


    Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission were found to be K(piperine) = 5.7 ± .2 × 10(5) M(-1) and K(piperine) = 9.3± .25 × 10(4) M(-1) which correspond to the free energy of -7.8 and -6.71 kcal M(-1)at 25 °C for HSA and AGP, respectively. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA due to partial destabilization of HSA-piperine complexes. Consequently, inference drawn from the site-specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), which was further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and free energy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the MD studies have showed that HSA-piperine complex reaches equilibration state at around 3 ns, which prove that the HSA-piperine complex is stable in nature.

  10. Interaction of the recently approved anticancer drug nintedanib with human acute phase reactant α 1-acid glycoprotein (United States)

    Abdelhameed, Ali Saber; Ajmal, Mohammad Rehan; Ponnusamy, Kalaiarasan; Subbarao, Naidu; Khan, Rizwan Hasan


    A comprehensive study of the interaction of the newly approved tyrosine kinase inhibitor, Nintedanib (NTB) and Alpha-1 Acid Glycoprotein (AAG) has been carried out by utilizing UV-Vis spectroscopy, fluorescence spectroscopy, circular dichroism, dynamic light scattering and molecular docking techniques. The obtained results showed enhancement of the UV-Vis peak of the protein upon binding to NTB with the fluorescence intensity of AAG is being quenched by NTB via the formation of ground state complex (i.e. Static quenching). Forster distance (Ro) obtained from fluorescence resonance energy transfer (FRET) is found to be 2.3 nm. The calculated binding parameters from the modified Stern-Volmer equation showed that NTB binds to AAG with a binding constant in the order of 103. Conformational alteration of the protein upon its binding to NTB was confirmed by the circular dichroism. Dynamic light scattering results showed that the binding interaction of NTB leads to the reduction in hydrodynamic radii of AAG. Dynamic molecular docking results showed that the NTB fits into the central binding cavity in AAG and hydrophobic interaction played the key role in the binding process also the docking studies were performed with methotrexate and clofarabine drugs to look into the common binding regions of these drugs on AAG molecule, it was found that five amino acid residues namely Phe 113, Arg 89, Tyr 126, Phe 48 and Glu 63 were common among the binding regions of three studied drugs this phenomenon of overlapping binding regions may influence the drug transport by the carrier molecule in turn affecting the metabolism of the drug and treatment outcome.

  11. Leucograma, proteína C reativa, alfa-1 glicoproteína ácida e velocidade de hemossedimentação na apendicite aguda Leucocyte count, C reactive protein, alpha-1 acid glycoprotein and erithrocyte sedimmentation rate in acute appendicitis

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    Bruno Ramalho de Carvalho


    ína ácida e velocidade de hemossedimentação mostraram-se pouco sensíveis e específicos. CONCLUSÕES: O leucograma e a proteína C reativa apresentam-se alterados de forma significativa nos casos de apendicite aguda, independentemente do sexo ou da faixa etária. O leucograma e, principalmente, a proteína C reativa devem ser exames considerados em indivíduos com tempo de evolução sintomática superior a 24 horas. Valores aumentados, entretanto, devem ser somados e não substituir a avaliação clínica do médico examinador. Dosagens de velocidade de hemossedimentação e da alfa-1 glicoproteína ácida não trazem auxílio ao diagnóstico da apendicite aguda.BACKGROUND: The diagnosis of acute appendicitis is clinic, but in some cases, it can present unusual symptoms. The diagnostic difficulties still lead surgeons to unnecessary laparotomies, which reach rates from 15% to 40%. Laboratory exams, then, may become important to complement appendicitis diagnosis. The leucocyte count seems to be the most important value, but measurement of acute phase proteins, specially, the C-reactive protein, is object of several studies. PATIENTS AND METHODS: This was a prospective study, involving 63 patients submitted to appendecectomies for acute appendicitis suspicion, in "Hospital das Clínicas", Federal University of Uberlândia, MG, Brazil, in whose blood were made dosages of acute phase proteins and the leucocyte count. RESULTS: The sample was composed by 44 male and 19 female patients, and the majority of them was between 11 and 30 years of age. The flegmonous type was the most freqüent (52.4%. The leucocyte count was altered in 74.6% of the cases and C-reactive protein elevation was observed in 88.9%. The alfa-1 acid glycoprotein and the erithrocyte sedimmentation rate were predominantly normal. The C-reactive protein was augmented in more than 80% of the cases in all ages. Leucocyte count and C-reactive protein were altered in 80% of the patients with the limit of 24

  12. Evaluación del efecto de la ingesta de una alta carga de ácidos grasos saturados sobre los niveles séricos de la proteína C reactiva, alfa1-antitripsina, fibrinógeno y alfa1-glicoproteína ácida en mujeres obesas Effect of a high saturated fatty acids load on serum concentrations of C-reactive protein, alpha1-antitrypsin, fibrinogen and alpha1-acid glycoprotein in obese women

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    M.ª M. Ramírez Alvarado


    en mujeres obesas. Los niveles séricos de PCR y fibrinógeno están incrementados en mujeres obesas y se correlacionan positivamente con el IMC.Obesity is associated with increased inflammation. Creactive protein (CRP and inflammation-sensitive plasma protein (ISPs are inflammatory markers. Proinflammatory process may be influenced by high saturated fatty acid intake. Objective: The aim of the present study was to evaluate the role of saturated fatty acids load on postprandial circulating levels of PCR and ISPs (alpha1-antitrypsin, alpha1-acid glucoprotein, and fibrinogen in obese women. Design: A total of 15 obese women (age = 31,7 ± 4,5 years, BMI = 37,9 ± 7,3 kg/m² and 15 lean controls women (age = 30,6 ± 4,6 years, BMI = 20,6 ± 2,6 kg/m² were recruited for this study. After and overnight fast subjects ate the fat load consisted of 75 g of fat (100% saturated fatty acid, 0% cholesterol, 5 g of carbohydrates, and 6 g of protein per m2 body surface area. Postprandial serum levels of CRP, alpha1-antitrypsin, alpha1-acid glucoprotein, and fibrinogen were measured. Anthropometry and blood biochemical parameters were measured in both groups. Results: The obese women had fasting serum PCR levels higher (p = 0,013 and fibrinogen (p = 0,04 than those of control women. Serum CRP and fibrinogen levels was positively related to body mass index (BMI in obese group. There weren't differences in fasting serum alpha1- antitrypsin levels (p = 0,40, and alpha1-acid glucoprotein (p = 0,28 levels in obese group in comparison to lean control group. Serum CRP, alpha1-antitrypsin, alpha1-acid glucoprotein, and fibrinogen did not change postprandially (p = > 0,05 difference to fasting levels. Conclusion: A high-saturated fatty acids load is not associated with serum CRP, alpha1-antitrypsin, alpha1-acid glucoprotein, and fibrinogen levels increase. Serum alpha1-antitripsin and alpha1-acid glucoprotein levels are not increased in obese women. Serum PCR and fibrinogen levels are

  13. Expression and structural-functional alterations of α-1-acid glycoprotein at the pathological state

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    Kulinich A. O.


    Full Text Available The review analyzes up-to-date knowledge on structure and biological functions of α-acid glycoprotein. The special attention is given to alterations of fucosylation, sialylation and branching of orosomucoid at the acute, chronic inflammation and oncotransformations.

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

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    Takayuki Asao


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

  15. Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

    DEFF Research Database (Denmark)

    Udby, Lene; Johnsen, Anders H; Borregaard, Niels


    CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular...

  16. Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein. (United States)

    van Dijk, W; Lasthuis, A M; van den Eijnden, D H


    CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.

  17. Zinc-alpha2-glycoprotein expression as a marker of differentiation in human oral tumors. (United States)

    Brysk, M M; Lei, G; Adler-Storthz, K; Chen, Z; Brysk, H; Tyring, S K; Arany, I


    Zinc-alpha2-glycoprotein (Znalpha2gp) is a soluble major histocompatibility complex homolog widespread in body fluids and in glandular epithelia; the authors recently demonstrated its presence in stratified epithelia. Znalpha2gp has been associated with tumor differentiation in breast cancers and other carcinomas. We compare here its gene expression in histopathologically graded oral squamous cell carcinomas and in their perilesional normals. Znalpha2gp levels are higher in the controls than in the tumors, and higher in well-differentiated tumors than in poorly differentiated ones. Markers of oral epithelial maturation (keratin K13 and involucrin) are less simply related to tumor histology.

  18. Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen

    DEFF Research Database (Denmark)

    Nieswandt, B; Brakebusch, C; Bergmeier, W


    Platelet adhesion on and activation by components of the extracellular matrix are crucial to arrest post-traumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin alpha2beta1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating...... subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of beta1 integrin on platelets has no significant effect on the bleeding time in mice. Aggregation of beta1-null platelets to native fibrillar collagen is delayed......, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, beta1-null platelets adhere to fibrillar, but not soluble collagen under static as well as low (150 s(-1)) and high (1000 s(-1)) shear flow conditions, probably through binding of alphaIIbbeta3 to von...

  19. 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. (United States)

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


    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 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 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 in amino acid metabolism caused by E. acervulina infection extended beyond reduced amino acid digestibility.

  20. Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

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    Anna Maisa

    Full Text Available BACKGROUND: Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor. CONCLUSIONS/SIGNIFICANCE: Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  1. Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

    DEFF Research Database (Denmark)

    Bergmeier, W; Bouvard, D; Eble, J A


    Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from...... that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation...... lacking both alpha(2)beta(1) integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin...

  2. Biopartitioning micellar chromatography with sodium dodecyl sulfate as a pseudo α(1)-acid glycoprotein to the prediction of protein-drug binding. (United States)

    Hadjmohammadi, Mohammadreza; Salary, Mina


    A simple and fast method is of urgent need to measure protein-drug binding affinity in order to meet the rapid development of new drugs. Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography (MLC) using micellar mobile phases in adequate experimental conditions, can be useful as an in vitro system in mimicking the drug-protein interactions. In this study, sodium dodecyl sulfate-micellar liquid chromatography (SDS-MLC) was used for the prediction of protein-drug binding based on the similar property of SDS micelles to α(1)-acid glycoprotein (AGP). The relationships between the BMC retention data of a heterogeneous set of 14 basic and neutral drugs and their plasma protein binding parameter were studied and the predictive ability of models was evaluated. Modeling of logk(BMC) of these compounds was established by multiple linear regression (MLR) and second-order polynomial models obtained in two different concentrations (0.07 and 0.09M) of SDS. The developed MLR models were characterized by both the descriptive and predictive ability (R(2)=0.882, R(CV)(2)=0.832 and R(2)=0.840, R(CV)(2)=0.765 for 0.07 and 0.09M SDS, respectively). The p values <0.01 also indicated that the relationships between the protein-drug binding and the logk(BMC) values were statistically significant at the 99% confidence level. The standard error of estimation showed the standard deviation of the regression to be 11.89 and 13.87 for 0.07 and 0.09M, respectively. The application of the developed model to a prediction set demonstrated that the model was also reliable with good predictive accuracy. The external and internal validation results showed that the predicted values were in good agreement with the experimental value.

  3. Trans-differentiation of prostatic stromal cells leads to decreased glycoprotein hormone alpha production. (United States)

    Rumpold, Holger; Mascher, Katarina; Untergasser, Gerold; Plas, Eugen; Hermann, Martin; Berger, Peter


    Age-related development of benign prostatic hyperplasia is an important health issue in developed countries. The histopathogenetic hallmark of this disease is the increase in relative and absolute numbers of smooth muscle cells (SMC). Glycoprotein hormone alpha-subunit (GPHalpha) is expressed in the human prostate, and, because of its structural similarities to other cystine knot growth factors, it has been considered to have growth regulatory functions of its own. Primary cell cultures allowing for selective cultivation of prostatic epithelial cells, fibroblasts, and SMC were established. Directed trans-differentiation and cellular homogeneity was pursued by confocal scanning laser microscopy with cell type-specific markers. GPHalpha production by these cells was assessed by immunofluorimetric assays. Its predominant source was young fibroblasts, whereas replicative senescent fibroblasts, SMC, and control fibroblasts from foreskin did not produce significant amounts. Functionally, GPHalpha reduced growth of stromal cells at concentrations of 10 and 100 nmol/liter as shown by cell viability assays. It is concluded that histogenetic reorganization over the adult lifetime, guided by endocrine factors like steroid hormones together with senescence of fibroblasts, leads to a decreased production of growth inhibitors, such as GPHalpha, favoring proliferation and the development of benign prostatic hyperplasia.

  4. Determination of competitive adsorption isotherm parameters of pindolol enantiomers on alpha1-acid glycoprotein chiral stationary phase. (United States)

    Zhang, Yan; Hidajat, Kus; Ray, Ajay K


    In this paper, inverse method (IM) was used to determine the binary competitive adsorption isotherm of pindolol enantiomers by a least-square fitting of the proposed model to the experimentally measured elution curves of racemic pindolol. The isotherm parameters were determined by minimizing the least-square error using an adaptation of genetic algorithm, non-dominated sorting genetic algorithm with jumping genes (NSGA-II-JG). An equilibrium dispersive (ED) model combined with bi-Langmuir isotherm was used in predicting the elution profiles. The determined parameters show good agreement with the experimental profiles at various experimental conditions such as sample volume, concentration and flow rates of the racemic mixture. Robustness and validity of the isotherm parameters were also verified by frontal analyses at various step inputs. Results from both the pulse tests and the frontal analysis indicate that adsorption isotherm derived from the inverse method is quite reliable. This method requires relatively less number of experiments to be performed and therefore, lower experimental costs confirming that inverse method is an attractive alternative approach of experimental technique in determining the competitive adsorption isotherm for binary systems.

  5. Relation between proteins tertiary structure, tryptophan fluorescence lifetimes and tryptophan S(o)→(1)L(b) and S(o)→(1)L(a) transitions. Studies on α1-acid glycoprotein and β-lactoglobulin. (United States)

    Albani, Jihad René


    We measured fluorescence lifetimes and fluorescence spectra (excitation and emission) of tryptophan residues of α(1)-acid glycoprotein (three Trp residues) and β-lactoglobulin (two Trp residues) in absence and presence of 450 μM progesterone. Progesterone binds only to α(1)-acid glycoprotein. In absence of progesterone, each of the two proteins displays three fluorescence lifetimes. Addition of progesterone induces a partial inhibition of the S(o) → (1)L(a) transition without affecting fluorescence lifetimes. The same experiments performed in presence of denatured proteins in 6 M guanidine show that addition of progesterone inhibits partially the S(o) → (1)L(a) transition and its peak is 15 nm shifted to the red compared to that obtained for native proteins. However, the S(o) → (1)L(b) transition position peak is not affected by protein denaturation. Thus, the tertiary structure of the protein plays an important role by modulating the tryptophan electronic transitions. Fluorescence emission decay recorded in absence and presence of progesterone yields three fluorescence lifetimes whether proteins are denatured or not. Thus, protein tertiary structure is not responsible for the presence of three fluorescence lifetimes. These characterize tryptophan substructures reached at the excited states and which population (pre-exponential values) depend on the tryptophan residues interaction with their microenvironment(s) and thus on the global conformation of the protein.

  6. Up-Regulation of Hepatic Alpha-2-HS-Glycoprotein Transcription by Testosterone via Androgen Receptor Activation

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    Jakob Voelkl


    Full Text Available Background/Aims: Fetuin-A (alpha-2-HS-glycoprotein, AHSG, a liver borne plasma protein, contributes to the prevention of soft tissue calcification, modulates inflammation, reduces insulin sensitivity and fosters weight gain following high fat diet or ageing. In polycystic ovary syndrome, fetuin-A levels correlate with free androgen levels, an observation pointing to androgen sensitivity of fetuin-A expression. The present study thus explored whether the expression of hepatic fetuin-A is modified by testosterone. Methods: HepG2 cells were treated with testosterone and androgen receptor antagonist flutamide, and were silenced with androgen receptor siRNA. To test the in vivo relevance, male mice were subjected to androgen deprivation therapy (ADT for 7 weeks. AHSG mRNA levels were determined by quantitative RT-PCR and fetuin-A protein abundance by Western blotting. Results: In HepG2 cells, AHSG mRNA expression and fetuin-A protein abundance were both up-regulated following testosterone treatment. The human alpha-2-HS-glycoprotein gene harbors putative androgen receptor response elements in the proximal 5 kb promoter sequence relative to TSS. The effect of testosterone on AHSG mRNA levels was abrogated by silencing of the androgen receptor in HepG2 cells. Moreover, treatment of HepG2 cells with the androgen receptor antagonist flutamide in presence of endogenous ligands in the medium significantly down-regulated AHSG mRNA expression and fetuin-A protein abundance. In addition, ADT of male mice was followed by a significant decrease of hepatic Ahsg mRNA expression and fetuin-A protein levels. Conclusions: Testosterone participates in the regulation of hepatic fetuin-A expression, an effect mediated, at least partially, by androgen receptor activation.

  7. Interaction of a novel Tn (GalNAc alpha 1-->Ser/Thr) glycoprotein with Gal, GalNAc and GlcNAc specific lectins. (United States)

    Wu, A M; Wu, J H; Shen, F


    A naturally occurring Tn glycoprotein (Native ASG-Tn) with GalNAc alpha 1-->Ser/Thr as the only carbohydrate side chains, has been prepared from armadillo submandibular glands. In a quantitative precipitin assay, this glycoprotein completely precipitated Maclura pomifera (MPA), Vicia villosa B4 (VVL-B4) and Artocarpus integrifolia (Jacalin, AIL). It also reacted well with Helix pomatia (HPL) and Wistaria floribunda (WFL) and precipitated over 75% of the lectin nitrogen added, but poorly with Ricinus communis agglutinin (RCA1), ricin, peanut (Arachis hypogaea, PNA), Abrus precatorius agglutinin (APA) and Triticum vulgaris (WGA). This finding suggests that this novel Tn-glycoprotein may serve as a useful reagent for differentiating Tn and T specific monoclonal antibodies and lectins.

  8. Decreased expression of alpha-2-HS glycoprotein in the sera of rats treated with Eurycoma longifolia extract

    Directory of Open Access Journals (Sweden)

    Yeng eChen


    Full Text Available Eurycoma longifolia is a Malaysian native herb that has been widely used as an aphrodisiac and a remedy for andropause. Although the physiological effects of the plant extract were predicted as a result of the alterations in protein expression, the key protein(s involved in these alterations are still unclear. In the present study, we have investigated the effect of standardized Eurycoma longifolia extract on serum protein expression up to 28 days following oral administration in rats. Serum protein profiles were analyzed by 2-dimensional electrophoresis, and altered proteins were identified via mass spectrometry. We observed that alpha-2-HS glycoprotein (AHS was significantly decreased in the serum of experimentally treated rats compared to controls. Moreover, reduction in AHS was confirmed using competitive enzyme-linked immunosorbent assay. AHS expression is known to be associated with insulin resistance and diabetes. Our data indicated that serum AHS was reduced in rats treated with standardized E. longifolia extract, and therefore form a prelude for further investigation into the effects of this natural extract in animal models involving infertility and diabetes.

  9. The role of Zn-alpha2 glycoprotein in sperm motility is mediated by changes in cyclic AMP. (United States)

    Qu, Fei; Ying, Xiaoqian; Guo, Wei; Guo, Qiangsu; Chen, Guowu; Liu, Yue; Ding, Zhide


    Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-alpha2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the beta(3)-adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90- or 120-min incubation (Pspermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway.

  10. Method for analysing glycoprotein isoforms by capillary electrophoresis


    Frutos, Mercedes de; Díez-Masa, José Carlos; Morales-Cid, Gabriel


    [EN] The present invention relates to a new method for the purification, concentration, separation and determination of the isoforms of alpha-1-acid glycoprotein (AGP) in human blood serum samples using capillary electrophoresis. The new method is based on the immunocapture and preconcentration of the sample within the separation capillary by using an immunoadsorbent phase magnetically immobilized within the electrophoresis capillary and the subsequent desorption and separation of the glycopr...

  11. Compensation for dystrophin-deficiency: ADAM12 overexpression in skeletal muscle results in increased alpha 7 integrin, utrophin and associated glycoproteins

    DEFF Research Database (Denmark)

    Moghadaszadeh, Behzad; Albrechtsen, Reidar; Guo, Ling T;


    , and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of alpha 7B integrin and utrophin, the functional homolog of dystrophin....... The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans....

  12. Differential mode of interaction of ThioflavinT with native β structural motif in human α 1-acid glycoprotein and cross beta sheet of its amyloid: Biophysical and molecular docking approach (United States)

    Ajmal, Mohammad Rehan; Nusrat, Saima; Alam, Parvez; Zaidi, Nida; Badr, Gamal; Mahmoud, Mohamed H.; Rajpoot, Ravi Kant; Khan, Rizwan Hasan


    The present study details the interaction mechanism of Thioflavin T (ThT) to Human α1-acid glycoprotein (AAG) applying various spectroscopic and molecular docking methods. Fluorescence quenching data revealed the binding constant in the order of 104 M-1 and the standard Gibbs free energy change value, ΔG = -6.78 kcal mol-1 for the interaction between ThT and AAG indicating process is spontaneous. There is increase in absorbance of AAG upon the interaction of ThT that may be due to ground state complex formation between ThT and AAG. ThT impelled rise in β-sheet structure in AAG as observed from far-UV CD spectra while there are minimal changes in tertiary structure of the protein. DLS results suggested the reduction in AAG molecular size, ligand entry into the central binding pocket of AAG may have persuaded the molecular compaction in AAG. Isothermal titration calorimetric (ITC) results showed the interaction process to be endothermic with the values of standard enthalpy change ΔH0 = 4.11 kcal mol-1 and entropy change TΔS0 = 10.82 kcal.mol- 1. Moreover, docking results suggested hydrophobic interactions and hydrogen bonding played the important role in the binding process of ThT with F1S and A forms of AAG. ThT fluorescence emission at 485 nm was measured for properly folded native form and for thermally induced amyloid state of AAG. ThT fluorescence with native AAG was very low, while on the other hand with amyloid induced state of the protein AAG showed a positive emission peak at 485 nm upon the excitation at 440 nm, although it binds to native state as well. These results confirmed that ThT binding alone is not responsible for enhancement of ThT fluorescence but it also required beta stacked sheet structure found in protein amyloid to give proper signature signal for amyloid. This study gives the mechanistic insight into the differential interaction of ThT with beta structures found in native state of the proteins and amyloid forms, this study reinforce

  13. Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey. (United States)

    Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L


    The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

  14. Complementary roles of glycoprotein VI and alpha2beta1 integrin in collagen-induced thrombus formation in flowing whole blood ex vivo

    DEFF Research Database (Denmark)

    Kuijpers, Marijke J E; Schulte, Valerie; Bergmeier, Wolfgang


    Platelets interact vigorously with subendothelial collagens that are exposed by injury or pathological damage of a vessel wall. The collagen-bound platelets trap other platelets to form aggregates, and they expose phosphatidylserine (PS) required for coagulation. Both processes are implicated...... in the formation of vaso-occlusive thrombi. We previously demonstrated that the immunoglobulin receptor glycoprotein VI (GPVI), but not integrin alpha2beta1, is essential in priming platelet-collagen interaction and subsequent aggregation. Here, we report that these receptors have yet a complementary function...... in ex vivo thrombus formation during perfusion of whole blood over collagen. With mice deficient in GPVI or blocking antibodies, we found that GPVI was indispensable for collagen-dependent Ca2+ mobilization, exposure of PS, and aggregation of platelets. Deficiency of integrin beta1 reduces the GPVI...

  15. Chromogranin A as serum marker for neuroendocrine neoplasia: comparison with neuron-specific enolase and the alpha-subunit of glycoprotein hormones. (United States)

    Nobels, F R; Kwekkeboom, D J; Coopmans, W; Schoenmakers, C H; Lindemans, J; De Herder, W W; Krenning, E P; Bouillon, R; Lamberts, S W


    Chromogranin A (CgA) is gaining acceptance as a serum marker of neuroendocrine tumors. Its specificity in differentiating between neuroendocrine and nonneuroendocrine tumors, its sensitivity to detect small tumors, and its clinical value, compared with other neuroendocrine markers, have not clearly been defined, however. The objectives of this study were to evaluate the clinical usefulness of CgA as neuroendocrine serum marker. Serum levels of CgA, neuron-specific enolase (NSE), and the alpha-subunit of glycoprotein hormones (alpha-SU) were determined in 211 patients with neuroendocrine tumors and 180 control subjects with nonendocrine tumors. The concentrations of CgA, NSE, and alpha-SU were elevated in 50%, 43%, and 24% of patients with neuroendocrine tumors, respectively. Serum CgA was most frequently increased in subjects with gastrinomas (100%), pheochromocytomas (89%), carcinoid tumors (80%), nonfunctioning tumors of the endocrine pancreas (69%), and medullary thyroid carcinomas (50%). The highest levels were observed in subjects with carcinoid tumors. NSE was most frequently elevated in patients with small cell lung carcinoma (74%), and alpha-SU was most frequently elevated in patients with carcinoid tumors (39%). Most subjects with elevated alpha-SU levels also had elevated CgA concentrations. A significant positive relationship was demonstrated between the tumor load and serum CgA levels (P NSE, and alpha-SU were present in, respectively, 7%, 35%, and 15% of control subjects. Markedly elevated serum levels of CgA, exceeding 300 micrograms/L, were observed in only 2% of control patients (n = 3) compared to 40% of patients with neuroendocrine tumors (n = 76). We conclude that CgA is the best general neuroendocrine serum marker available. It has the highest specificity for the detection of neuroendocrine tumors compared to the other neuroendocrine markers, NSE and alpha-SU. Elevated levels are strongly correlated with tumor volume; therefore, small tumors may

  16. Proteomic analysis of coronary sinus serum reveals leucine-rich alpha2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure.

    LENUS (Irish Health Repository)

    Watson, Chris J


    BACKGROUND: Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF. METHODS AND RESULTS: Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich alpha2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P<\\/=0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-alpha (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-betaR1 (P<0.001) and alpha-smooth muscle actin (P=0.025) expression. CONCLUSIONS: LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, beta-blocker therapy, and BNP.

  17. Systematic determination of the peptide acceptor preferences for the human UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase). (United States)

    Perrine, Cynthia; Ju, Tongzhong; Cummings, Richard D; Gerken, Thomas A


    Mucin-type protein O-glycosylation is initiated by the addition of alpha-GalNAc to Ser/Thr residues of a polypeptide chain. The addition of beta-Gal to GalNAc by the UDP-Gal:glycoprotein-alpha-GalNAc beta 3 galactosyltransferase (T-synthase), forming the Core 1 structure (beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr), is a common and biologically significant subsequent step in O-glycan biosynthesis. What dictates the sites of Core 1 glycosylation is poorly understood; however, the peptide sequence and neighboring glycosylation effects have been implicated. To systematically address the role of the peptide sequence on the specificity of T-synthase, we used the oriented random glycopeptide: GAGAXXXX(T-O-GalNAc)XXXXAGAG (where X = G, A, P, V, I, F, Y, S, N, D, E, H, R, and K) as a substrate. The Core 1 glycosylated product was isolated on immobilized PNA (Arachis hypogaea) lectin and its composition determined by Edman amino acid sequencing for comparison with the initial substrate composition, from which transferase preferences were obtained. From these studies, elevated preferences for Gly at the +1 position with moderately high preferences for Phe and Tyr in the +3 position relative to the acceptor Thr-O-GalNAc were found. A number of smaller Pro enhancements were also observed. Basic residues, i.e., Lys, Arg, and His, in any position were disfavored, suggesting electrostatic interactions as an additional important component modulating transferase specificity. This work suggests that there are indeed subtle specific and nonspecific protein-targeting sequence motifs for this transferase.

  18. Alfa-2-glicoproteína rica en leucina urinaria en pacientes con apendicitis aguda (Urinary leucine-rich alpha-2-glycoprotein in patients with acute appendicitis

    Directory of Open Access Journals (Sweden)

    Heberto Machado-Montero


    Full Text Available The objective of research was to determine diagnostic efficacy of urinary concentrations of leucine-rich alpha-2-glycoprotein (A2GRL in patients with acute appendicitis. There were included patients with high suspicion of acute appendicitis (group A; cases, n = 30. In group B (controls, n = 30 were included patients with non-surgical abdominal pain who attended to Hospital Central “Dr. Urquinaona”, Maracaibo, Zulia. General characteristics, urinary concentrations of A2GRL and diagnostic efficacy of assay were evaluated. Mean age of patients in group A was 36.3 +/- 8.8 years-old and in group B was 35.8 +/- 9.6 years-old (p = ns. There were not found significant differences in sex distribution, weight and height between groups (p = ns. Urinary concentrations of A2GRL were significantly higher in group A (1543.8 +/- 762.7 ng/mL compared with mean value in patients of group B (774.1 +/- 356.1 ng/mL; p < 0.0001. A2GRL presented a value below curve 0.81. A cut-off value of 1000 ng/mL, showed sensivity 73.3%, specificity 70.9%, positive predictive value 72.4% and negative predictive value 72.4%. It is concluded that urinary concentrations of A2GRL have a high diagnostic efficacy in patients with acute appendicitis

  19. Evaluation of Zinc-alpha-2-Glycoprotein and Proteasome Subunit beta-Type 6 Expression in Prostate Cancer Using Tissue Microarray Technology.

    LENUS (Irish Health Repository)


    Prostate cancer (CaP) is a significant cause of illness and death in males. Current detection strategies do not reliably detect the disease at an early stage and cannot distinguish aggressive versus nonaggressive CaP leading to potential overtreatment of the disease and associated morbidity. Zinc-alpha-2-glycoprotein (ZAG) and proteasome subunit beta-Type 6 (PSMB-6) were found to be up-regulated in the serum of CaP patients with higher grade tumors after 2-dimensional difference gel electrophoresis analysis. The aim of this study was to investigate if ZAG and PSMB-6 were also overexpressed in prostatic tumor tissue of CaP patients. Immunohistochemical analysis was performed on CaP tissue microarrays with samples from 199 patients. Confirmatory gene expression profiling for ZAG and PSMB-6 were performed on 4 cases using Laser Capture Microdissection and TaqMan real-time polymerase chain reaction. ZAG expression in CaP epithelial cells was inversely associated with Gleason grade (benign prostatic hyperplasia>G3>G4\\/G5). PSMB-6 was not expressed in either tumor or benign epithelium. However, strong PSMB-6 expression was noted in stromal and inflammatory cells. Our results indicate ZAG as a possible predictive marker of Gleason grade. The inverse association between grade and tissue expression with a rising serum protein level is similar to that seen with prostate-specific antigen. In addition, the results for both ZAG and PSMB-6 highlight the challenges in trying to associate the protein levels in serum with tissue expression.

  20. Influence of Alpha-1 Glycoprotein Acid Concentrations and Variants on Atazanavir Pharmacokinetics in HIV-Infected Patients Included in the ANRS 107 Trial▿ (United States)

    Barrail-Tran, A.; Mentré, F.; Cosson, C.; Piketty, C.; Chazallon, C.; Gérard, L.; Girard, P. M.; Taburet, A. M.


    Atazanavir is an HIV-1 protease inhibitor with high protein binding in human plasma. The objectives were first to determine the in vitro binding characteristics of atazanavir and second to evaluate whether plasma protein binding to albumin and alpha-1 glycoprotein acid (AAG) influences the pharmacokinetics of atazanavir in HIV-infected patients. For the in vitro study, atazanavir protein binding characteristics were determined in AAG- and albumin-containing purified solutions. Atazanavir was found to bind AAG on a high-affinity saturable site (association constant, 4.61 × 105 liters/mol) and albumin on a low-affinity nonsaturable site. For the in vivo study, blood samples from 51 patients included in trial ANRS 107—Puzzle 2 were drawn prior to drug intake at week 6. For 10 patients included in the pharmacokinetic substudy, five additional blood samples were collected during one dosing interval at week 6. Atazanavir concentrations were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Albumin concentrations, AAG concentrations, and phenotypes were also measured in these patients. Concentrations of atazanavir were modeled using a population approach. A one-compartment model with first-order absorption and elimination best described atazanavir pharmacokinetics. Atazanavir pharmacokinetic parameters and their interindividual variabilities were as follows: absorption rate constant (ka), 0.73 h−1 (139.3%); apparent clearance (CL/F), 13.3 liters/h (26.7%); and apparent volume of distribution (V/F), 79.7 liters (27.0%). Atazanavir CL/F decreased significantly when alanine aminotransferase and/or AAG levels increased (P < 0.01). The ORM1*S phenotype also significantly increased atazanavir V/F (P < 0.05). These in vivo results indicate that atazanavir pharmacokinetics is moderately influenced by its protein binding, especially to AAG, without expected clinical consequences. PMID:19995932

  1. Influence of alpha-1 glycoprotein acid concentrations and variants on atazanavir pharmacokinetics in HIV-infected patients included in the ANRS 107 trial. (United States)

    Barrail-Tran, A; Mentré, F; Cosson, C; Piketty, C; Chazallon, C; Gérard, L; Girard, P M; Taburet, A M


    Atazanavir is an HIV-1 protease inhibitor with high protein binding in human plasma. The objectives were first to determine the in vitro binding characteristics of atazanavir and second to evaluate whether plasma protein binding to albumin and alpha-1 glycoprotein acid (AAG) influences the pharmacokinetics of atazanavir in HIV-infected patients. For the in vitro study, atazanavir protein binding characteristics were determined in AAG- and albumin-containing purified solutions. Atazanavir was found to bind AAG on a high-affinity saturable site (association constant, 4.61x10(5) liters/mol) and albumin on a low-affinity nonsaturable site. For the in vivo study, blood samples from 51 patients included in trial ANRS 107--Puzzle 2 were drawn prior to drug intake at week 6. For 10 patients included in the pharmacokinetic substudy, five additional blood samples were collected during one dosing interval at week 6. Atazanavir concentrations were assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Albumin concentrations, AAG concentrations, and phenotypes were also measured in these patients. Concentrations of atazanavir were modeled using a population approach. A one-compartment model with first-order absorption and elimination best described atazanavir pharmacokinetics. Atazanavir pharmacokinetic parameters and their interindividual variabilities were as follows: absorption rate constant (ka), 0.73 h(-1) (139.3%); apparent clearance (CL/F), 13.3 liters/h (26.7%); and apparent volume of distribution (V/F), 79.7 liters (27.0%). Atazanavir CL/F decreased significantly when alanine aminotransferase and/or AAG levels increased (P<0.01). The ORM1*S phenotype also significantly increased atazanavir V/F (P<0.05). These in vivo results indicate that atazanavir pharmacokinetics is moderately influenced by its protein binding, especially to AAG, without expected clinical consequences.

  2. 应用CLSI EP10-A2指南评价国产α1-酸性糖蛋白试剂%Evaluation by CLSI EP10-A2 Document on α1-Acid Glycoprotein Reagent

    Institute of Scientific and Technical Information of China (English)

    孙艳虹; 高玲; 曾智杰; 姚真荣; 龙伟清; 姜傥


    Objective Preliminary evaluation of alphal acid glycoprotein (AGP) kit of Shanghai Jingyuan medical devices Co. Ltd. for clinical application performance using Clinical and Laboratory Standards Institute (CLSI) EP10 A2. Methods High and low quality control materials were provided by the manufacturer of reagent and middle quality control material was made by laboratory. The control materials were measured continuously at the order of medium,high,low,medium,medium,low, low,high,high,medium every day for 5 days. The first measurment only was as the starting value,and was not included in statistics. Any of the 9 behind data failed,then all data should be discarded and redetermined. All data were calculated by sta tistical software to get the bias,imprecision and other statistical parameters. Results The deviations of the low,medium and high quality control of AGP were 0. 093,0. 061 and 0. 269 mg/L seperately,and all of them were lower than the calibration error. The total imprecision of the three concentrations were 3. 87% ,1. 27% and 3. 19% seperately,lower than the maximum allowable total imprecision. Linear regression equation Y= 0. 071 6X2 +0. 563 7X +0. 586 7, correlation coefficient R2 = 0. 992 7,greater than 0. 95,indicating that the linear range of evaluation meets the requirements. Slope: 1. 958 (P>0. 01) ; carryover:0. 007 (P>0. 01); linear: -0. 131 (P>0. 01) ; Drift:0. 002(P<0. 01). Conclusion Evaluated by CLSI EP10 A2 document on Medical Devices Co. Ltd. Shanghai Jingyuan production of al acid glycoprotein (AGP) ,the linear,bias,impre cision and the capacity of anti total cross contamination were up to standard. The reagent had a good linear relationship with in a certain range,low carryover and stable can meet routine clinical testing and research needs.%目的 利用CLSI EP10-A2指南初步评价上海景源医疗器械有限公司生产的α1-酸性糖蛋白(AGP)试剂盒的临床应用性能.方法 厂家提供高、低值质控品,自

  3. 升清胶囊对胆汁促成核蛋白及生化指标影响的临床研究%Clinical study on effects of Shengqing capsule on nucleoprotein α1-acid glycoprotein and biochemical indicators of bile in patients with cholelithiasis

    Institute of Scientific and Technical Information of China (English)

    杨吉勇; 张静喆


    Objective To investigate effects and clinical significance of Chinese herbal drugs Shengqing capsule on nucleoprotein α1~acid glycoprotein and biochemical indicators of bile in patient with cholelithiasis. Methods A retrospective analysis was conducted in 40 patients with cholecystolithiasis or cholangiolithiasis definitely diagnosed by B-mode ultrasonic diagnostic equipment or computerized tomography between June 2007 and March 2009. T tube was placed via biliary drainage operation or ERCP+ENBD in biliary surgery. All patients were randomly divided into experimental group and control group, each group had 20 patients. Two groups received conventional treatments (including antibiotic treatments), and experimental group took Shengqing capsule after the first day of operation, T tube patients took Shengqing capsule for 14 d, ENBD patients took Shengqing capsule for 5 d. Then, bile from T tube and ENBD were collected on 1st, 3rd, 5th, 14th after operation respectively, and α1-acid glycoprotein, total bilirubin, un-conjugated bilirubin, cholesterol, phospholipid, total bile acid, calcium of bile were detected. Results Shengqing capsule reduced AAG in experimental group, there was a significant difference between two groups (/k0.05). Compared with control group, Shengqing capsule in experimental group reduced TBIL、 UCB、 CH、 Ca2* (P<0.05), and increased PL ? TBA of bile (P<0.05). Conclusion Shengqing capsule can reduce nucleoprotein AAG content in patients bile with cholelithiasis significantly, which improve biochemical indicators efficiently, and inhibit the formation of calculus in bile.%目的 探讨中药升清胶囊对胆汁中重要的促成核蛋白α1-酸性糖蛋白(α1-acid glycoprotein,AAG)及生化指标的影响及其临床意义.方法 对2007年6月至2009年3月期间收治的40例胆囊结石或肝内外胆管结石患者进行回顾性分析.术前经B超或CT等检查确诊.术中行胆道手术放置T管或行内镜逆行胰胆管造影和

  4. Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Pamela D.; Sakwe, Amos [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Koumangoye, Rainelli [Division of Surgical Oncology and Endocrine Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Yarbrough, Wendell G. [Division of Otolaryngology, Departments of Surgery and Pathology and Yale Cancer Center, Yale University, New Haven, CT 06520 (United States); Ochieng, Josiah [Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208 (United States); Marshall, Dana R., E-mail: [Department of Pathology, Anatomy and Cell Biology, Meharry Medical College, Nashville, TN 37208 (United States)


    This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head

  5. Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Positive (ER+ and Negative (ER- Human Breast Cancer Tissues by LC-LTQ/FT-ICR Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Suzan M. Semaan, Xu Wang, Alan G. Marshall, Qing-Xiang Amy Sang


    Full Text Available Breast cancer is the second most fatal cancer in American women. To increase the life expectancy of patients with breast cancer new diagnostic and prognostic biomarkers and drug targets must be identified. A change in the glycosylation on a glycoprotein often causes a change in the function of that glycoprotein; such a phenomenon is correlated with cancerous transformation. Thus, glycoproteins in human breast cancer estrogen receptor positive (ER+ tissues and those in the more advanced stage of breast cancer, estrogen receptor negative (ER- tissues, were compared. Glycoproteins showing differences in glycosylation were examined by 2-dimensional gel electrophoresis with double staining (glyco- and total protein staining and identified by reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/ Fourier transform ion cyclotron resonance mass spectrometer. Among the identified glycosylated proteins are alpha 1 acid glycoprotein, alpha-1-antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor that were further verified by Western blotting for both ER+ and ER- human breast tissues. Results show the presence of a possible glycosylation difference in alpha-1-antitrypsin, a potential tumor-derived biomarker for breast cancer progression, which was expressed highest in the ER- samples.

  6. Comparative structure analyses of cystine knot-containing molecules with eight aminoacyl ring including glycoprotein hormones (GPH alpha and beta subunits and GPH-related A2 (GPA2 and B5 (GPB5 molecules

    Directory of Open Access Journals (Sweden)

    Combarnous Yves


    Full Text Available Abstract Background Cystine-knot (cys-knot structure is found in a rather large number of secreted proteins and glycoproteins belonging to the TGFbeta and glycoprotein hormone (GPH superfamilies, many of which are involved in endocrine control of reproduction. In these molecules, the cys-knot is formed by a disulfide (SS bridge penetrating a ring formed by 8, 9 or 10 amino-acid residues among which four are cysteine residues forming two SS bridges. The glycoprotein hormones Follicle-Stimulating Hormone (FSH, Luteinizing Hormone (LH, Thyroid-Stimulating Hormone (TSH and Chorionic Gonadotropin (CG are heterodimers consisting of non-covalently associated alpha and beta subunits that possess cys-knots with 8-amino-acyl (8aa rings. In order to get better insight in the structural evolution of glycoprotein hormones, we examined the number and organization of SS bridges in the sequences of human 8-aa-ring cys-knot proteins having 7 (gremlins, 9 (cerberus, DAN, 10 (GPA2, GPB5, GPHα and 12 (GPHβ cysteine residues in their sequence. Discussion The comparison indicated that the common GPH-alpha subunit exhibits a SS bridge organization ressembling that of DAN and GPA2 but possesses a unique bridge linking an additional cysteine inside the ring to the most N-terminal cysteine residue. The specific GPHbeta subunits also exhibit a SS bridge organization close to that of DAN but it has two additional C-terminal cysteine residues which are involved in the formation of the "seat belt" fastened by a SS "buckle" that ensures the stability of the heterodimeric structure of GPHs. GPA2 and GPB5 exhibit no cys residue potentially involved in interchain SS bridge and GPB5 does not possess a sequence homologous to that of the seatbelt in GPH β-subunits. GPA2 and GPB5 are thus not expected to form a stable heterodimer at low concentration in circulation. Summary The 8-aa cys-knot proteins GPA2 and GPB5 are expected to form a heterodimer only at concentrations above 0

  7. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G. (La Jolla, CA); Wang, Lei (San Diego, CA); Zhang, Zhiwen (San Diego, CA)


    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.

  8. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Schultz, Peter G. (La Jolla, CA); Wang, Lei (San Diego, CA); Zhang, Zhiwen (San Diego, CA)


    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.

  9. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    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.


    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.

  10. The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes

    DEFF Research Database (Denmark)

    Benfield, T L; Lundgren, Bettina; Levine, S J


    Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia. In order to investigate whether the major surface antigen (MSG) of human P. carinii is capable of eliciting...... the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG. MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0...... with 0.2 to 5 microg of MSG/ml (P

  11. The relationship between renal function and plasma concentration of the cachectic factor zinc-alpha2-glycoprotein (ZAG) in adult patients with chronic kidney disease. (United States)

    Pelletier, Caroline C; Koppe, Laetitia; Alix, Pascaline M; Kalbacher, Emilie; Croze, Marine L; Hadj-Aissa, Aoumeur; Fouque, Denis; Guebre-Egziabher, Fitsum; Soulage, Christophe O


    Zinc-α2-glycoprotein (ZAG), a potent cachectic factor, is increased in patients undergoing maintenance dialysis. However, there is no data for patients before initiation of renal replacement therapy. The purpose of the present study was to assess the relationship between plasma ZAG concentration and renal function in patients with a large range of glomerular filtration rate (GFR). Plasma ZAG concentration and its relationship to GFR were investigated in 71 patients with a chronic kidney disease (CKD) stage 1 to 5, 17 chronic hemodialysis (HD), 8 peritoneal dialysis (PD) and 18 non-CKD patients. Plasma ZAG concentration was 2.3-fold higher in CKD stage 5 patients and 3-fold higher in HD and PD patients compared to non-CKD controls (Prenal disease.

  12. The major surface glycoprotein of Pneumocystis carinii induces release and gene expression of interleukin-8 and tumor necrosis factor alpha in monocytes

    DEFF Research Database (Denmark)

    Benfield, T L; Lundgren, Bettina; Levine, S J;


    with 0.2 to 5 microg of MSG/ml (P TNF-alpha from MSG-stimulated monocytes at 20 h was inhibited by 60 and 86%, respectively, after coincubation with soluble yeast mannan (P = 0.01). With an RNase protection assay, increases in steady-state mRNA levels for IL-8 and TNF......Recent studies suggest that interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) may play a central role in host defense and pathogenesis during Pneumocystis carinii pneumonia. In order to investigate whether the major surface antigen (MSG) of human P. carinii is capable of eliciting...... the release of IL-8 and TNF-alpha, human monocytes were cultured in the presence of purified MSG. MSG-stimulated cells released significant amounts of IL-8 within 4 h, and at 20 h, cells stimulated with MSG released 45.5 +/- 9.3 ng of IL-8/ml versus 3.7 +/- 1.1 ng/ml for control cultures (P = 0...

  13. Are alpha-gliadins glycosylated? (United States)

    Turner, J B; Garner, G V; Gordon, D B; Brookes, S J; Smith, C A


    Alpha-gliadins isolated by carboxymethylcellulose chromatography contain noncovalently bound glucose probably due to contaminating proteoglycans and to material shed from the column. Traces of carbohydrate remain strongly bound to alpha-gliadins even after harsh denaturation, but our results indicate alpha-gliadins are not glycoproteins. Suggestions that gliadins are glycoproteins are probably due to contamination with this glucose and the presence of these proteoglycans.

  14. Zinc-alpha 2-glycoprotein gene expression in adipose tissue is related with insulin resistance and lipolytic genes in morbidly obese patients.

    Directory of Open Access Journals (Sweden)

    Lourdes Garrido-Sánchez

    Full Text Available OBJECTIVE: Zinc-α(2 glycoprotein (ZAG stimulates lipid loss by adipocytes and may be involved in the regulation of adipose tissue metabolism. However, to date no studies have been made in the most extreme of obesity. The aims of this study are to analyze ZAG expression levels in adipose tissue from morbidly obese patients, and their relationship with lipogenic and lipolytic genes and with insulin resistance (IR. METHODS: mRNA expression levels of PPARγ, IRS-1, IRS-2, lipogenic and lipolytic genes and ZAG were quantified in visceral (VAT and subcutaneous adipose tissue (SAT of 25 nondiabetic morbidly obese patients, 11 with low IR and 14 with high IR. Plasma ZAG was also analyzed. RESULTS: The morbidly obese patients with low IR had a higher VAT ZAG expression as compared with the patients with high IR (p = 0.023. In the patients with low IR, the VAT ZAG expression was greater than that in SAT (p = 0.009. ZAG expression correlated between SAT and VAT (r = 0.709, p<0.001. VAT ZAG expression was mainly predicted by insulin, HOMA-IR, plasma adiponectin and expression of adiponectin and ACSS2. SAT ZAG expression was only predicted by expression of ATGL. CONCLUSIONS: ZAG could be involved in modulating lipid metabolism in adipose tissue and is associated with insulin resistance. These findings suggest that ZAG may be a useful target in obesity and related disorders, such as diabetes.

  15. The adsorption and lubrication behavior of synovial fluid proteins and glycoproteins on the bearing-surface materials of hip replacements. (United States)

    Roba, Marcella; Naka, Marco; Gautier, Emanuel; Spencer, Nicholas D; Crockett, Rowena


    The selectivity of synovial fluid protein adsorption onto ultra-high molecular weight polyethylene (UHMWPE) and alumina (Al(2)O(3)), and in particular the ability of glycoproteins to adsorb in the presence of all the other synovial fluid proteins, was investigated by means of fluorescence microscopy and gel electrophoresis (SDS-PAGE). The non-specific nature of protein adsorption from synovial fluid indicated that the lubrication of artificial hip-joint materials may not be attributable to a single protein as has been frequently suggested. The friction behavior of polyethylene (PE) sliding against Al(2)O(3) in solutions of bovine serum albumin (BSA), alpha-1-acid glycoprotein (AGP) and alpha-1-antitrypsin (A1AT) was investigated by means of colloidal probe atomic force microscopy. BSA was shown to be a poorer boundary lubricant than the phosphate buffered saline used as a control. This was attributed to denaturation of the BSA upon adsorption, which provided a high-shear-strength layer at the interface, impairing the lubrication. Interestingly, both the glycoproteins AGP and A1AT, despite their low concentrations, improved lubrication. The lubricating properties of AGP and A1AT were attributed to adsorption via the hydrophobic backbone, allowing the hydrophilic carbohydrate moieties to be exposed to the aqueous solution, thus providing a low-shear-strength fluid film that lubricated the system. The amount of glycoprotein adsorbed on hydrophobic surfaces was determined by means of optical waveguide lightmode spectroscopy (OWLS), allowing conclusions to be drawn about the conformation of the glycan residues following adsorption.

  16. Suplementação de N-acetilcisteína em pacientes infectados pelo HIV submetidos ao primeiro tratamento anti-retroviral: Avaliação do efeito sobre a carga viral, TNF-α, IL-6, IL-8, β2-microglobulina, IgA, IgG e IgM, haptoglobina e α1-glicoproteína ácida N-acetylcysteine supplementation of HIV-infected patients under the first anti-retroviral treatment: Evaluation of the effect on viral load, TNF-α, IL-6, IL-8, β2-microglobulin, IgA, IgG, IgM, haptoglobin and α1-acid glycoprotein

    Directory of Open Access Journals (Sweden)

    Aricio Treitinger


    alterations are characterized by elevated levels of tumor necrosis factor alpha (TNF-α, interleukin 8 (IL-8, β2-microglobulin, IgA, IgG, IgM, haptoglobin and a1-acid glycoprotein. The goal of this double blind placebo-controlled study was to evaluate the effect of N-acetylcysteine supplementation on virological, immunological and inflammatory markers in 24 HIVinfected individuals who were taking their first anti-retroviral therapy. Eleven individuals were treated with anti-retroviral therapy plus placebo supplementation and thirteen were treated with anti-retroviral therapy plus 600 mg/day of Nacetylcysteine. The levels of the studied markers were evaluated at the day before and after 60, 120 and 180 days of treatment. In both groups a significant decrease in serum levels of TNF-α (p=0.0001, IL-6 (p>0.05, IL-8 (p=0.0001, b2 microglobulin (p=0.0005, IgA (p=0.007, IgG (p=0.001, IgM (p=0.0001, haptoglobin (p=0.0001 e α1-acid glycoprotein (p=0.012 was found due to anti-retroviral therapy. N-acetylcysteine supplementation had no additive or synergistic effects on the studied parameters. In conclusion, N-acetylcysteine had no additional beneficial effects, at least at the dose used in this study, on the treatment of HIV-infected patients under anti-retroviral therapy.

  17. Glycoproteins: Occurrence and Significance (United States)

    Wittmann, Valentin

    Protein glycosylation is regarded as the most complex form of post-translational modification leading to a heterogeneous expression of glycoproteins as mixtures of glycoforms. This chapter describes the structure and occurrence of glycoproteins with respect to their glycan chains. Discussed are different carbohydrate-peptide linkages including GPI anchors, common structures of N- and O-glycans, and the structure of glycosaminoglycans contained in proteoglycans. Also covered are the bacterial cell wall polymer peptidoglycan and the glycopeptide antibiotics of the vancomycin group. Properties and functions of the glycans contained in glycoproteins are dealt with in the next chapter of this book.

  18. Determinação sérica de haptoglobina, ceruloplasmina, α1-glicoproteína ácida, transferrina e α1-antitripsina, em equinos com cólica Determination of serum haptoglobin, ceruloplasmin, α1-acid glycoprotein, transferrin and α1-antitrypsin in colic horses

    Directory of Open Access Journals (Sweden)

    Paula Alessandra Di Filippo


    Full Text Available Foram examinados 46 equinos adultos, 6 hígidos (G1 e 40 com cólica, submetidos à laparotomia. Vinte apresentavam lesões no intestino grosso (G2 e 20 no intestino delgado (G3. Avaliaram-se os teores séricos das proteínas de fase aguda: haptoglobina, ceruloplasmina, antitripsina, transferrina e glicoproteína ácida, antes e até sete dias após a laparotomia. Após centrifugação e fracionamento das amostras, as proteínas de fase aguda foram separadas por eletroforese em gel de poliacrilamida contendo SDS-PAGE, e suas concentrações determinadas por densitometria computadorizada. Constatou-se elevação dos valores das proteínas de fase aguda nos animais com cólica, antes e após laparotomia, porém com valores mais elevados, precoces e persistentes nos animais do G3. Os resultados deveram-se ao processo inflamatório intestinal, desencadeado pela lesão entérica e indicam que o proteinograma sérico pode auxiliar na identificação do segmento intestinal obstruído e, consequentemente, na elaboração do prognóstico de equinos com cólica.Forty six equines were examined, 6 were healthy (G1 and 40 with colic, submitted to laparotomy. Twenty were showing lesions on the large intestine (G2 and 20 lesions on the small intestine (G3. The serum concentrations of acute phase proteins: haptoglobin, ceruloplasmin, antitrypsin, transferrin, and - acid glycoprotein before and until 7 days after laparotomy. After centrifugation and fractioning of the samples, the acute phase proteins were individualized by electrophoresis on polyacrylamide gel containing SDS-PAGE, and the concentrations were determined by computerized densitometry. The increase was verified on the levels of acute phase proteins from animals with colic, before and after laparotomy, however with higher levels, premature and persistent on animals from G3. The results were due to the process of intestinal inflammation, caused by enteric injury and indicate that the serum protein

  19. Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives. (United States)

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


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

  20. Serum alpha2-Heremans-Schmid glycoprotein levels in subjects with simple overweight and obesity%单纯性超重和肥胖人群血清胎球蛋白 A水平的变化

    Institute of Scientific and Technical Information of China (English)

    周中卫; 居会祥; 孙明忠; 丁福万; 江冬梅; 季禹乔; 季建伟


    目的:探讨单纯性超重和肥胖人群血清胎球蛋白A( AHSG)水平的变化及其与相关临床指标的关系。方法从随机数字表法抽取的体检者中选取肥胖者49例,超重者176例,正常体重者327名;收集相关临床资料,酶联免疫法( ELISA)测定血清AHSG及脂联素水平;采用Pearson相关分析及多元线性回归模型分析AHSG与其它临床指标间的关系。结果血清AHSG水平在超重组及肥胖组中均明显高于正常体重组[(276.30和302.10对241.60)μg/ml],肥胖组高于超重组(P<0.05或P<0.01);在正常体重组中,AHSG与空腹血糖、稳态模型评估的胰岛素抵抗指数、C反应蛋白( CRP)、三酰甘油及游离脂肪酸(FFA)呈正相关,与年龄及脂联素呈负相关(P<0.05或P<0.01);在超重和肥胖组中,AHSG除与上述指标呈显著相关性,与体重指数、腰臀比及低密度脂蛋白胆固醇亦呈正相关(P<0.05或P<0.01);多元线性回归模型中,超重和肥胖组中的AHSG与腰臀比、CRP及FFA呈独立正相关,与脂联素呈独立负相关( P<0.05或P<0.01)。结论超重及肥胖者体内升高的AHSG可能是肥胖相关疾病的高危因素。%Objective To investigate serum alpha2-Heremans-Schmid glycoprotein ( AHSG ) level and its relationship with associated clinical parameters in subjects with simple overweight and obesity. Methods Forty-nine subjects with obesity ( OB group) , 176 subjects with overweight ( OW group) , and 327 individuals with normal weight ( NW group) were randomly enrolled. The clinical data were collected and serum levels of AHSG and adiponectin ( APN) were determined by ELISA. The associations of serum AHSG level with other clinical parameters were assessed by Pearson correlation analysis and multiple linear regressive model. Results Serum AHSG levels were higher in OW and OB groups than that in NW group[(276. 30 and 302. 10 vs 241. 60)μg/ml], being especially higher in OB group (P<0. 05 or P<0. 01). Serum AHSG level

  1. Engineered CHO cells for production of diverse, homogeneous glycoproteins

    DEFF Research Database (Denmark)

    Yang, Zhang; Wang, Shengjun; Halim, Adnan;


    genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like alpha 2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic...

  2. Platelet membrane glycoproteins and their function: an overview. (United States)

    Kunicki, T J


    The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.

  3. KDN-containing glycoprotein from loach skin mucus. (United States)

    Nakagawa, H; Hama, Y; Sumi, T; Li, S C; Li, Y T


    It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc.

  4. Defining carbohydrate specificity of Ricinus communis agglutinin as Gal beta 1-->4GlcNAc (II) > Gal beta 1-->3GlcNAc (I) > Gal alpha 1-->3Gal (B) > Gal beta 1-->3GalNAc (T). (United States)

    Wu, J H; Herp, A; Wu, A M


    To define carbohydrate specificity of Ricinus communis agglutinin (RCA1), the combining site of RCA1 was further characterized by quantitative precipitin (QPA) and precipitin-inhibition assays (QPIA). Among the oligosaccharides tested for QPIA, Gal beta 1-->4GlcNAc (II, human blood group type II precursor sequence) was found to be 7.1 times more active than Gal beta 1-->3GalNAc (T, Thomsen-Friedenreich sequence) and about 1.7 times more active than the other three disaccharides tested--Gal beta 1-->4Man, Gal beta 1-->3DAra and Gal beta 1-->6GalNAc. Gal alpha 1-->4Gal, the receptor of the uropathogenic E. coli ligand was 3.6 times less active than the II sequence. These results indicate that the beta 1-->4 linkage of the terminal Gal to subterminal GlcNAc is important as this beta 1-->4GlcNAc sequence is at least 1.6 times more active than other types of disaccharides. Among the glycoproteins examined for QPA, native and desialized bovine submandibular glycoproteins, native and desialized human plasma alpha 1-acid glycoproteins, as well as crude hog stomach mucin and its three mild acid hydrolyzed products reacted well with the lectin. These glycoproteins precipitated over 75% of the lectin nitrogen added indicating that RCA1 has the ability to recognize Gal beta 1-->4/3GlcNAc and/or the related residues at the non-reducing ends and at positions in the interior of the chains. However, Tn (GalNAc alpha 1-->Ser/Thr sequence) rich glycoproteins such as desialized ovine submandibular glycoprotein and desialized armadillo salivary glycoprotein, in which over 90% of the carbohydrate side chains are Tn determinants with none or only a trace of I/II or T determinants, precipitated poorly with RCA1. From the present and previous results obtained, the carbohydrate specificity of RCA1 can be constructed and summarized in decreasing order by lectin determinants as follows: II (Gal beta 1-->4GlcNAc) > I (Gal beta 1-->3GlcNAc) > E (Gal alpha 1-->4Gal) and B (Gal alpha 1-->3Gal

  5. Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy. (United States)

    Lamblin, G; Boersma, A; Klein, A; Roussel, P; van Halbeek, H; Vliegenthart, J F


    The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.

  6. Determinação sérica de haptoglobina, ceruloplasmina e alfa-glicoproteína ácida em cães com gastrenterite hemorrágica Determination of serum haptoglobin, ceruloplasmin and acid alpha-glycoprotein in dogs with haemorrhagic gastroenteritis

    Directory of Open Access Journals (Sweden)

    Márcia Mery Kogika


    Full Text Available As proteínas de fase aguda (PFA são proteínas plasmáticas, cujo estímulo à síntese ocorre de forma rápida e marcante em resposta à injúria tecidual. Estas proteínas permitem o diagnóstico de processos inflamatórios em animais com supressão ou depressão medular. Além disso, são úteis na monitorização da resolução tecidual de traumas ou inflamação e também na avaliação da resposta orgânica ao tratamento. Uma vez que a leucopenia é observada nos estádios iniciais da parvovirose canina, a dosagem das PFA pode permitir a avaliação do processo inflamatório sob estas condições. Considerando-se estas hipóteses, foram determinados os níveis séricos das PFA (haptoglobina, ceruloplasmina e alfa-glicoproteína ácida em 11 cães saudáveis e 11 cães leucopênicos com gastrenterite hemorrágica, com suspeita clínica de parvovirose canina. A avaliação estatística mostrou diferença significativa, com intervalo de confiabilidade de 99% (PAcute phase proteins (APP are serum proteins whose stimulus for the synthesis happens in a quick and intense manner in response to tissue injury. Those proteins allow the diagnosis of inflammatory process in animals with bone marrow depression and, also, they are useful in the follow up of tissue resolution of traumas or inflammation, as well as in the evaluation of the organic response of the treatment. As leukopenia is observed in the initial stage of the canine parvovirus infection, the dosage of APP can allow the evaluation of the inflammatory process under these conditions. According to these hypothesis, serum APP levels (haptoglobin, ceruloplasm and a-acid-glycoprotein in 11 healthy dogs and 11 leukopenic dogs with haemorrhagic gastroenteritis, clinically suspected of canine parvovirus infection, were measured. There was a significant difference, with confidence interval of 99% (P <0.01 for the haptoglobin (p <0.0064 and the acid alpha-glycoprotein (p <0.0042 and 95% (P <0.05 of

  7. 1Alpha,25-dihydroxyvitamin D3 up-regulates P-glycoprotein via the vitamin D receptor and not farnesoid X receptor in both fxr(-/-) and fxr(+/+) mice and increased renal and brain efflux of digoxin in mice in vivo. (United States)

    Chow, Edwin C Y; Durk, Matthew R; Cummins, Carolyn L; Pang, K Sandy


    Secondary farnesoid X receptor (FXR) effects, in addition to vitamin D receptor (VDR) effects, were observed in the rat liver after treatment with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the natural ligand of VDR, caused by increased bile acid absorption as a consequence of apical sodium-dependent bile acid transporter induction. To investigate whether the increased multidrug resistance protein 1 (Mdr1)/P-glycoprotein (P-gp) expression in the rat liver and kidney was caused by the VDR and not the FXR, we examined changes in Mdr1/P-gp expression in fxr(+/+) and fxr(-/-) mice after intraperitoneal dosing of vehicle versus 1,25(OH)(2)D(3) (0 or 2.5 μg/kg every other day for 8 days). Renal and brain levels of Mdr1 mRNA and P-gp protein were significantly increased in both fxr(+/+) and fxr(-/-) mice treated with 1,25(OH)(2)D(3), confirming that Mdr1/P-gp induction occurred independently of the FXR. Increased P-gp function was evident in 1,25(OH)(2)D(3)-treated fxr(+/+) mice given intravenous bolus doses of the P-gp probe, [(3)H]digoxin (0.1 mg/kg). Decreased blood (24%) and brain (29%) exposure, estimated as reduced areas under the curve, caused by increased renal (74%) and total body (34%) clearances of digoxin, were observed in treated mice. These events were predicted by physiologically based pharmacokinetic modeling that showed increased renal secretory intrinsic clearance (3.45-fold) and brain efflux intrinsic clearance (1.47-fold) in the 1,25(OH)(2)D(3)-treated mouse, trends that correlated well with increases in P-gp protein expression in tissues. The clearance changes were less apparent because of the high degree of renal reabsorption of digoxin. The observations suggest an important role of the VDR in the regulation of P-gp in the renal and brain disposition of P-gp substrates.

  8. Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

    Energy Technology Data Exchange (ETDEWEB)

    Tropea, J.E.; Molyneux, R.J.; Kaushal, G.P.; Pan, Y.T.; Mitchell, M.; Elbein, A.D. (Univ. of Texas Health Science Center, San Antonio (USA))


    Australine is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, the authors tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the {alpha}-glucosidase amyloglucosidase (50% inhibition at 5.8 {mu}M), but it did not inhibit {beta}-glucosidase, {alpha}- or {beta}-mannosidase, or {alpha}- or {beta}-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc{sub 3}Man{sub 7-9}(GlcNAc){sub 2}-oligosaccharides.

  9. Envelope glycoprotein of arenaviruses. (United States)

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


    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.

  10. Envelope Glycoprotein of Arenaviruses

    Directory of Open Access Journals (Sweden)

    Antonella Pasquato


    Full Text Available 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.

  11. Alpha Thalassemia (United States)

    Alpha Thalassemia Physicians often mistake alpha thalassemia trait for iron deficiency anemia and incorrectly prescribe iron supplements that have no effect 1 on the anemia. αα αα Normal alpha ...

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

    Directory of Open Access Journals (Sweden)

    Beata Olejnik


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

  13. Interaction of native and asialo rat sublingual glycoproteins with lectins. (United States)

    Wu, A M; Herp, A; Song, S C; Wu, J H; Chang, K S


    The binding properties of the rat sublingual glycoprotein (RSL) and its asialo product with lectins were characterized by quantitative precipitin(QPA) and precipitin inhibition(QPIA) assays. Among twenty lectins tested for QPA, native RSL reacted well only with Artocarpus integrifolia (jacalin), but weakly or not at all with the other lectins. However, its asialo product (asialo-RSL) reacted strongly with many Gal and GalNAc specific lectins-it bound best to three of the GalNAc alpha 1-->Ser/Thr (Tn) and/or Gal beta 1-->4GlcNAc (II) active lectins [jacalin, Wistaria floribunda and Ricinus communis agglutinins] and completely precipitated each of these three lectins. Asialo-RSL also reacted well with Abrus precatorius, Glycine max, Bauhinia purpurea alba, and Maclura pomifera agglutinins, and abrin-a, but not with Arachis hypogeae and Dolichos biflorus agglutinins. The interaction between asialo-RSL and lectins were inhibited by either Gal beta 1-->4GlcNAc, p-NO2-phenyl alpha-GalNAc or both. The mapping of the precipitation and inhibition profiles leads to the conclusion that the asialo rat sublingual glycoprotein provides important ligands for II (Gal beta 1-->4GlcNAc beta 1-->) and Tn (GalNAc alpha 1-->Ser/Thr) active lectins.

  14. Recent Progress in Electrochemical Biosensors for Glycoproteins

    Directory of Open Access Journals (Sweden)

    Uichi Akiba


    Full Text Available This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements.

  15. Recent Progress in Electrochemical Biosensors for Glycoproteins. (United States)

    Akiba, Uichi; Anzai, Jun-Ichi


    This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements.

  16. Specific glycosylation of α1-acid glycoprotein characterises patients with familial Mediterranean fever and obligatory carriers of MEFV

    NARCIS (Netherlands)

    Poland, D.C.W.; Drenth, J.P.H.; Rabinovitz, E.; Livneh, A.; Bijzet, J.; Van het Hof, B.; Van Dijk, W.


    Background - Familial Mediterranean fever (FMF) is a periodic febrile disorder, characterised by fever and serositis. The acute phase response during attacks of FMF results from the release of cytokines, which in turn induce increased expression and changed glycosylation of acute phase proteins. A r

  17. Pig α1-Acid Glycoprotein: Characterization and First Description in Any Species as a Negative Acute Phase Protein

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Miller, Ingrid; Sørensen, Nanna Skall;


    concentrations of pig AGP were in the range of 1-3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2-5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative...... acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase...

  18. Alpha fetoprotein (United States)

    Fetal alpha globulin; AFP ... Greater than normal levels of AFP may be due to: Cancer in testes , ovaries, biliary (liver secretion) tract, stomach, or pancreas Cirrhosis of the liver Liver cancer ...

  19. Mannosidase IA is in Quality Control Vesicles and Participates in Glycoprotein Targeting to ERAD. (United States)

    Ogen-Shtern, Navit; Avezov, Edward; Shenkman, Marina; Benyair, Ron; Lederkremer, Gerardo Z


    Endoplasmic reticulum-associated degradation (ERAD) of a misfolded glycoprotein in mammalian cells requires the removal of 3-4 alpha 1,2 linked mannose residues from its N-glycans. The trimming and recognition processes are ascribed to ER Mannosidase I, the ER-degradation enhancing mannosidase-like proteins (EDEMs), and the lectins OS-9 and XTP3-B, all residing in the ER, the ER-derived quality control compartment (ERQC), or quality control vesicles (QCVs). Folded glycoproteins with untrimmed glycans are transported from the ER to the Golgi complex, where they are substrates of other alpha 1,2 mannosidases, IA, IB, and IC. The apparent redundancy of these enzymes has been puzzling for many years. We have now determined that, surprisingly, mannosidase IA is not located in the Golgi but resides in QCVs. We had recently described this type of vesicles, which carry ER α1,2 mannosidase I (ERManI). We show that the overexpression of alpha class I α1,2 mannosidase IA (ManIA) significantly enhances the degradation of ERAD substrates and its knockdown stabilizes it. Our results indicate that ManIA trims mannose residues from Man9GlcNAc2 down to Man5GlcNAc2, acting in parallel with ERManI and the EDEMs, and targeting misfolded glycoproteins to ERAD.

  20. A sheep hydatid cyst glycoprotein as receptors for three toxic lectins, as well as Abrus precatorius and Ricinus communis agglutinins. (United States)

    Wu, A M; Song, S C; Wu, J H; Pfüller, U; Chow, L P; Lin, J Y


    The binding properties of a glycoprotein with blood group P1 specificity isolated from sheep hydatid cyst fluid with Gal and GalNAc specific lectins was investigated by quantitative precipitin and precipitin inhibition assays. The glycoprotein completely precipitated Ricinus communis agglutinin (RCA1), Abrus precatorius agglutinin (APA) and Mistletoe toxic lectin-I (ML-I). Only 1.0 microgram of P1 glycoprotein was required to precipitate 50% of 5.1 micrograms ML-I nitrogen. It also reacted well with abrin-a and ricin, precipitating over 73% of the lectin nitrogen added, but poorly or weakly with Dolichos biflorus (DBL), Vicia villosa (VVL, a mixture of A4, A2B2 and B4), VVL-B4, Arachis hypogaea (PNA), Maclura pomifera (MPL), Bauchinia purpurea alba (BPL) and Wistaria floribunda (WFL) lectins. When an inhibition assay in the range of 5.1 micrograms N to 5.9 micrograms N of lectins (ML-I, abrin-a; ricin, RCA1, and APA, and 10 micrograms P1 active glycoprotein interaction was performed; from 76 to 100% of the precipitations were inhibited by 0.44 and 0.52 mumol of Gal alpha 1-->4Gal and Gal beta 1-->4GlcNAc, respectively, but not or insignificantly with 1.72 mumol of GlcNAc. The Gal alpha 1-->4Gal disaccharide found in this P1 active glycoprotein is a frequently occurring sequence of many glycosphingolipids located at the surface of mammalian cell membranes, especially human erythrocytes and intestinal cells for ligand binding and microbial toxin attachment. The present finding suggests that the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in this P1 active glycoprotein is one of the best glycoprotein receptors for three toxic lectins (ricin, abrin-a, and ML-I) as well as for APA, and RCA1, and the result of inhibition assay implies that these lectins are recognizing part or all of the Gal alpha 1-->4Gal beta 1-->4GlcNAc sequence in the P1 active glycoprotein.

  1. $\\alpha_s$ review (2016)

    CERN Document Server

    d'Enterria, David


    The current world-average of the strong coupling at the Z pole mass, $\\alpha_s(m^2_{Z}) = 0.1181 \\pm 0.0013$, is obtained from a comparison of perturbative QCD calculations computed, at least, at next-to-next-to-leading-order accuracy, to a set of 6 groups of experimental observables: (i) lattice QCD "data", (ii) $\\tau$ hadronic decays, (iii) proton structure functions, (iv) event shapes and jet rates in $e^+e^-$ collisions, (v) Z boson hadronic decays, and (vi) top-quark cross sections in p-p collisions. In addition, at least 8 other $\\alpha_s$ extractions, usually with a lower level of theoretical and/or experimental precision today, have been proposed: pion, $\\Upsilon$, W hadronic decays; soft and hard fragmentation functions; jets cross sections in pp, e-p and $\\gamma$-p collisions; and photon F$_2$ structure function in $\\gamma\\,\\gamma$ collisions. These 14 $\\alpha_s$ determinations are reviewed, and the perspectives of reduction of their present uncertainties are discussed.

  2. Glycomic analysis of sialic acid linkages in glycans derived from blood serum glycoproteins. (United States)

    Alley, William R; Novotny, Milos V


    A number of alterations to the normal glycomic profile have been previously described for a number of diseases and disorders, thus underscoring the medical importance of studying the glycans associated with proteins present in biological samples. An important alteration in cancer progression is an increased level of alpha2,6-sialylation, which aids in increasing the metastatic potential of tumor cells. Here we report a glycomic method that selectively amidates alpha2,6-linked sialic acids, while those that are alpha2,3-linked undergo spontaneous lactonization. Following subsequent permethylation, MALDI-TOF MS analysis revealed that many sialylated glycans present on glycoproteins found in blood serum featured increased levels of alpha2,6-sialylation in breast cancer samples. On the basis of the altered ratios of alpha2,3-linked to alpha2,6-linked sialic acids, many of these glycans became diagnostically relevant when they did not act as such indicators when based on traditional glycomic profiling alone.

  3. Salivary agglutinin/glycoprotein-340/DMBT1

    DEFF Research Database (Denmark)

    Ligtenberg, Antoon J M; Veerman, Enno C I; Nieuw Amerongen, Arie V;


    Salivary agglutinin (SAG), lung glycoprotein-340 (gp-340) and Deleted in Malignant Brain Tumours 1 (DMBT1) are three names for identical proteins encoded by the dmbt1 gene. DMBT1/SAG/gp-340 belongs to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins, a superfamily of secreted o...

  4. Isolation of glycoproteins from brown algae

    DEFF Research Database (Denmark)


    The present invention relates to a novel process for the isolation of unique anti-oxidative glycoproteins from the pH precipitated fractions of enzymatic extracts of brown algae. Two brown seaweeds viz, Fucus serratus and Fucus vesiculosus were hydrolysed by using 3 enzymes viz, Alcalase, Viscozyme...

  5. Human alpha galactosidase and alpha 1,2fucosyltransferase concordantly inhibit xenoreactivity of NIH 3T3 cells with human serum

    Institute of Scientific and Technical Information of China (English)

    YANJing-Lian; YULu-Yang; GUOLi-He


    AIM: To study the influence of the expression of human alpha galactosidase and alphal,2 fucosyltransferase on Galalpha 1,3 Gal and consequent xenoreactivity in NIH3T3 cells. METHODS: The expression levels of G antigen andH antigen and binding of human natural antibodies (IgG and IgM) and complement (C3c) to NIH3T3 cells wereanalyzed by flow cytometry. Western blot was employed to further determine the expression of glycoproteins of Gantigen. Cytolysis assay with normal human serum was performed by MTT assay. RESULTS: Western blotshowed that glycoproteins with molecular weight of 107 kDa, 98 kDa, 88 kDa, 56 kDa, 40 kDa, and 37 kDa wereinhibited and even abrogated totally in alpha galactosidase transfectants and alpha 1,2 fucosyltransferase transfectants.The combined transfection of the two enzymes led to a much stronger inhibition of the glycoproteins. The bindingof Gs-IB4 was decreased by 57.4% in alpha galactosidase transfectants, 28.8% in alpha 1,2 fucosyltransferasetransfectants, and 72.1% in combined transfectants, respectively. In contrast, UEA-1 binding was increased about6.7-fold, 6.0-fold, and 8.0-fold respectively. The xenoreactivity with human IgG was also reduced by 61.4%, 67.0%,and 73.4%, respectively in the three kinds of transfectants. The resistance to cytolysis mediated by human serumwas enhanced by 42.4% in alpha galactosidase transfectants, 51.9% in alpha 1,2 fucosyltranferase, and even65.5% in the combined transfectants. CONCLUSION: Although alpha galactosidase and alpha 1,2 fucosyltransferasehad different biochemical properties, they could inhibit the expression of Gal alpha 1,3 Gal synergistically, leading tostronger resistance of xenograft against cytolysis.

  6. The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids. (United States)

    Roberts, D D; Haverstick, D M; Dixit, V M; Frazier, W A; Santoro, S A; Ginsburg, V


    The human platelet glycoprotein thrombospondin (TSP) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). Binding of 125I-TSP to lipids from sheep and human erythrocytes and human platelets resolved on thin layer chromatograms indicates that sulfatides are the only lipids in the membrane which bind TSP. Binding to less than 2 ng of sulfatide could be detected. TSP failed to bind to other purified lipids including cholesterol 3-sulfate, phospholipids, neutral glycolipids, and gangliosides. Binding of 125I-TSP was inhibited by unlabeled TSP, by low pH, and by reduction of intersubunit disulfide bonds with dithiothreitol. A monoclonal antibody against TSP (A2.5), which inhibits hemagglutination and agglutination of fixed activated platelets by TSP, strongly inhibited TSP binding to sulfatides. A second monoclonal antibody (C6.7), which inhibits hemagglutination and aggregation of thrombin-activated live platelets, weakly inhibited sulfatide binding. Binding was inhibited by high ionic strength and by some monosaccharide sulfates including methyl-alpha-D-GlcNAc-3-sulfate. Neutral sugars did not inhibit. Fucoidan, a sulfated fucan, strongly inhibited binding with 50% inhibition at 0.3 micrograms/ml fucoidan. Other sulfated polysaccharides including heparin and dextran sulfates were good inhibitors, whereas hyaluronic acid and keratan sulfate were very weak.

  7. Protein and glycoprotein abnormalities in platelets from human Chediak-Higashi syndrome: polyacrylamide gel electrophoretic study of platelets from five patients. (United States)

    Ledezma, E; Apitz-Castro, R


    Polyacrylamide electrophoretic analysis of proteins and Tritium-labelled glycoproteins of the platelets from five patients with Chediak-Higashi Syndrome shows the existence of marked quantitative differences when compared to normal platelets. While the glycoprotein abnormalities are solely related to the plasma membrane, some of the abnormalities detected in the Coomasie blue pattern are probably representative of defects related to the dense bodies and the alpha-granules. Some of the abnormalities found may, in part, explain the variability of aggregatory responses described in these patients, as well as the marked tendency towards desaggregation exhibited by platelets from humans with the Chediak-Higashi Syndrome.

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

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


    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

  9. [Glycoproteins, inherited diseases of platelets, and the role of platelets in wound healing]. (United States)

    Nurden, Alan T; Nurden, Paquita


    Recognition that platelets have a glycocalyx rich in membrane glycoproteins prompted the discovery in France that inherited bleeding syndromes due to defects of platelet adhesion and aggregation were caused by deficiencies in major receptors at the platelet surface. Identification of the alpha IIb beta3 integrin prompted the development of powerful anti-thrombotic drugs that have gained worldwide use. Since these discoveries, the genetic causes of many other defects of platelet function and production have been elucidated, with the identification of an ADP receptor, P2 Y12, another widespread target for anti-thrombotic drugs. Discovery of the molecular basis of a rare disease of storage of biologically active proteins in platelet alpha-granules has been accompanied by the recognition of the roles of platelets in inflammation, the innate immune system and tissue repair, opening new avenues for therapeutic advances.

  10. Fraction A of armadillo submandibular glycoprotein and its desialylated product as sialyl-Tn and Tn receptors for lectins. (United States)

    Wu, A M; Shen, F; Herp, A; Song, S C; Wu, J H


    Fraction A of the armadillo submandibular glycoprotein (ASG-A) is one of the simplest glycoproteins among mammalian salivary mucins. The carbohydrate side chains of this mucous glycoprotein have one-third of the NeuAc alpha 2-->6GalNAc (sialyl-Tn) sequence and two thirds of Tn (GalNAc alpha-->Ser/Thr) residues. Those of the desialylated product (ASG-Tn) are almost exclusively unsubstituted GalNAc residues (Tn determinant). When the binding properties of these glycoproteins were tested by a precipitin assay with Gal, GalNAc and GlcNAc specific lectins, it was found that ASG-Tn reacted strongly with all of the Tn-active lectins and completely precipitated Vicia villosa (VVL both B4 and mixture of A and B), Maclura pomifera (MPA), and Artocarpus integrifolia (jacalin) lectins. However, it precipitated poorly or negligibly with Ricinus communis (RCA1); Dolichos biflorus (DBA); Viscum album, ML-I; Arachis hypogaea (PNA), and Triticum vulgaris (WGA). The reactivity of ASG-A (sialyl-Tn) was as active as that of ASG-Tn with MPA and less or slightly less active than that of ASG-Tn with VVL-A+B, VVL-B4, HPA, WFA, and jacalin, as one-third of its Tn was sialylated. These findings indicate that ASG-A and its desialylated product (ASG-Tn) are highly useful reagents for the differentiation of Tn, T (Gal beta 1-->3GalNAc), A (GalNAc alpha 1-->3Gal) or Gal specific lectins and monoclonal antibodies against such epitopes.

  11. Analysis on effect of separation and purification of glycoprotein extracted from Camellia seeds and its functional activity as basis for the economic development of Camellia oleifera industry

    Directory of Open Access Journals (Sweden)

    Feng Aiguo


    Full Text Available Taking Camellia oleifera seeds as raw materials, this study explored extraction and purification of glycoprotein separated from Camellia seeds as well as its antitumor activity, aiming to provide a theoretical basis for the economic development of Camellia oleifera industry. Key impact factors of Camellia seed glycoprotein were extracted using buffer solution method and water extraction method and a regression model was set up. Methyl thiazolyl tetrazolium was used to evaluate the in vitro antitumor activity of glycoprotein extracted from Camellia seeds and Differential Scanning Calorimetry (DSC was used to measure its denaturation enthalpy value. Results indicated that protein and sugar yields were 8.96% and 17.05% respectively under optimal conditions when water extraction method was used. Crude glycoprotein extracted from Camellia oleifera had a certain inhibitory effect on human hepatoma cell HepG2, gastric cancer cell MGC-803 and breast cancer cell MCF-7 and crude glycoprotein extracted from Camellia oleifera by water-extraction and alcohol-precipitation method had a strong antitumor effect. Crude glycoprotein obtained in the two different ways was capable of scavenging DPPH, •OH and O2g- free radicals and also showed good reducing capacity. DSC measurement results revealed that specific rotation of COGP2a[α]n20${\\rm{COGP}}2{\\rm{a}}\\left[ \\alpha \\right]_n^{20} $ was - 32.5. Antitumor experiment in vitro showed that glycoprotein extracted from Camellia seeds in the two different ways had a certain inhibitory effect on HepG2, MGC-803 and MCF-7, which has important theoretical and realistic significances to promoting utilization value of camellia resources, strengthening Camellia oleifera’s comprehensive development and utilization of high added value as well as enriching types and functions of active glycoprotein.

  12. Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein

    NARCIS (Netherlands)

    Gagneten, S; Gout, O; Dubois-Dalcq, M; Rottier, P; Rossen, J; Holmes, K V


    In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions

  13. Glycoprotein degradation in the blind loop syndrome: identification of glycosidases in jejunal contents. (United States)

    Prizont, R


    Contents obtained from jejunum of normal controls, self-emptying and self-filling blind loop rats were analyzed for the presence of glycoprotein-degrading glycosidases. The blind loop syndrome was documented by the increased fat excretion and slower growth rate of self-filling blind loop rats 6 wk after surgery. With p-nitrophenylglycosides as substrate, the specific activity of alpha-N-acetylgalactosaminidase, a potential blood group A destroying glycosidase, was 0.90+/-0.40 mU/mg of protein. This level was 23-fold higher than the specific activity of normal controls. In partially purified self-filling blind loop contents, the activity of alpha-N-acetylgalactosaminidase was 9- to 70-fold higher than activities of self-emptying and normal controls. Antibiotic treatment with chloromycetin and polymyxin decreased 24-fold the glycosidase levels in self-filling blind loops. In experiments with natural substrate, the blood group A titer of a20,000g supernate from normal jejunal homogenates decreased 128-fold after 24-h incubation with blind loop contents. Normal contents failed to diminish the blood group reactivity of the natural substrate. Furthermore, blind loop contents markedly decreased the blood group A titer of isolated brush borders. Incubation between blind loop bacteria and mucosal homogenates or isolated brush borders labeled with d-[U-(14)C]glucosamine revealed increased production of labeled ether extractable organic acids. Likewise, intraperitoneal injection of d-[U-(14)C]glucosamine into self-filling blind loop rats resulted in incorporation of the label into luminal short chain fatty acids. These results suggest that glycosidases may provide a mechanism by which blind loop bacteria obtain sugars from intestinal glycoproteins. The released sugars are used and converted by bacteria into energy and organic acids. This use of the host's glycoproteins would allow blind loop bacteria to grow and survive within the lumen independent of exogenous sources.

  14. Ab initio alpha-alpha scattering

    CERN Document Server

    Elhatisari, Serdar; Rupak, Gautam; Epelbaum, Evgeny; Krebs, Hermann; Lähde, Timo A; Luu, Thomas; Meißner, Ulf-G


    Processes involving alpha particles and alpha-like nuclei comprise a major part of stellar nucleosynthesis and hypothesized mechanisms for thermonuclear supernovae. In an effort towards understanding alpha processes from first principles, we describe in this letter the first ab initio calculation of alpha-alpha scattering. We use lattice effective field theory to describe the low-energy interactions of nucleons and apply a technique called the adiabatic projection method to reduce the eight-body system to an effective two-cluster system. We find good agreement between lattice results and experimental phase shifts for S-wave and D-wave scattering. The computational scaling with particle number suggests that alpha processes involving heavier nuclei are also within reach in the near future.

  15. Native and asialo-Tamm-Horsfall glycoproteins as important ligands for the detection of GalNAc beta 1-->and Gal beta 1-->4GlcNAc active lectins. (United States)

    Wu, A M; Watkins, W M; Song, S C; Herp, A; Wu, J H


    The binding properties of human Tamm-Horsfall Sd(a+) urinary glycoprotein(THGP) and asialo-THGP with various applied lectins was investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins completely precipitated Abrus precatorius agglutinin(APA). They also reacted well with Wistaria floribunda (WFA), Glycine max (soybean, SBA), and Ricinus communis agglutinins and precipitated over 78% of the lectin nitrogen added, but reacted poorly or weakly with all alpha-anomeric GalNAc specific lectins, such as Helix pomatia (HPA), Phaseolus lunatus (lima bean, LBL), and Maclura pomifera (MPL) lectins. The glycoprotein-lectin interaction was inhibited by GalNAc beta 1-->, Gal beta 1-->4GlcNAc, or by both. The findings suggest that Sd (a+) THGP and asialo-THGP are among the best water-soluble glycoprotein ligands for GalNAc beta 1-->and Gal beta 1-->4GlcNAc active lectins.

  16. The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. (United States)

    Kounnas, M Z; Morris, R E; Thompson, M R; FitzGerald, D J; Strickland, D K; Saelinger, C B


    The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

  17. Primary structure of human alpha 2-macroglobulin. V. The complete structure

    DEFF Research Database (Denmark)

    Sottrup-Jensen, Lars; Stepanik, Terrence M; Kristensen, Torsten


    The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven...... in the activation cleavage area (the "bait" region) are located in the sequence: -Arg681-Val-Gly-Phe-Tyr-Glu-. The molecular weight of the unmodified alpha 2-macroglobulin subunit is 160,837 and approximately 179,000, including the carbohydrate groups. The presence of possible internal homologies within the alpha 2...

  18. Isolation and partial characterization of rat gastric mucous glycoprotein

    NARCIS (Netherlands)

    Spee-Brand, R.; Strous, G.J.A.M.; Kramer, M.F.


    Mucus glycoproteins from the rat stomach were characterized after their isolation from homogenates of the superficial gastric mucosa by equilibrium centrifugation in CsCl density gradients. Water-soluble as well as water-insoluble glycoproteins were studied. The latter were solubilized by 2-mercapto

  19. Faddeev calculation of 3 alpha and alpha alpha Lambda systems using alpha alpha resonating-group method kernel

    CERN Document Server

    Fujiwara, Y; Kohno, M; Suzuki, Y; Baye, D; Sparenberg, J M


    We carry out Faddeev calculations of three-alpha (3 alpha) and two-alpha plus Lambda (alpha alpha Lambda) systems, using two-cluster resonating-group method kernels. The input includes an effective two-nucleon force for the alpha alpha resonating-group method and a new effective Lambda N force for the Lambda alpha interaction. The latter force is a simple two-range Gaussian potential for each spin-singlet and triplet state, generated from the phase-shift behavior of the quark-model hyperon-nucleon interaction, fss2, by using an inversion method based on supersymmetric quantum mechanics. Owing to the exact treatment of the Pauli-forbidden states between the clusters, the present three-cluster Faddeev formalism can describe the mutually related, alpha alpha, 3 alpha and alpha alpha Lambda systems, in terms of a unique set of the baryon-baryon interactions. For the three-range Minnesota force which describes the alpha alpha phase shifts quite accurately, the ground-state and excitation energies of 9Be Lambda are...

  20. Solubilization of glycoproteins of envelope viruses by detergents

    Energy Technology Data Exchange (ETDEWEB)

    Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.; Isaeva, E.S.; Zhdanov, V.M.


    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. 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.

  1. P-glycoprotein acts as an immunomodulator during neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Gijs Kooij

    Full Text Available BACKGROUND: Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system in which autoreactive myelin-specific T cells cause extensive tissue damage, resulting in neurological deficits. In the disease process, T cells are primed in the periphery by antigen presenting dendritic cells (DCs. DCs are considered to be crucial regulators of specific immune responses and molecules or proteins that regulate DC function are therefore under extensive investigation. We here investigated the potential immunomodulatory capacity of the ATP binding cassette transporter P-glycoprotein (P-gp. P-gp generally drives cellular efflux of a variety of compounds and is thought to be involved in excretion of inflammatory agents from immune cells, like DCs. So far, the immunomodulatory role of these ABC transporters is unknown. METHODS AND FINDINGS: Here we demonstrate that P-gp acts as a key modulator of adaptive immunity during an in vivo model for neuroinflammation. The function of the DC is severely impaired in P-gp knockout mice (Mdr1a/1b-/-, since both DC maturation and T cell stimulatory capacity is significantly decreased. Consequently, Mdr1a/1b -/- mice develop decreased clinical signs of experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis. Reduced clinical signs coincided with impaired T cell responses and T cell-specific brain inflammation. We here describe the underlying molecular mechanism and demonstrate that P-gp is crucial for the secretion of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Importantly, the defect in DC function can be restored by exogenous addition of these cytokines. CONCLUSIONS: Our data demonstrate that P-gp downmodulates DC function through the regulation of pro-inflammatory cytokine secretion, resulting in an impaired immune response. Taken together, our work highlights a new physiological role for P-gp as an immunomodulatory molecule and reveals a possible

  2. Characterization of salivary alpha-amylase binding to Streptococcus sanguis

    Energy Technology Data Exchange (ETDEWEB)

    Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.; Levine, M.J. (State Univ. of New York, Buffalo (USA))


    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.

  3. Pumping of drugs by P-glycoprotein

    DEFF Research Database (Denmark)

    Litman, Thomas; Skovsgaard, Torben; Stein, Wilfred D


    The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different...... levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows...... that Kapp for inhibition should increase linearly with the amount of pumps present in the membrane for a reverser that inhibits pumping from the cytoplasmic face. In contrast, if the reverser acts by blocking transport from the outer face, i.e., preemptively, Kapp should be independent of the number...

  4. Separation and characterization of mares' milk alpha(s1)-, beta-, kappa-caseins, gamma-casein-like, and proteose peptone component 5-like peptides. (United States)

    Egito, A S; Miclo, L; López, C; Adam, A; Girardet, J M; Gaillard, J L


    The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, presented strong identities with regions of alpha(s1)-CN primary structures of other species. In particular, 51, 48, 43, and 40% identities were obtained with corresponding regions of sow, dromedary, cow, and human alpha(s1)-CN, respectively. On the other hand, trace amounts of equine gamma-CN-like and proteose peptone component 5-like peptides were found in the whole CN. They were identified by microsequencing and corresponded to beta-CN peptides generated by plasmin action on the whole CN. The equine alpha(s1), beta-, and kappa-CN were separated by bidimensional electrophoresis in numerous isoelectric variants with apparent isoelectric points distributed between pH 4.4 to 6.3, 4.4 to 5.9, and 3.5 to 5.5, respectively. The beta- and kappa-CN displayed a more acidic character in the mare than in the cow.

  5. Immunological aspects of pregnancy-associated glycoproteins. (United States)

    Dosogne, H; Massart-Leën, A M; Burvenich, C


    The incidence of severe cases of acute E. coli mastitis in dairy cows is highest during early lactation. This phenomenon has been associated with a decreased function and decreased numbers of circulating polymorphonuclear neutrophil leukocytes (PMN). The cause of this impaired function and decreased number is poorly understood. Stress, hormonal and metabolic alterations around parturition and the onset of lactation may play a role in this phenomenon. Several molecules, such as cortisol and beta-hydroxybutyrate have been found to alter the oxidative burst activity of circulating PMN around parturition. Pregnancy-Associated Glycoprotein (bPAG) could also be involved. The theory of immunosuppression by bPAG was investigated because analogous glycoproteins produced by the placenta of other species exert local immunosuppression in order to maintain the histoincompatible feto-maternal unit. The production and subsequent release into the maternal circulation of bPAG is ensured by the binucleate cells from the trophoblast and starts already at implantation. However, peak levels are only reached 1 week before parturition. Due to the long half-life time of this molecule, high levels are found in plasma until 2 weeks after calving. The co-occurrence of the impairment of PMN oxidative burst activity in the early postpartum period and a peak in plasma bPAG concentrations might support the hypothesis of an immunosuppressive effect of PAG. Moreover, an inhibitory effect of bPAG on the proliferation of bovine bone marrow progenitor cells has been found recently in our laboratory. bPAG occurs in colostrum, but its effect on milk cells has not been clarified. It is concluded that interaction between the physiology of reproduction and lactation on the one side and immune function on the other side in dairy cattle requires further research.

  6. Core Structure of S2 from the Human Coronavirus NL63 Spike Glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Zheng,Q.; Deng, Y.; Liu, J.; van der Hoek, L.; Berkhout, B.; Lu, M.


    Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an {alpha}-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 {sup o}C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.

  7. The determination of $\\alpha_s$ by the ALPHA collaboration

    CERN Document Server

    Bruno, Mattia; Fritzsch, Patrick; Korzec, Tomasz; Ramos, Alberto; Schaefer, Stefan; Simma, Hubert; Sint, Stefan; Sommer, Rainer


    We review the ALPHA collaboration strategy for obtaining the QCD coupling at high scale. In the three-flavor effective theory it avoids the use of perturbation theory at $\\alpha > 0.2$ and at the same time has the physical scales small compared to the cutoff $1/a$ in all stages of the computation. The result $\\Lambda_\\overline{MS}^{(3)}=332(14)$~MeV is translated to $\\alpha_\\overline{MS}(m_Z)=0.1179(10)(2)$ by use of (high order) perturbative relations between the effective theory couplings at the charm and beauty quark "thresholds". The error of this perturbative step is discussed and estimated as $0.0002$.

  8. [Glycoprotein hexoses in feces of infants with lactose intolerance]. (United States)

    Filippvskiĭ, G K; Klimov, L Ia


    A modified method for estimation of total glycoprotein hexoses in feces, based on their measurements in the blood serum, is presented. Sixty-six nursing children with lactose intolerance, breastfed or formula fed, were examined; formula fed babies were kept on mixtures with high and low lactose content. Glycoprotein hexose parameters were as follows (X +/- m): 13.51 +/- 1.93, 12.05 +/- 2.20, and 3.69 +/- 0.47 g/l feces. In control children without lactose intolerance (n = 33) this value was 3.6 +/- 0.79 g/l. Increased glycoprotein excretion is connected with glycocalix and small intestinal enterocyte alteration.

  9. Lectin-based glycoproteomics to explore and analyze hepatocellular carcinoma-related glycoprotein markers. (United States)

    Dai, Zhi; Zhou, Jian; Qiu, Shuang-Jian; Liu, Yin-Kun; Fan, Jia


    More and more new diagnostic biomarkers of hepatocellular carcinoma (HCC) have been found in association with advances in the standardization of 2-DE coupled with MS analysis. However, the diagnosis of HCC is still detected in the late stages of the disease, when treatment options are limited and prognosis is poor. The glycosylation of proteins is known to change in tumor cells during the development of HCC as the result of alterations in the levels of glycosyltransferases, such as increased fucosylation of Golgi Protein 73 and alpha-fetoprotein. These structural changes can influence the function or physiochemical properties of a protein, resulting in abnormal cancer cell behavior. Therefore, identification of HCC-related glycoprotein markers and analysis of glycan structural alterations might assist in the early detection of HCC. Here, we summarize lectin-based glycoproteomic strategies for the discovery of relevant biomarkers of HCC. The carbohydrate-binding specificities of different lectins offer a biological affinity approach that complements existing MS capabilities. These strategies involve the enrichment of glycoproteins or glycopeptides by lectins, followed by releasing carbohydrates with peptide-N-glycosidase F or reductive beta-elimination. The obtained glycopeptides are then identified by automated MS/MS and structural analysis of glycans is performed through modern methods such as quadrupole IT-TOF, MALDI-TOF/TOF and lectin microarray. These strategies will lead to faster and more clinically adaptable tests with greater sensitivity and specificity.

  10. Synthesis of mucin-type glycoprotein; Muchingata to tanbakushitsu no gosei

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, M. [Tokyo Inst. of Tech., Tokyo (Japan)


    A mucin-type glycoprotein has a configuration in which serine or threonine is bonded at 1-position of N-acetyl galactosamine (GalNAc) with a sugar chain of galactose or sialic acid bonded thereto. The mucin-type glycoprotein is paid attention in researches in respect to the progress and dislocation of carcinoma. A method used in conventional synthesis of such a kind of compounds is to introduce an amino acid residue after the whole sugar chain portion is constructed. However, there is risk in this synthesis that great loss in yield may occur in the final stage. Danishefsky et al. noticed that, for a mucin-type protein, the first sugar bonded to the amino acid is GalNAc, and therefore suggested a new synthesizing method named mucin-type cassette approach. Specifically, this method is characterized in introducing serine or threonine to 1-position of a GalNAc derivative to obtain GalNAc{alpha}l-Ser/Thr derivative, and then extending a sugar chain thereto. As a consequence, cassette approach shows effectiveness in syntheses of mucin-type proteins. (NEDO)

  11. Cholesterol-mediated activation of P-glycoprotein: distinct effects on basal and drug-induced ATPase activities. (United States)

    Belli, Sara; Elsener, Priska M; Wunderli-Allenspach, Heidi; Krämer, Stefanie D


    Cholesterol promotes basal and verapamil-induced ATPase activity of P-glycoprotein (P-gp). We investigated whether these effects are related to each other and to the impact of the sterol on bilayer fluidity and verapamil membrane affinity. P-gp was reconstituted in egg-phosphatidylcholine (PhC) liposomes with or without cholesterol, 1,2-dipalmitoyl-phosphatidylcholine (DPPC), alpha-tocopherol (alpha-Toc) or 2,2,5,7,8-pentamethyl-6-chromanol (PMC). Basal and verapamil-induced ATPase activities were studied with an enzymatic assay. Membrane fluidity was characterized with diphenyl-hexatriene anisotropy measurements and membrane affinity by equilibrium dialysis. DPPC (70% mol/mol) decreased the fluidity of PhC bilayers to the same level as 20% cholesterol. PMC (20%) and alpha-Toc (20%) decreased the fluidity to lesser extents. alpha-Toc and PMC, but not DPPC increased the verapamil membrane affinity. While 20% cholesterol strikingly enhanced the basal ATPase activity, none of the other constituents had a similar effect. In contrast, verapamil stimulation of P-gp ATPase activity was not only enabled by cholesterol but also by alpha-Toc and DPPC. PMC had no effect. In conclusion, cholesterol exerts distinct effects on basal and verapamil-induced ATPase activity. The influence on basal ATPase activity is sterol-specific while its effect on verapamil-induced ATPase activity is unspecific and not related to its influence on membrane fluidity and on verapamil membrane affinity.

  12. Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.). (United States)

    Ali, M S; Mitsui, T; Akazawa, T


    Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.

  13. Overview of varicella-zoster virus glycoproteins gC, gH and gL. (United States)

    Grose, Charles; Carpenter, John E; Jackson, Wallen; Duus, Karen M


    The VZV genome is smaller than the HSV genome and only encodes nine glycoproteins. This chapter provides an overview of three VZV glycoproteins: gH (ORF37), gL (ORF60), and gC (ORF14). All three glycoproteins are highly conserved among the alpha herpesviruses. However, VZV gC exhibits unexpected differences from its HSV counterpart gC. In particular, both VZV gC transcription and protein expression are markedly delayed in cultured cells. These delays occur regardless of the virus strain or the cell type, and may account in part for the aberrant assembly of VZV particles. In contrast to VZV gC, the general properties of gH and gL more closely resemble their HSV homologs. VZV gL behaves as a chaperone protein to facilitate the maturation of the gH protein. The mature gH protein in turn is a potent fusogen. Its fusogenic activity can be abrogated when infected cultures are treated with monoclonal anti-gH antibodies.

  14. Detection of glycoproteins in the Acanthamoeba plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Paatero, G.I.L. (Abo Akademi (Finland)); Gahmberg, C.G. (Univ. of Helsinki (Finland))


    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 presence 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.

  15. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

    Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

  16. New ALPHA-2 magnet

    CERN Multimedia

    Anaïs Schaeffer


    On 21 June, members of the ALPHA collaboration celebrated the handover of the first solenoid designed for the ALPHA-2 experiment. The magnet has since been successfully installed and is working well.   Khalid Mansoor, Sumera Yamin and Jeffrey Hangst in front of the new ALPHA-2 solenoid. “This was the first of three identical solenoids that will be installed between now and September, as the rest of the ALPHA-2 device is installed and commissioned,” explains ALPHA spokesperson Jeffrey Hangst. “These magnets are designed to allow us to transfer particles - antiprotons, electrons and positrons - between various parts of the new ALPHA-2 device by controlling the transverse size of the particle bunch that is being transferred.” Sumera Yamin and Khalid Mansoor, two Pakistani scientists from the National Centre for Physics in Islamabad, came to CERN in February specifically to design and manufacture these magnets. “We had the chance to work on act...

  17. Lyman Alpha Control

    CERN Document Server

    Nielsen, Daniel Stefaniak


    This document gives an overview of how to operate the Lyman Alpha Control application written in LabVIEW along with things to watch out for. Overview of the LabVIEW code itself as well as the physical wiring of and connections from/to the NI PCI-6229 DAQ box is also included. The Lyman Alpha Control application is the interface between the ALPHA sequencer and the HighFinesse Wavelength Meter as well as the Lyman Alpha laser setup. The application measures the wavelength of the output light from the Lyman Alpha cavity through the Wavelength Meter. The application can use the Wavelength Meter’s PID capabilities to stabilize the Lyman Alpha laser output as well as switch between up to three frequencies.

  18. Interpreting EEG alpha activity. (United States)

    Bazanova, O M; Vernon, D


    Exploring EEG alpha oscillations has generated considerable interest, in particular with regards to the role they play in cognitive, psychomotor, psycho-emotional and physiological aspects of human life. However, there is no clearly agreed upon definition of what constitutes 'alpha activity' or which of the many indices should be used to characterize it. To address these issues this review attempts to delineate EEG alpha-activity, its physical, molecular and morphological nature, and examine the following indices: (1) the individual alpha peak frequency; (2) activation magnitude, as measured by alpha amplitude suppression across the individual alpha bandwidth in response to eyes opening, and (3) alpha "auto-rhythmicity" indices: which include intra-spindle amplitude variability, spindle length and steepness. Throughout, the article offers a number of suggestions regarding the mechanism(s) of alpha activity related to inter and intra-individual variability. In addition, it provides some insights into the various psychophysiological indices of alpha activity and highlights their role in optimal functioning and behavior.

  19. Alpha Shapes and Proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Sterner, Henrik; Sterner, Peter


    We provide a unified description of (weighted) alpha shapes, beta shapes and the corresponding simplicialcomplexes. We discuss their applicability to various protein-related problems. We also discuss filtrations of alpha shapes and touch upon related persistence issues.We claim that the full...... potential of alpha-shapes and related geometrical constructs in protein-related problems yet remains to be realized and verified. We suggest parallel algorithms for (weighted) alpha shapes, and we argue that future use of filtrations and kinetic variants for larger proteins will need such implementation....

  20. Effect of the carbohydrate moiety on the secondary structure of beta 2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins. (United States)

    Walsh, M T; Watzlawick, H; Putnam, F W; Schmid, K; Brossmer, R


    By use of six highly purified exoglycosidases with well-defined specificity, the oligosaccharide units of human plasma beta 2-glycoprotein I (beta 2I) were modified by sequential enzymatic degradation. The released monosaccharides (NeuAc, Gal, GlcNAc, and Man) were quantified, and the carbohydrate compositions of the resulting glycoprotein (gp) derivatives were determined. The gp was found to be both partially sialylated and galactosylated. These findings which are in agreement with earlier reports suggest that the carbohydrate moiety of beta 2I possesses more bi- than tri-antennas, probably three of the former and two of the latter carbohydrate units. Circular dichroic (CD) spectra of native beta 2I and its derivatives were measured in aqueous buffer and 2-chloroethanol (2-CE). Analysis of these spectra for elements of secondary structure showed beta 2I and most of the derivatives to contain predominantly beta-sheet and beta-turn structures. The lack of alpha-helical structures in aqueous buffer was noted. Removal of a large portion of the carbohydrate moiety did not alter the CD spectra or secondary structure of beta 2I in either aqueous buffer or in 2-CE. However, after enzymatic removal of approximately 96% of the carbohydrate moiety, large significant changes in the spectra and secondary structures were observed. In aqueous buffer a shift in the wavelength minimum occurred, accompanied by an increase in the magnitude of the molar ellipticity and the amount of beta-turn, with a reduction in random coil. One-third of the amino acids which were originally in random coil conformation assumed beta-turns after removal of 96% of the carbohydrate moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid: re-examination of the Ustilago maydis glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.; Champ, D.R.; Young, J.L.; Grant, C.E.


    Holloman reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.

  2. P-glycoprotein targeted nanoscale drug carriers

    KAUST Repository

    Li, Wengang


    Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug carrying processes that shuttle the drugs out of tumor cells. Thus, P -gp inhibitors have attracted a lot of attention as they can stop cancer drugs from being pumped out of target cells with the consumption of ATP. Using quantitive structure activity relationship (QSAR), we have successfully synthesized a series of novel P -gp inhibitors. The obtained dihydropyrroloquinoxalines series were fully characterized and then tested against bacterial and tumor assays with over-expressed P -gps. All compounds were bioactive especially compound 1c that had enhanced antibacterial activity. Furthermore, these compounds were utilized as targeting vectors to direct drug delivery vehicles such as silica nanoparticles (SNPs) to cancerous Hela cells with over expressed P -gps. Cell uptake studies showed a successful accumulation of these decorated SNPs in tumor cells compared to undecorated SNPs. The results obtained show that dihydropyrroloquinoxalines constitute a promising drug candidate for targeting cancers with MDR. Copyright © 2013 American Scientific Publishers All rights reserved.

  3. Differential sensitivity of human, avian, and equine influenza A viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants. (United States)

    Rogers, G N; Pritchett, T J; Lane, J L; Paulson, J C


    Human and animal (avian and equine) influenza A virus isolates of the H3 serotype exhibit marked differences in their ability to bind specific sialyloligosaccharide sequences that serve as cell surface receptor determinants (G. Rogers and J. Paulson, 1983, Virology 127, 361-373). Whereas human isolates of this subtype strongly agglutinate enzymatically modified human erythrocytes containing the terminal SA alpha 2,6Gal sequence, avian and equine isolates preferentially agglutinate erythrocytes bearing the SA alpha 2, 3Gal sequence. As shown in this report, a glycoprotein found in horse serum, alpha 2-macroglobulin, is a potent inhibitor of viral adsorption to the cell surface for human H3 isolates. In contrast, avian and equine isolates are poorly inhibited suggesting a correlation between receptor specificity and inhibitor sensitivity. Growth of a human H3 isolate (A/Memphis/102/72) on MDCK cells in the presence of horse serum resulted in an overall shift in the virus receptor specificity from preferential binding of the SA alpha 2,6Gal linkage to preferential binding of the SA alpha 2,3Gal linkage characteristic of avian and equine isolates. Clonally isolated variants of A/Memphis/102/72 grown in the presence or absence of horse serum exhibited binding properties that account for those observed in the field isolates. Clones which preferentially bound the SA alpha 2,6Gal linkage, like the parent human virus, were very sensitive to inhibition of hemagglutination by horse serum and equine alpha 2-macroglobulin. In contrast, receptor variants which preferentially bound the SA alpha 2,3Gal linkage, like the avian and equine isolate, were insensitive to such inhibitors. None of the variants was very sensitive to inhibition of hemagglutination by human alpha 2-macroglobulin. These results suggest that the presence, in vivo, of a glycoprotein inhibitor such as equine alpha 2-macroglobulin could suppress infection of influenza viruses bearing an H3 hemagglutinin with a SA

  4. An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

    Energy Technology Data Exchange (ETDEWEB)

    Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B. (Univ. of Alabama, Birmingham (USA))


    UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ((beta-35S)UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with (beta-35S). UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue.

  5. Buffett’s Alpha

    DEFF Research Database (Denmark)

    Frazzini, Andrea; Kabiller, David; Heje Pedersen, Lasse

    Berkshire Hathaway has realized a Sharpe ratio of 0.76, higher than any other stock or mutual fund with a history of more than 30 years, and Berkshire has a significant alpha to traditional risk factors. However, we find that the alpha becomes insignificant when controlling for exposures to Betting...

  6. Therapeutic effect of anti-feline TNF-alpha monoclonal antibody for feline infectious peritonitis. (United States)

    Doki, Tomoyoshi; Takano, Tomomi; Kawagoe, Kohei; Kito, Akihiko; Hohdatsu, Tsutomu


    Feline infectious peritonitis virus (FIPV) replication in macrophages/monocytes induced tumor necrosis factor (TNF)-alpha production, and that the TNF-alpha produced was involved in aggravating the pathology of FIP. We previously reported the preparation of a feline TNF-alpha (fTNF-alpha)-neutralizing mouse monoclonal antibody (anti-fTNF-alpha mAb). This anti-fTNF-alpha mAb 2-4 was confirmed to inhibit the following fTNF-alpha-induced conditions in vitro. In the present study, we investigated whether mAb 2-4 improved the FIP symptoms and survival rate of experimentally FIPV-inoculated SPF cats. Progression to FIP was prevented in 2 out of 3 cats treated with mAb 2-4, whereas all 3 cats developed FIP in the placebo control group. Plasma alpha1-glycoprotein and vascular endothelial growth factor levels were improved by the administration of mAb 2-4, and the peripheral lymphocyte count also recovered. These results strongly suggested that the anti-fTNF-alpha antibody is effective for the treatment of FIP.

  7. N-glycoprotein analysis discovers new up-regulated glycoproteins in colorectal cancer tissue. (United States)

    Nicastri, Annalisa; Gaspari, Marco; Sacco, Rosario; Elia, Laura; Gabriele, Caterina; Romano, Roberto; Rizzuto, Antonia; Cuda, Giovanni


    Colorectal cancer is one of the leading causes of death due to cancer worldwide. Therefore, the identification of high-specificity and -sensitivity biomarkers for the early detection of colorectal cancer is urgently needed. Post-translational modifications, such as glycosylation, are known to play an important role in cancer progression. In the present work, we used a quantitative proteomic technique based on (18)O stable isotope labeling to identify differentially expressed N-linked glycoproteins in colorectal cancer tissue samples compared with healthy colorectal tissue from 19 patients undergoing colorectal cancer surgery. We identified 54 up-regulated glycoproteins in colorectal cancer samples, therefore potentially involved in the biological processes of tumorigenesis. In particular, nine of these (PLOD2, DPEP1, SE1L1, CD82, PAR1, PLOD3, S12A2, LAMP3, OLFM4) were found to be up-regulated in the great majority of the cohort, and, interestingly, the association with colorectal cancer of four (PLOD2, S12A2, PLOD3, CD82) has not been hitherto described.

  8. Laser assisted {alpha} decay

    Energy Technology Data Exchange (ETDEWEB)

    Castaneda Cortes, Hector Mauricio


    Excited or short-lived nuclei often decay by emitting alpha particles that are assumed to be preformed inside the nucleus and confined in the nuclear potential well. In this picture, {alpha} decay refers to the tunneling of the alpha particle through the potential barrier. In this thesis we investigate for the first time how strong laser fields can assist the tunneling of the alpha particle and thus influence the nuclear decay. Generally speaking, laser-assisted {alpha} decay can be described as laser-assisted tunneling of a quasistationary state, i.e, a slowly decaying state. Our theoretical treatment is developed starting from the complex trajectory formulation of the well-known strong-field approximation used to describe laser-induced ionization. We extend this formulation and develop a method to treat the decay of quasistationary states. The effect of both static and optical and X-ray monochromatic fields on the lifetimes and {alpha}-particle emission spectra are investigated for a number of {alpha}-emitting nuclei. We find that even at strong intensities, the laser-induced acceleration of the {alpha} decay is negligible, ranging from a relative modification in the decay rate of 10{sup -3} for static fields of electric field strengths of 10{sup 15} V/m, to 10{sup -8} for strong optical fields with intensities of 10{sup 22} W/cm{sup 2}, and to 10{sup -6} for strong X-ray fields with laser intensities around 10{sup 24} W/cm{sup 2}. However, the effect of the external field is visible in the spectrum of emitted alpha particles, leading in the case of optical fields even to rescattering phenomena for intensities approaching 6 x 10{sup 22} W/cm{sup 2}. The dynamics of the alpha particle in laser fields of intensities below the rescattering limit is investigated.

  9. Role of zona pellucida glycoproteins during fertilization in humans. (United States)

    Gupta, Satish Kumar


    In the last decade, scientific investigations pertaining to the role of zona pellucida (ZP) glycoproteins during fertilization in humans have led to new insights. This has been achieved using purified native/recombinant human zona proteins and transgenic mice expressing human ZP glycoproteins. The proposed model in mice of ZP glycoprotein-3 (ZP3) acting as primary sperm receptor and ZP glycoprotein-2 (ZP2) as secondary sperm receptor has been modified for sperm-egg binding in humans. ZP glycoprotein-1 (ZP1), ZP3, and ZP glycoprotein-4 (ZP4) have been shown to bind to the capacitated human sperm. ZP2 binds to the acrosome-reacted human spermatozoa. Further, the eggs obtained from transgenic mice expressing human ZP2 alone or in conjunction with other human instead of mouse zona proteins showed binding of human sperm, suggesting that ZP2 might also play a role in sperm-egg binding. This function has been mapped to a domain corresponding to amino acid residues 51-144 of ZP2. In contrast to mice, where ZP3 is the primary agonist for inducing the acrosome reaction, in humans, the acrosome reaction can be mediated by ZP1, ZP3, and ZP4. The effect of mutations in the genes encoding zona proteins on the ZP morphology and infertility has not been established. Further, the role of autoantibodies against ZP in women with 'unexplained infertility' leading to poor outcome of in vitro fertilization is currently controversial and needs further investigations. Understanding the role of ZP glycoproteins during human fertilization facilitates the development of new contraceptives and strategies to overcome the problem of infertility.

  10. Acrosome reaction: relevance of zona pellucida glycoproteins

    Institute of Scientific and Technical Information of China (English)

    Satish K Gupta; Beena Bhandari


    During mammalian fertilisation,the zona pellucida(ZP)matrix surrounding the oocyte is responsible for the binding of the spermatozoa to the oocyte and induction of the acrosome reaction(AR)in the ZP-bound spermatozoon.The AR is crucial for the penetration of the ZP matrix by spermatozoa.The ZP matrix in mice is composed of three glycoproteins designated ZP1,ZP2 and ZP3,whereas in humans,it is composed of four(ZP1,ZP2,ZP3 and ZP4).ZP3 acts as the putative primary sperm receptor and is responsible for AR induction in mice,whereas in humans(in addition to ZP3),ZP1 and ZP4 also induce the AR.The ability of ZP3 to induce the AR resides in its C-terminal fragment.O-linked glycans are critical for the murine ZP3-mediated AR.However,N-linked glycans of human ZP1,ZP3 and ZP4 have important roles in the induction of the AR.Studies with pharmacological inhibitors showed that the ZP3-induced AR involves the activation of the G1-coupled receptor pathway,whereas ZP1-and ZP4-mediated ARs are independent of this pathway.The ZP3-induced AR involves the activation of T-type voltage-operated calcium channels(VOCCs),whereas ZP1-and ZP4-induced ARs involve both T-and L-type VOCCs.To conclude,in mice,ZP3 is primarily responsible for the binding of capacitated spermatozoa to the ZP matrix and induction of the AR,whereas in humans(in addition to ZP3),ZP1 and ZP4 also participate in these stages of fertilisation.

  11. Genetics Home Reference: alpha thalassemia (United States)

    ... Understand Genetics Home Health Conditions alpha thalassemia alpha thalassemia Enable Javascript to view the expand/collapse boxes. Download PDF Open All Close All Description Alpha thalassemia is a blood disorder that reduces the production ...

  12. Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris

    Institute of Scientific and Technical Information of China (English)

    ZHAO Li-hui; YU Xiang-hui; JIANG Chun-lai; WU Yong-ge; SHEN Jia-cong; KONG Wei


    To obtain a sufficient amount of glycoprotein for further studying the structure and function of HIV-1 envelope glycoprotein, amplified and modified HIV-1 envelope glycoprotein gene which recombined subtypes(850amino acids) from Guangxi in China was inserted into Pichiapastoris expression vector pPICZaB; then the recombinant plasmid was transported into the yeast cells to induce the expression of Env protein with methanol. The results of SDS-PAGE and Western blot indicate that the envelope glycoprotein could be expressed in Pichia pastoris with productions of a 120000 glycoprotein and a 41000 glycoprotein, which showed satisfactory immunogenicity by indirect ELISA.

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

    Directory of Open Access Journals (Sweden)

    Fun-In Wang


    Full Text Available 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.

  14. Glycoprotein labeling with click chemistry (GLCC) and carbohydrate detection. (United States)

    Wu, Zhengliang L; Huang, Xinyi; Burton, Andrew J; Swift, Karl A D


    Molecular labeling and detection techniques are essential to research in life science. Here, a method for glycoprotein labeling/carbohydrate detection through glycan replacement, termed glycoprotein labeling with click chemistry (GLCC), is described. In this method, a glycoprotein is first treated with specific glycosidases to remove certain sugar residues, a procedure that creates acceptor sites for a specific glycosyltransferase. A 'clickable' monosaccharide is then installed onto these sites by the glycosyltransferase. This modified glycoprotein is then conjugated to a reporter molecule using a click chemistry reaction. For glycoproteins that already contain vacant glycosylation sites, deglycosylation is not needed before the labeling step. As a demonstration, labeling on fetal bovine fetuin, mouse immunoglobulin IgG and bacterial expressed human TNFα and TNFβ are shown. Compared to traditional ways of protein labeling, labeling at glycosylation sites with GLCC is considerably more specific and less likely to have adverse effects, and, when utilized as a method for carbohydrate detection, this method is also highly specific and sensitive.

  15. Proteolysis of specific porcine zona pellucida glycoproteins by boar acrosin. (United States)

    Dunbar, B S; Dudkiewicz, A B; Bundman, D S


    The morphologic and biochemical effects on the structure and constituent glycoproteins of the zona pellucida (ZP) by a specific sperm enzyme, acrosin, and a nonsperm enzyme, trypsin, have been evaluated. Intact porcine ZP matricies, exposed to either acrosin or trypsin, were analyzed microscopically. Changes in specific glycoproteins were monitored by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and the silver-based color stain, GELCODE. Although these enzymes did not alter the macroscopic properties of the ZP matrix, the 2D-PAGE ZP protein patterns were markedly altered. The high molecular weight glycoprotein families (II and III) were sensitive to proteolytic digestion, whereas the major glycoprotein family (I) of the porcine zona was only partially proteolyzed by acrosin and trypsin. Furthermore, it was demonstrated that acrosin had unique substrate specificity compared to that of trypsin, since the ZP peptide patterns were found to be different. These studies are the first to demonstrate which integral glycoproteins of the native porcine ZP matrix are specifically proteolyzed by acrosin from the homologous species and that this proteolysis occurs without the dissolution of the native porcine matrix.

  16. alpha_s from tau decays revisited

    CERN Document Server

    Boito, D; Golterman, M; Jamin, M; Maltman, K; Osborne, J; Peris, S


    Being a determination at low energies, the analysis of hadronic tau decay data provides a rather precise determination of the strong coupling alpha_s after evolving the result to M_Z. At such a level of precision, even small non-perturbative effects become relevant for the central value and error. While those effects had been taken into account in the framework of the operator product expansion, contributions going beyond it, so-called duality violations, have previously been neglected. The following investigation fills this gap through a finite-energy sum rule analysis of tau decay spectra from the OPAL experiment, including duality violations and performing a consistent fit of all appearing QCD parameters. The resulting values for alpha_s(M_tau) are 0.307(19) in fixed-order perturbation theory and 0.322(26) in contour-improved perturbation theory, which translates to the n_f=5 values 0.1169(25) and 0.1187(32) at M_Z, respectively.

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

    Energy Technology Data Exchange (ETDEWEB)

    Namazue, J.; Campo-Vera, H.; Kitamura, K.; Okuno, T.; Yamanishi, K.


    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 gp 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.

  18. Retroviral Env Glycoprotein Trafficking and Incorporation into Virions

    Directory of Open Access Journals (Sweden)

    Tsutomu Murakami


    Full Text Available Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.

  19. Alpha-1 Antitrypsin Deficiency (United States)

    ... by blood tests showing the low levels of alpha-1 antitrypsin and abnormal liver tests. Other tests such as ultrasound imaging or tests using specialized X-ray techniques may be necessary. A liver biopsy may ...

  20. Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.

    Directory of Open Access Journals (Sweden)

    Marisa J Fortunato

    Full Text Available Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5-2, 29-5, and 45-3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

  1. AlphaACT (United States)


    CBR can be found in the world around us (e.g., a doctor’s diagnosis based on a prior patient case, a lawyer preparing arguments based on legal ... metaphors in decision research. Judgment & Decision Making, 3(3), 195-204. Payne, J., Bettman, J., & Johnson, E. (1993). The Adaptive Decision Maker...alphaact. com 64 AlphaACT HAZMAT User Guide 8.3 Changing Measurement Units AlphaACT HAZMAT lets you show distances in either metric or English

  2. Square-wave voltammetry assays for glycoproteins on nanoporous gold (United States)

    Pandey, Binod; Bhattarai, Jay K.; Pornsuriyasak, Papapida; Fujikawa, Kohki; Catania, Rosa; Demchenko, Alexei V.; Stine, Keith J.


    Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A – ALP (or soybean agglutinin – ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A–ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL−1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay. PMID:24611035

  3. ALPHA-2: the sequel

    CERN Multimedia

    Katarina Anthony


    While many experiments are methodically planning for intense works over the long shutdown, there is one experiment that is already working at full steam: ALPHA-2. Its final components arrived last month and will completely replace the previous ALPHA set-up. Unlike its predecessor, this next generation experiment has been specifically designed to measure the properties of antimatter.   The ALPHA team lower the new superconducting solenoid magnet into place. The ALPHA collaboration is working at full speed to complete the ALPHA-2 set-up for mid-November – this will give them a few weeks of running before the AD shutdown on 17 December. “We really want to get some experience with this device this year so that, if we need to make any changes, we will have time during the long shutdown in which to make them,” says Jeffrey Hangst, ALPHA spokesperson. “Rather than starting the 2014 run in the commissioning stage, we will be up and running from the get go.&...

  4. Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

    DEFF Research Database (Denmark)

    Kovacs, J A; Powell, F; Edman, J C;


    this antigen is a good candidate for development as a vaccine to prevent or control P. carinii infection. We have cloned and sequenced seven related but unique genes encoding the major surface glycoprotein of rat P. carinii. Partial amino acid sequencing confirmed the identity of these genes. Based on Southern...... hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development...

  5. A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

    Directory of Open Access Journals (Sweden)

    Broder Christopher C


    Full Text Available Abstract Background Hendra virus (HeV and Nipah virus (NiV are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4 containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP gene encoding human immunodeficiency virus type-1 (HIV-1 genome in conjunction with the HeV and NiV fusion (F and attachment (G glycoproteins. Results Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2 peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F

  6. Structure of a trimeric variant of the Epstein–Barr virus glycoprotein B



    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 str...

  7. Structural and quantitative comparison of cerebrospinal fluid glycoproteins in Alzheimer's disease patients and healthy individuals.

    NARCIS (Netherlands)

    Sihlbom, C.; Davidsson, P.; Sjogren, M.; Wahlund, L.O.; Nilsson, C.L.


    Glycoproteins in cerebrospinal fluid (CSF) are altered in Alzheimer's Disease (AD) patients compared to control individuals. We have utilized albumin depletion prior to 2D gel electrophoresis to enhance glycoprotein concentration for image analysis as well as structural glycoprotein determination wi

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

    Energy Technology Data Exchange (ETDEWEB)

    Korecka, Lucie [Department of Analytical Chemistry, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic)]. E-mail:; Jezova, Jana [Department of Analytical Chemistry, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Bilkova, Zuzana [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Benes, Milan [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Namesti 2, 162 06 Prague (Czech Republic); Horak, Daniel [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Namesti 2, 162 06 Prague (Czech Republic); Hradcova, Olga [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Slovakova, Marcela [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Laboratoire Physicochimie Curie, UMR 168 CNRS/Institute Curie, Paris Cedex 05 (France); Viovy, Jean-Louis [Laboratoire Physicochimie Curie, UMR 168 CNRS/Institute Curie, Paris Cedex 05 (France)


    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.

  9. Determination of site-specific glycan heterogeneity on glycoproteins

    DEFF Research Database (Denmark)

    Kolarich, Daniel; Jensen, Pia Hønnerup; Altmann, Friedrich;


    and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco...

  10. Glycoprotein Ibalpha signalling in platelet apoptosis and clearance

    NARCIS (Netherlands)

    van der Wal, E.


    Storage of platelets at low temperature reduces bacterial growth and might better preserve the haemostatic function of platelets than current procedures. Incubation at 0C is known to expose ?-N-acetyl-D-glucosamine-residues on glycoprotein (GP)Ibalpha inducing receptor-clustering and platelet destru

  11. Glycoprotein Ibα clustering in platelet storage and function

    NARCIS (Netherlands)

    Gitz, E.


    Platelets are anucleated, discoid-shaped cells that play an essential role in the formation of a hemostatic plug to prevent blood loss from injured vessels. Initial platelet arrest at the damaged arterial vessel wall is mediated through the interaction between the platelet receptor glycoprotein (GP)

  12. Glycoprotein expression by adenomatous polyps of the colon (United States)

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


    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.

  13. Folding of viral envelope glycoproteins in the endoplasmic reticulum

    NARCIS (Netherlands)

    Braakman, L.J.; Anken, E. van


    Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality con

  14. Spinosad is a potent inhibitor of canine P-glycoprotein

    NARCIS (Netherlands)

    Schrickx, Johannes A


    Inhibition of the drug transporter P-glycoprotein (P-gp) by the oral flea preventative spinosad has been suggested as the underlying cause of the drug-drug interaction with ivermectin. In this study, an in vitro model consisting of canine cells was validated to describe the inhibitory effect of drug

  15. beta(2)-Glycoprotein I : evolution, structure and function

    NARCIS (Netherlands)

    de Groot, P. G.; Meijers, J. C. M.


    beta(2)-Glycoprotein I (beta(2)-GPI) is a protein that circulates in blood at high concentrations. The function of beta(2)-GPI has long been an enigma. More than 20 years ago, it was discovered that beta(2)-GPI is the major antigen for the circulating antibodies in the antiphospholipid syndrome. How

  16. Inhibition of mucin glycosylation by aryl-N-acetyl-alpha-galactosaminides in human colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuan, S.F.; Byrd, J.C.; Basbaum, C.; Kim, Y.S. (Veterans Administration Medical Center, San Francisco, CA (USA))


    Specific inhibitors of the glycosylation of O-glycosidically linked glycoproteins have not previously been described. When tested for their effects on mucin glycosylation in a mucin-producing colon cancer cell line, LS174T, benzyl-, phenyl-, and p-nitrophenyl-N-acetyl-alpha-galactosaminide inhibited the formation of fully glycosylated mucin in a dose-dependent manner. Free aryl-oligosaccharides were found in the medium of treated cells labeled with ({sup 3}H)glucosamine, ({sup 3}H)galactose, ({sup 3}H)fucose, ({sup 3}H)mannosamine, or phenyl-alpha-(6-{sup 3}H) N-acetylgalactosamine. UDP-Gal:GalNAc-beta 1,3-galactosyltransferase was inhibited by aryl-N-acetyl-alpha-galactosaminides but not by a number of other aryl-glycosides. Treatment with these inhibitors also causes reversible morphologic changes including formation of intercellular cysts. Aryl-N-acetyl-alpha-galactosaminides can be useful for the structural and functional studies of mucin macromolecules and other O-linked glycoproteins.

  17. The rainbow trout TLR9 gene and its role in the immune responses elicited by a plasmid encoding the glycoprotein G of the viral haemorrhagic septicaemia rhabdovirus (VHSV). (United States)

    Ortega-Villaizan, M; Chico, V; Falco, A; Perez, L; Coll, J M; Estepa, A


    The aim of this work was to improve the knowledge about the factors contributing to the immunogenicity of the DNA vaccines based on the viral haemorrhagic septicaemia virus glycoprotein G gene, through identifying the rainbow trout Toll-like receptor 9 (Omtlr9) gene that curiously contains an insertion of an incomplete transposon at the 5'-end of the third intron. Concerning the role played by this receptor in the fish innate defence, in response to the injection of a plasmid (pAE6) encoding or not the viral haemorrhagic septicaemia rhabdovirus (VHSV) glycoprotein G gene (pAE6-G), the presence of Omtlr9 transcripts remained unchanged in the fish secondary lymphoid organs while was highly increased at the injection site (muscle). The level of Omtlr9 transcripts correlated with those of cluster of differentiation 83 (cd83) and CXC chemokine receptor 4 (cxcr4), suggesting the recruitment of dendritic-like cells into the muscle as the source of Omtlr9 expressing cells. Transcription of tumour necrosis factor-alpha (tnf alpha) and interleukin-6 (il6) genes, two cytokines directly related to TLR9 induction with unmethylated CpG oligodeoxynucleotides (CpG ODNs), was solely observed in head kidney and spleen of the fish immunised with pAE6-G. Thus, the glycoprotein G of VHSV could be more implicated in triggering the pathways for TNF-alpha and IL6 production than the recognition of the unmethylated CpG motifs of the plasmid backbone by OmTLR9. Therefore, our results seem to indicate that OmTLR9-mediated recognition of plasmid DNA is not the key of the innate immune recognition of the adjuvant elements of fish DNA vaccines.

  18. ALPHA MIS: Reference manual

    Energy Technology Data Exchange (ETDEWEB)

    Lovin, J.K.; Haese, R.L.; Heatherly, R.D.; Hughes, S.E.; Ishee, J.S.; Pratt, S.M.; Smith, D.W.


    ALPHA is a powerful and versatile management information system (MIS) initiated and sponsored and by the Finance and Business Management Division of Oak Ridge National Laboratory, who maintain and develop it in concert with the Business Systems Division for its Information Center. A general-purpose MIS, ALPHA allows users to access System 1022 and System 1032 databases to obtain and manage information. From a personal computer or a data terminal, Energy Systems employees can use ALPHA to control their own report reprocessing. Using four general commands (Database, Select, Sort, and Report) they can (1) choose a mainframe database, (2) define subsets within it, (3) sequentially order a subset by one or more variables, and (4) generate a report with their own or a canned format.

  19. Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor

    DEFF Research Database (Denmark)

    Tran, E H; Kuziel, W A; Owens, T


    Macrophage inflammatory protein (MIP)-1alpha is a chemokine that is associated with Th1 cytokine responses. Expression and antibody blocking studies have implicated MIP-1alpha in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). We examined the role of MIP-1alpha...... and its CCR5 receptor in the induction of EAE by immunizing C57BL / 6 mice deficient in either MIP-1alpha or CCR5 with myelin oligodendrocyte glycoprotein (MOG). We found that MIP-1alpha-deficient mice were fully susceptible to MOG-induced EAE. These knockout animals were indistinguishable from wild...... chemoattractant protein-1, MIP-1beta, MIP-2, lymphotactin and T cell activation gene-3 during the course of the disease. CCR5-deficient mice were also susceptible to disease induction by MOG. The dispensability of MIP-1alpha and CCR5 for MOG-induced EAE in C57BL / 6 mice supports the idea that differential...

  20. Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells


    Arenas-Del Ángel, Maria; Legorreta-Herrera, Martha; Mendoza-Hernández, Guillermo; Garfias, Yonathan; Chávez, Raul; Zenteno, Edgar; Lascurain,Ricardo


    The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electroph...

  1. AlphaSphere


    Place, A.; Lacey, L.; Mitchell, T.


    The AlphaSphere is an electronic musical instrument featuring a series of tactile, pressure sensitive touch pads arranged in a spherical form. It is designed to offer a new playing style, while allowing for the expressive real-time modulation of sound available in electronic-based music. It is also designed to be programmable, enabling the flexibility to map a series of different notational arrangements to the pad-based interface.\\ud \\ud The AlphaSphere functions as an HID, MIDI and OSC devic...

  2. Platelet receptor expression and shedding: glycoprotein Ib-IX-V and glycoprotein VI. (United States)

    Gardiner, Elizabeth E; Andrews, Robert K


    Quantity, quality, and lifespan are 3 important factors in the physiology, pathology, and transfusion of human blood platelets. The aim of this review is to discuss the proteolytic regulation of key platelet-specific receptors, glycoprotein(GP)Ib and GPVI, involved in the function of platelets in hemostasis and thrombosis, and nonimmune or immune thrombocytopenia. The scope of the review encompasses the basic science of platelet receptor shedding, practical aspects related to laboratory analysis of platelet receptor expression/shedding, and clinical implications of using the proteolytic fragments as platelet-specific biomarkers in vivo in terms of platelet function and clearance. These topics can be relevant to platelet transfusion regarding both changes in platelet receptor expression occurring ex vivo during platelet storage and/or clinical use of platelets for transfusion. In this regard, quantitative analysis of platelet receptor profiles on blood samples from individuals could ultimately enable stratification of bleeding risk, discrimination between causes of thrombocytopenia due to impaired production vs enhanced clearance, and monitoring of response to treatment prior to change in platelet count.

  3. Mass spectrometry and /sup 13/C nuclear magnetic resonance spectroscopy of compounds modeling the glycopeptide linkage of glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Blumberg, K.; Bush, C.A.


    The properties of several compounds useful as models for three-dimensional conformational studies and the investigation of the chemical degradation of glycopeptide linkages both of the N- and O-glycosidic type are described. Using the method of differential chemical shift in H/sub 2/O and D/sub 2/O as solvents, the carbon NMR spectrum of N-acetylglucosaminylasparagine, 1-N-acetyl-..beta..-D-glucopyranosylamine, and 1-N-acetyl-2-acetamido-..beta..-D-glycopyranosylamine has been assigned. Electron impact mass spectra of the peracetylated derivatives of the latter two compounds show a peak apparently unique to glycopyranosylamides at m/e = 269, no analog of which is observed in the mass spectra of other peracetylated sugars. As models of the ..cap alpha..-O-glycosidic linkage, fully assigned carbon NMR spectra of ..cap alpha..-methyl-N-acetylgalactosamine (GalNAc), ..cap alpha..-methyl-3-O-methyl GalNAc, and -GlcNAc as well as the disaccharide Glc-..beta..-l ..-->.. 3 GalNAc are reported. Because certain anomalies in the chemical shifts and /sup 1/J/sub CH/ observed in the disaccharide and in O-glycosylated glycoproteins are not observed in the simple model compounds, they may result from conformational interactions in the glycopeptides.

  4. Proteomics computational analyses suggest that the bornavirus glycoprotein is a class III viral fusion protein (γ penetrene

    Directory of Open Access Journals (Sweden)

    Garry Robert F


    Full Text Available Abstract Background Borna disease virus (BDV is the type member of the Bornaviridae, a family of viruses that induce often fatal neurological diseases in horses, sheep and other animals, and have been proposed to have roles in certain psychiatric diseases of humans. The BDV glycoprotein (G is an extensively glycosylated protein that migrates with an apparent molecular mass of 84,000 to 94,000 kilodaltons (kDa. BDV G is post-translationally cleaved by the cellular subtilisin-like protease furin into two subunits, a 41 kDa amino terminal protein GP1 and a 43 kDa carboxyl terminal protein GP2. Results Class III viral fusion proteins (VFP encoded by members of the Rhabdoviridae, Herpesviridae and Baculoviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Proteomics computational analyses suggest that the structural/functional motifs that characterize class III VFP are located collinearly in BDV G. Structural models were established for BDV G based on the post-fusion structure of a prototypic class III VFP, vesicular stomatitis virus glycoprotein (VSV G. Conclusion These results suggest that G encoded by members of the Bornavirdae are class III VFPs (gamma-penetrenes.

  5. Alpha Antihydrogen Experiment

    CERN Document Server

    Fujiwara, M C; Ashkezari, M D; Baquero-Ruiz, M; Bertsche, W; Bray, C C; Butler, E; Cesar, C L; Chapman, S; Charlton, M; Cesar, C L; Fajans, J; Friesen, T; Gill, D R; Hangst, J S; Hardy, W N; Hayano, R S; Hayden, M E; Humphries, A J; Hydomako, R; Jonsell, S; Kurchaninov, L; Lambo, R; Madsen, N; Menary, S; Nolan, P; Olchanski, K; Olin, A; Povilus, A; Pusa, P; Robicheaux, F; Sarid, E; Silveira, D M; So, C; Storey, J W; Thompson, R I; van der Werf, D P; Wilding, D; Wurtele, J S; Yamazaki, Y


    ALPHA is an experiment at CERN, whose ultimate goal is to perform a precise test of CPT symmetry with trapped antihydrogen atoms. After reviewing the motivations, we discuss our recent progress toward the initial goal of stable trapping of antihydrogen, with some emphasis on particle detection techniques.

  6. Case Study - Alpha

    Directory of Open Access Journals (Sweden)

    Stephen Leybourne


    Full Text Available This case study was developed from an actual scenario by Dr. Steve Leybourne of Boston University.  The case documents the historical evolution of an organization, and has been used successfully in courses dealing with organizational and cultural change, and the utilization of ‘soft skills’ in project-based management. This is a short case, ideal for classroom use and discussion.  The issues are easily accessible to students, and there is a single wide ranging question that allows for the inclusion of many issues surrounding strategic decision-making, and behavioural and cultural change. Alpha was one of the earlier companies in the USA to invest in large, edge-of-town superstores, with plentiful free vehicle parking, selling food and related household products. Alpha was created in the 1950s as a subsidiary of a major publicly quoted retail group.  It started business by opening a string of very large discount stores in converted industrial and warehouse premises in the south of the United States. In the early days shoppers were offered a limited range of very competitively priced products. When Alpha went public in 1981 it was the fourth largest food retailer in the US, selling an ever-widening range of food and non-food products.  Its success continued to be based on high volume, low margins and good value for money, under the slogan of ‘Alpha Price.’

  7. Alpha-mannosidosis

    DEFF Research Database (Denmark)

    Borgwardt, Line; Stensland, Hilde Monica Frostad Riise; Olsen, Klaus Juul;


    of the three subgroups of genotype/subcellular localisation and the clinical and biochemical data were done to investigate the potential relationship between genotype and phenotype in alpha-mannosidosis. Statistical analyses were performed using the SPSS software. Analyses of covariance were performed...

  8. Purification and characterization of the human platelet. cap alpha. /sub 2/-adrenergic receptor

    Energy Technology Data Exchange (ETDEWEB)

    Shreeve, S.M.; Kerlavage, A.R.; Fraser, C.M.; Mariani, A.P.; Venter, J.C.


    The ..cap alpha../sub 2/-receptor (..cap alpha../sub 2/-R) from human platelets has been purified to homogeneity using a four step process. An affinity column was prepared by coupling p-aminoclonidine to CH-Sepharose 4B via the p-NH/sub 2/ group. Digitonin solubilized ..cap alpha../sub 2/-R bound to the affinity matrix were eluted with 100 phentolamine and directly applied to a DEAE-Sepharose column. Bound receptors were eluted with a linear gradient of 0-500 mM NaCl, pooled and chromatographed on HPLC size exclusion columns. Three peaks of ..cap alpha../sub 2/-R binding were eluted from HPLC columns (t = 33, 42, 47 min). Radioiodination of HPLC eluates and analysis by SDS-PAGE indicated that ..cap alpha../sub 2/-R binding was associated with a 75-85 kDa protein. These data suggest that the ..cap alpha../sub 2/-R may exist in monomeric and oligomeric forms in the purified state and support previous target size data which indicate that the ..cap alpha../sub 2/-R exists as a dimer in the native membrane. The pure radioiodinated ..cap alpha../sub 2/-R (77-85 kDa) is a glycoprotein with terminal sialic acid or N-acetylglucosamine residues and has a pI of 4.1 on column isoelectric focusing. These data are consistent with those previously reported on the partially purified ..cap alpha../sub 2/-R. Electron micrographs confirm the oligomeric nature and size of the pure ..cap alpha../sub 2/-R.

  9. Prominent 85-kDa oligomannosidic glycoproteins of rat brain are signal regulatory proteins and include the SHP substrate-1 for tyrosine phosphatases. (United States)

    Bartoszewicz, Z P; Jaffe, H; Sasaki, M; Möller, J R; Stebbins, J W; Gebrekristos, H; Quarles, R H


    The glycoprotein component in rat brain reacting most strongly with Galanthus nivalis agglutinin (GNA) on western blots migrates as an 85-kDa band. GNA identifies mannose-rich oligosaccharides because it is highly specific for terminal alpha-mannose residues. After purification of this 85-kDa glycoprotein band by chromatography on GNA-agarose and preparative gel electrophoresis, binding of other lectins demonstrated the presence of fucose and a trace of galactose, but no sialic acid. Treatment with N-Glycanase or endoglycosidase H produced a 65-kDa band, indicating that it consisted of about one-fourth N-linked oligomannosidic carbohydrate moieties. High-performance anion-exchange chromatography and fluorescence-assisted carbohydrate electrophoresis indicated that the major carbohydrate moiety is a heptasaccharide with the structure Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc (Man5GlcNAc2). Determination of amino acid sequences of peptides produced by endoproteinase digestion demonstrated that this 85-kDa mannose-rich glycoprotein component contained the SHP substrate-1 for phosphotyrosine phosphatases and at least one other member of the signal-regulatory protein (SIRP) family. The unusually high content of oligomannosidic carbohydrate moieties on these receptor-like members of the immunoglobulin superfamily in neural tissue could be of functional significance for intercellular adhesion or signaling.

  10. Magnetic immunoassay coupled with inductively coupled plasma mass spectrometry for simultaneous quantification of alpha-fetoprotein and carcinoembryonic antigen in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xing; Chen, Beibei; He, Man; Zhang, Yiwen; Xiao, Guangyang; Hu, Bin, E-mail:


    The absolute quantification of glycoproteins in complex biological samples is a challenge and of great significance. Herein, 4-mercaptophenylboronic acid functionalized magnetic beads were prepared to selectively capture glycoproteins, while antibody conjugated gold and silver nanoparticles were synthesized as element tags to label two different glycoproteins. Based on that, a new approach of magnetic immunoassay-inductively coupled plasma mass spectrometry (ICP-MS) was established for simultaneous quantitative analysis of glycoproteins. Taking biomarkers of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) as two model glycoproteins, experimental parameters involved in the immunoassay procedure were carefully optimized and analytical performance of the proposed method was evaluated. The limits of detection (LODs) for AFP and CEA were 0.086 μg L{sup −1} and 0.054 μg L{sup −1} with the relative standard deviations (RSDs, n = 7, c = 5 μg L{sup −1}) of 6.5% and 6.2% for AFP and CEA, respectively. Linear range for both AFP and CEA was 0.2–50 μg L{sup −1}. To validate the applicability of the proposed method, human serum samples were analyzed, and the obtained results were in good agreement with that obtained by the clinical chemiluminescence immunoassay. The developed method exhibited good selectivity and sensitivity for the simultaneous determination of AFP and CEA, and extended the applicability of metal nanoparticle tags based on ICP-MS methodology in multiple glycoprotein quantifications. - Highlights: • 4-Mercaptophenylboronic acid functionalized magnetic beads were prepared and characterized. • ICP-MS based magnetic immunoassay approach was developed for quantification of glycoproteins. • AFP and CEA were quantified simultaneously with Au and Ag NPs as element tags. • The developed method exhibited good selectivity and sensitivity for target glycoproteins.

  11. A defined peptide that inhibits the formation of the glycoprotein IIb and IIIa complex. (United States)

    Chiang, Thomas M; Zhu, Jiaqian


    Collagen-platelet interaction plays an important role in hemostasis and pathological thrombosis. The proposed mechanism of the interaction was the activation of platelets-->releasing of contents from granules-->aggregation. The common end point is the platelets and fibrin aggregates. Platelet glycoprotein (GP) IIb/IIIa (the alphaIIbbeta3 integrin) complexes serve as a receptor for the binding of fibrinogen to form firmed aggregates. Blockading of GP IIb/IIIa has been proposed to prevent platelet aggregation independent of the substance(s) responsible for activating the platelets. The development of various forms of GP IIb/IIIa inhibitor has resulted in the inhibition of platelet aggregation, although studies of alphaIIbbeta3 receptor function and various GP IIb/IIIa inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function as well as having nonplatelet effects. This study investigated platelet inhibition provided by blocking the GP IIb/IIIa complex formation by using a peptide derived from the GP IIIa molecule. The peptide inhibits both types I and III collagen-induced platelet aggregation in a dose-dependent manner. The defined peptide interferes with the formation of the GP IIb/IIIa complex by inhibiting the binding of FITC-PAC-1 onto ADP-, type I collagen-, and type III collagen-activated platelets. However, P-selectin secretion is not affected by the peptide. In addition, the peptide is not interfering with the binding of FITC-PAC-1 to platelets that were preincubated with indomethacin. Results from this study may suggest that the defined peptide is an effective agent to block the interaction of types I and III collagen with platelets.

  12. HB Hillingdon [alpha46(CE4)Phe-->Val (alpha1 Or alpha2)]: a new alpha chain hemoglobin variant. (United States)

    Babb, Anna; Solaiman, Susannah; Green, Brian N; Mantio, Debbie; Patel, Ketan


    Routine antenatal hemoglobinopathy screening detected a new alpha chain variant that eluted with Hb A(2) on cation exchange high performance liquid chromatography (HPLC) in a lady of Sri Lankan origin who had normal hematological indices. The mutation was identified by electrospray ionization mass spectrometry (ESI-MS) as alpha46(CE4)Phe-->Val, inferring that the variant was due to a single base change at codon 46 (TTC>GTC) of the alpha1- or alpha2-globin genes.

  13. Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin

    Energy Technology Data Exchange (ETDEWEB)

    An, Xiuli; Gauthier, Emilie; Zhang, Xihui; Guo, Xinhua; Anstee, David; Mohandas, Narla; Anne Chasis, Joel


    The Lutheran (Lu) and Lu(v13) blood group glycoproteins function as receptors for extracellular matrix laminins. Lu and Lu(v13) are linked to the erythrocyte cytoskeleton through a direct interaction with spectrin. However, neither the molecular basis of the interaction nor its functional consequences have previously been delineated. In the present study, we defined the binding motifs of Lu and Lu(v13) on spectrin and identified a functional role for this interaction. We found that the cytoplasmic domains of both Lu and Lu(v13) bound to repeat 4 of the spectrin chain. The interaction of full-length spectrin dimer to Lu and Lu(v13) was inhibited by repeat 4 of {alpha}-spectrin. Further, resealing of this repeat peptide into erythrocytes led to weakened Lu-cytoskeleton interaction as demonstrated by increased detergent extractability of Lu. Importantly, disruption of the Lu-spectrin linkage was accompanied by enhanced cell adhesion to laminin. We conclude that the interaction of the Lu cytoplasmic tail with the cytoskeleton regulates its adhesive receptor function.

  14. Collagen can selectively trigger a platelet secretory phenotype via glycoprotein VI.

    Directory of Open Access Journals (Sweden)

    Véronique Ollivier

    Full Text Available Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these "non classical" functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI, we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.

  15. Comparison of glycoprotein expression between ovarian and colon adenocarcinomas

    DEFF Research Database (Denmark)

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


    OBJECTIVE: 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. DESIGN: 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. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both...


    Directory of Open Access Journals (Sweden)

    R. N. Bogdanovich


    Full Text Available Abstract. The level of trophoblastic β1 – glycoprotein (SP–1 was determined in the blood sera of 200 healthy pregnant women and 184 women with threatened abortions in term till 20 weeks of pregnancy. In group of women experiencing recurrent abortions in 38 % cases antibodies to chorionic gonadotropin, in 39,5 % cases antibodies to phospholipids, in 25,5 % – antibodies to tireoglobulin were revealed in significant amounts. In 20,65 % lupus anticoagulant was found. The majority of women in this group had changes in homeostasis. The presence of autoantibodies during pregnancy is the unfavourable factor in the development of placental insufficiency. This is proved by the decreased secretion of trophoblastic β1 – glycoprotein – a marker of the fetal part of placenta. (Med. Immunol., 2005, vol.7, № 1, pp. 85588

  17. Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions

    Directory of Open Access Journals (Sweden)

    Makoto Ujike


    Full Text Available The envelopes of coronaviruses (CoVs contain primarily three proteins; the two major glycoproteins spike (S and membrane (M, and envelope (E, a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER-Golgi intermediate compartment (ERGIC. For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions.

  18. Rheologic studies on middle ear effusions and their mucus glycoproteins. (United States)

    FitzGerald, J E; Green, G G; Birchall, J P; Pearson, J P


    The properties of pooled thick and thin middle ear effusions, from children with otitis media with effusion, were studied by viscometry. Mucus glycoproteins were responsible for effusion viscosity. Their percentage by weight in thick and thin effusions was 25% and 8.2%, respectively. N-acetylcysteine and 0.2 mol/L of mercaptoethanol caused a 39% viscosity drop in a 5-mg/mL glycoprotein solution, whereas S-carboxymethylcysteine had no effect. Treatment of thick effusions with 0.2 mol/L of mercaptoethanol initially caused a viscosity decrease followed by a gradual increase. Higher reducing agent concentrations (0.5 mol/L) caused a more rapid decrease followed by a rapid increase, presumably by causing nonspecific aggregation of reduced protein molecules. These results suggest that the concentration of and the time that a mucolytic is in the middle ear would be of prime importance in achieving the desired decrease in viscosity.

  19. $\\alpha$-Representation for QCD


    Tuan, Richard Hong


    An $\\alpha$-parameter representation is derived for gauge field theories.It involves, relative to a scalar field theory, only constants and derivatives with respect to the $\\alpha$-parameters. Simple rules are given to obtain the $\\alpha$-representation for a Feynman graph with an arbitrary number of loops in gauge theories in the Feynman gauge.

  20. An analysis of amino acid sequences surrounding archaeal glycoprotein sequons


    Mehtap Abu-Qarn; Jerry Eichler


    Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, t...

  1. alpha-1-antitrypsin in breast milk of healthy Nigerian mothers. (United States)

    Omeme, J A; Lantos, J D; Ihongbe, J C


    Alpha-1-antitryspin (x-1-AT) may play a possible role as effector of immunological stasis. This study examines the levels of this glycoprotein in 73 breast milk samples from 60 healthy Nigerian mothers. Levels of x-1-AT were measured by single radial immunodiffusion according to the method of Mancini. Serum protein was measured by Lowry's method, albumin by Doumas' method. Highest mean levels of x-1-AT were found in colostrum (25 mg/dl). The level was significantly higher compared to transitional milk (14.2 mg/dl) or mature milk (165 mg/dl) (p0.001). Breast milk contains substantial amounts of x-1-AT which is not destroyed by pasturization at 56 degrees Centigrade. The immunological protective properties of breast milk are ideal for newborn babies, particularly those who are low birthweight and are thus most susceptible to neonatal necrotizing enterocolitis.

  2. A double responsive smart upconversion fluorescence sensing material for glycoprotein. (United States)

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


    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.

  3. Characterization of an estrogen-induced oviduct membrane glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Poola, I.; Lucas, J.J.


    During estrogen-induced chick oviduct differentiation a number of N-linked membrane glycoproteins are induced as judged by GDP-(/sup 14/C)Man labeling of endogenous acceptors, /sup 125/I-con A labeling as well as coomassie blue and PAS staining of SDS polyacrylamide gels. The authors have begun to characterize one of these glycoproteins having an M/sub r/ of 91 KDa. The protein has been purified via preparative SDS-PAGE and electroelution. The purified protein migrates as a single band on analytical SDS-PAGE and comigrates with an endogenous membrane glycoprotein labeled with GDP-(/sup 14/C)Man. Amino acid analysis indicates a high proportion of GLU and ASP residues (110 and 66 moles respectively). N-terminal sequence analysis by gas phase instrumentation yielded the following: X-X-VAL-ASP-VAL-ASP-ALA-THR-VAL-GLU-GLU-ASP-GLU. The protein contains about 2% neutral sugar including 6 mol Man, 2 mol Gal, 1 mol Fuc, 4 mol GlcNAc, 1 mol GalNAc and 1 mol sialic acid per mole of protein. The presence of the GalNAc residue suggests the protein contains an O-linked oligosaccharide moiety in addition to the N-linked chain(s). The detailed structure of the carbohydrate moieties is currently under investigation.

  4. Role of sialidase in glycoprotein utilization by Tannerella forsythia. (United States)

    Roy, Sumita; Honma, Kiyonobu; Douglas, C W Ian; Sharma, Ashu; Stafford, Graham P


    The major bacterial pathogens associated with periodontitis include Tannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth of T. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host-pathogen interactions in vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing that T. forsythia can utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with a nanH mutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of the nanH mutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a key in vivo nutrient source for T. forsythia when growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.

  5. Alpha and Beta Determinations

    CERN Document Server

    Dunietz, Isard


    Because the Bd -> J/psi Ks asymmetry determines only sin(2 beta), a discrete ambiguity in the true value of beta remains. This note reviews how the ambiguity can be removed. Extractions of the CKM angle alpha are discussed next. Some of the methods require very large data samples and will not be feasible in the near future. In the near future, semi-inclusive CP-violating searches could be undertaken, which are reviewed last.

  6. Combining Alphas via Bounded Regression

    Directory of Open Access Journals (Sweden)

    Zura Kakushadze


    Full Text Available We give an explicit algorithm and source code for combining alpha streams via bounded regression. In practical applications, typically, there is insufficient history to compute a sample covariance matrix (SCM for a large number of alphas. To compute alpha allocation weights, one then resorts to (weighted regression over SCM principal components. Regression often produces alpha weights with insufficient diversification and/or skewed distribution against, e.g., turnover. This can be rectified by imposing bounds on alpha weights within the regression procedure. Bounded regression can also be applied to stock and other asset portfolio construction. We discuss illustrative examples.

  7. Orthopositronium lifetime. Analytic results in O ({alpha}) and O ({alpha}{sup 3} ln {alpha})

    Energy Technology Data Exchange (ETDEWEB)

    Kniehl, B.A.; Kotikov, A.V.; Veretin, O.L. [Hamburg Univ. (Germany). 2. Inst. fuer Theoretische Physik


    We present the O({alpha}) and O({alpha}{sup 3}ln {alpha}) corrections to the total decay width of orthopositronium in closed analytic form, in terms of basic transcendental numbers, which can be evaluated numerically to arbitrary precision. (orig.)

  8. Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors

    Energy Technology Data Exchange (ETDEWEB)

    Koener, J.F.; Leong, J.A.C. (Oregon State Univ., Corvallis (United States))


    A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots.

  9. Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins


    Kaariainen, L; Hashimoto, K.; Saraste, J; Virtanen, I; Penttinen, K


    Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the muta...

  10. Identification and antigenicity of the major envelope glycoprotein of lymphadenopathy-associated virus

    Energy Technology Data Exchange (ETDEWEB)

    Montagnier, L.; Clavel, F.; Krust, B.; Chamaret, S.; Rey, F.; Barre-Sinoussi, F.; Chermann, J.C.


    The major envelope glycoprotein of the causative agent of Acquired Immune Deficiency Syndrome (AIDS) lymphadenopathy-associated virus (LAV) has been identified and characterized. The glycoprotein has an apparent molecular weight of 110,000-120,000 under denaturing conditions in polyacrylamide gel electrophoresis. Upon deglycosylation by a specific endoglycosydase, its size is reduced to 80,000. Cellular precursors of this glycoprotein have been detected with apparent molecular weight of 150,000 and 135,000. Nearly all AIDS and pre-AIDS patients have detectable antibodies against this viral glycoprotein.

  11. Alpha-mannosidosis

    Directory of Open Access Journals (Sweden)

    Nilssen Øivind


    Full Text Available Abstract Alpha-mannosidosis is an inherited lysosomal storage disorder characterized by immune deficiency, facial and skeletal abnormalities, hearing impairment, and intellectual disability. It occurs in approximately 1 of 500,000 live births. The children are often born apparently normal, and their condition worsens progressively. Some children are born with ankle equinus or develop hydrocephalus in the first year of life. Main features are immune deficiency (manifested by recurrent infections, especially in the first decade of life, skeletal abnormalities (mild-to-moderate dysostosis multiplex, scoliosis and deformation of the sternum, hearing impairment (moderate-to-severe sensorineural hearing loss, gradual impairment of mental functions and speech, and often, periods of psychosis. Associated motor function disturbances include muscular weakness, joint abnormalities and ataxia. The facial trait include large head with prominent forehead, rounded eyebrows, flattened nasal bridge, macroglossia, widely spaced teeth, and prognathism. Slight strabismus is common. The clinical variability is significant, representing a continuum in severity. The disorder is caused by lysosomal alpha-mannosidase deficiency. Alpha-mannosidosis is inherited in an autosomal recessive fashion and is caused by mutations in the MAN2B1 gene located on chromosome 19 (19 p13.2-q12. Diagnosis is made by measuring acid alpha-mannosidase activity in leukocytes or other nucleated cells and can be confirmed by genetic testing. Elevated urinary secretion of mannose-rich oligosaccharides is suggestive, but not diagnostic. Differential diagnoses are mainly the other lysosomal storage diseases like the mucopolysaccharidoses. Genetic counseling should be given to explain the nature of the disease and to detect carriers. Antenatal diagnosis is possible, based on both biochemical and genetic methods. The management should be pro-active, preventing complications and treating

  12. Alpha phoenix公司

    Institute of Scientific and Technical Information of China (English)


    德国Alpha phoenix公司成立于2009年.专注于制药领域清洗机器的研发和生产,产品符合cGMP的要求。公司的人员是制药领域清洗机器研发和生产的专家,他们具有20多年的全自动清洗机器的设计和生产经验。

  13. Robust estimation of Cronbach's alpha



    Cronbach’s alpha is a popular method to measure reliability, e.g. in quantifying the reliability of a score to summarize the information of several items in questionnaires. The alpha coefficient is known to be non-robust. We study the behavior of this coefficient in different settings to identify situations, which can easily occur in practice, but under which the Cronbach’s alpha coefficient is extremely sensitive to violations of the classical model assumptions. Furthermore, we construct a r...

  14. Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins. (United States)

    Kääriäinen, L; Hashimoto, K; Saraste, J; Virtanen, I; Penttinen, K


    Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 mug/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 mug/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of

  15. Evidence for the absence of intron H of the histidine-rich glycoprotein (HRG) gene: Genetic mapping and in situ localization of HRG to chromosome 3q28-q29

    Energy Technology Data Exchange (ETDEWEB)

    Hennis, B.C.; Poort, E.W. van der; Kluft, C.; Frants, R.R.; Bakker, E.; Vossen, R.H.A.M.; Blonden, L.A.; Khan, P.M. (Leiden Univ. (Netherlands)); Cox, S.; Spurr, N.K. (Imperial Cancer Research Fund, London (United Kingdom))


    Histidine-rich glycoprotein (HRG) belongs to the cystatin superfamily and appears to be a potential risk factor for thrombosis. An increased prevalence of elevated HRG plasma levels in patients with venous thrombosis and families with thrombophilia has been reported. It is interesting to note that the genes of four different members of the cystatin superfamily are located on the distal section of the long arm of chromosome 3: Stefin A (STF1) on 3q21, Kininogen (KNG) on 3q26-qter, [alpha]-2-HS-glycoprotein (AHSG) on 3q27-q28, and HRG on 3q21-qter. To further investigate the evolutionary relationship between HRG and members of the cystatin superfamily, the authors isolated a cosmid that was used to refine the chromosomal localization of HRG by in situ hybridization. In addition, they used a dinucleotide repeat polymorphism to localize HRG on the linkage map of chromosome 3q. 10 refs., 2 figs.

  16. PDFs, $\\alpha_s$, and quark masses from global fits

    CERN Document Server

    Alekhin, S; Moch, S; Placakyte, R


    The strong coupling constant $\\alpha_s$ and the heavy-quark masses, $m_c$, $m_b$, $m_t$ are extracted simultaneosly with the parton distribution functions (PDFs) in the updated ABM12 fit including recent data from CERN-SPS, HERA, Tevatron, and the LHC. The values of \\begin{eqnarray} \

  17. Molecular characterization of alpha 1- and alpha 2-adrenoceptors. (United States)

    Harrison, J K; Pearson, W R; Lynch, K R


    Three 'alpha 1-adrenoceptors' and three 'alpha 2-adrenoceptors' have now been cloned. How closely do these receptors match the native receptors that have been identified pharmacologically? What are the properties of these receptors, and how do they relate to other members of the cationic amine receptor family? Kevin Lynch and his colleagues discuss these questions in this review.

  18. Hypoglycosylation of alpha-dystroglycan in patients with hereditary IBM due to GNE mutations. (United States)

    Huizing, Marjan; Rakocevic, Goran; Sparks, Susan E; Mamali, Ioanna; Shatunov, Alexey; Goldfarb, Lev; Krasnewich, Donna; Gahl, William A; Dalakas, Marinos C


    Hereditary inclusion body myopathy (HIBM) is an adult onset neuromuscular disorder associated with mutations in the gene UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), whose product is the rate limiting bi-functional enzyme catalyzing the first two steps of sialic acid biosynthesis. Loss of GNE activity in HIBM is thought to impair sialic acid production and interfere with proper sialylation of glycoconjugates, but it remains unclear how such a defect would lead to muscle destruction and muscle weakness. Hypoglycosylation of alpha-dystroglycan, a central protein of the skeletal muscle dystrophin-glycoprotein complex, results in disturbed interactions with extracellular matrix proteins. This has recently been identified as the pathomechanism involved in several congenital muscular dystrophies. We examined the glycosylation status of alpha-dystroglycan in muscle biopsies of four HIBM patients of non-Iranian Jewish origin (one American, two Indians, and one Greek). Two of these patients carry novel compound heterozygous GNE mutations on exon 2 and exon 9. All four muscle biopsies showed absent or markedly reduced immunolabeling with two different antibodies (VIA4 and IIH6) to glycosylated epitopes of alpha-dystroglycan. Normal labeling was found using antibodies to the core alpha-dystroglycan protein, beta-dystroglycan, and laminin alpha-2. These findings resemble those found for other congenital muscular dystrophies, suggesting that HIBM may be a "dystroglycanopathy," and providing an explanation for the muscle weakness of patients with GNE mutations.

  19. Alpha-mannosidosis

    DEFF Research Database (Denmark)

    Borgwardt, Line Gutte; Danielsen, E R; Thomsen, Carsten;


    Alpha-mannosidosis (AM) (OMIM 248500) is a rare lysosomal storage disease. The understanding of the central nervous system (CNS) pathology is limited. This study is the first describing the CNS pathology and the correlation between the CNS pathology and intellectual disabilities in human AM. Thirty......-protein, glial fibrillary acidic protein and neurofilament light protein in 97 patients, 74% and 41% of CSF samples, respectively. A negative correlation between CSF-biomarkers and cognitive function and CSF-oligosaccharides and cognitive function was found. The combination of MRS/MRI changes, elevated...... concentrations of CSF-biomarkers and CSF-oligosaccharides suggests gliosis and reduced myelination, as part of the CNS pathology in AM. Our data demonstrate early neuropathological changes, which may be taken into consideration when planning initiation of treatment....

  20. Characterization of monomeric intermediates during VSV glycoprotein structural transition.

    Directory of Open Access Journals (Sweden)

    Aurélie A Albertini


    Full Text Available 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 (G(th, 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, G(th 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.

  1. Surface Glycoproteins of Exosomes Shed by Myeloid-Derived Suppressor Cells Contribute to Function. (United States)

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


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

  2. Structural analysis of N- and O-glycans released from glycoproteins

    DEFF Research Database (Denmark)

    Jensen, Pia Hønnerup; Karlsson, Niclas G; Kolarich, Daniel;


    This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically...

  3. Investigating the biomarker potential of glycoproteins using comparative glycoprofiling - application to tissue inhibitor of metalloproteinases-1

    DEFF Research Database (Denmark)

    Thøgersen, Ida; Lademann, Ulrik Axel; Offenberg, Hanne Kjær;


    Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential...... tool for biomarker investigation of low-abundant glycoproteins....

  4. Histochemical and structural analysis of mucous glycoprotein secreted by the gill of Mytilus edulis

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Hae-Young.


    Studies were carried out to characterized various mucous cells in the gill filament, to ascertain structural characteristics of the secreted mucous glycoproteins, and to determine the ability of the gill epithelium to incorporate ({sup 14}C)glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins. Using histochemical staining techniques, mucous cells containing neutral and acidic mucins were found in the lateral region, whereas mucous cells containing primarily neutral or sulfated mucins were found in the postlateral region. Serotonin, but not dopamine, stimulated the mucous secretion. In tissues pretreated with ({sup 14}C)glucosamine, the secreted glycoproteins contain incorporated radiolabel. Analysis by column chromatography using Bio-Gel P-2 and P-6 shows that the secretion contains two glycoprotein populations. Glycoprotein II has a molecular weight of 2.3 {times} 10{sup 4} daltons. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of glycoprotein I, about 70% of the radiolabel was removed from the protein. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contains N-acetylglucosamine (GluNAc), N-acetylgalactosamine (GalNAc), and galactose, fucose and mannose. Amino acid analysis shows that the glycoproteins are rich in serine, threonine and proline.

  5. Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication. (United States)

    Schlie, Katrin; Maisa, Anna; Lennartz, Frank; Ströher, Ute; Garten, Wolfgang; Strecker, Thomas


    Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.

  6. Glycoproteins from sugarcane plants regulate cell polarity of Ustilago scitaminea teliospores. (United States)

    Millanes, Ana-María; Fontaniella, Blanca; Legaz, María-Estrella; Vicente, Carlos


    Saccharum officinarum, cv. Mayarí, is a variety of sugarcane resistant to smut disease caused by Ustilago scitaminea. Sugarcane naturally produces glycoproteins that accumulate in the parenchymatous cells of stalks. These glycoproteins contain a heterofructan as polysaccharide moiety. The concentration of these glycoproteins clearly increases after inoculation of sugarcane plants with smut teliospores, although major symptoms of disease are not observed. These glycoproteins induce homotypic adhesion and inhibit teliospore germination. When glycoproteins from healthy, non-inoculated plants are fractionated, they inhibit actin capping, which occurs before teliospore germination. However, inoculation of smut teliospores induce glycoprotein fractions that promote teliospore polarity and are different from those obtained from healthy plants. These fractions exhibit arginase activity, which is strongly enhanced in inoculated plants. Arginase from healthy plants binds to cell wall teliospores and it is completely desorpted by sucrose, but only 50% of arginase activity from inoculated plants is desorpted by the disaccharide. The data presented herein are consistent with a model of excess arginase entry into teliospores. Arginase synthesized by sugarcane plants as a response to the experimental infection would increase the synthesis of putrescine, which impedes polarization at concentration values higher than 0.05 mM. However, smut teliospores seem to be able to change the pattern of glycoprotein production by sugarcane, thereby promoting the synthesis of different glycoproteins that activate polarization after binding to their cell wall ligand.


    Recent DFT optimization studies on alpha-maltose improved our understanding of the preferred conformations of alpha-maltose and the present study extends these studies to alpha-maltotriose with three alpha-D-glucopyranose residues linked by two alpha-[1-4] bridges, denoted herein as DP-3's. Combina...

  8. The ALPHA antihydrogen trapping apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Amole, C. [Department of Physics and Astronomy, York University, Toronto ON Canada, M3J 1P3 (Canada); Andresen, G.B. [Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus C (Denmark); Ashkezari, M.D. [Department of Physics, Simon Fraser University, Burnaby, BC Canada, V5A 1S6 (Canada); Baquero-Ruiz, M. [Department of Physics, University of California at Berkeley, Berkeley, CA 94720-7300 (United States); Bertsche, W. [Department of Physics, College of Science, Swansea University, Swansea SA2 8PP (United Kingdom); School of Physics and Astronomy, University of Manchester, Manchester M13 9PL (United Kingdom); The Cockcroft Institute, Warrington WA4 4AD (United Kingdom); Bowe, P.D. [Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus C (Denmark); Butler, E. [Physics Department, CERN, CH-1211 Geneva 23 (Switzerland); Capra, A. [Department of Physics and Astronomy, York University, Toronto ON Canada, M3J 1P3 (Canada); Carpenter, P.T. [Department of Physics, Auburn University, Auburn, AL 36849-5311 (United States); Cesar, C.L. [Instituto de Física, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-972 (Brazil); Chapman, S. [Department of Physics, University of California at Berkeley, Berkeley, CA 94720-7300 (United States); Charlton, M.; Deller, A.; Eriksson, S. [Department of Physics, College of Science, Swansea University, Swansea SA2 8PP (United Kingdom); Escallier, J. [Brookhaven National Laboratory, Upton, NY 11973 (United States); Fajans, J. [Department of Physics, University of California at Berkeley, Berkeley, CA 94720-7300 (United States); Friesen, T. [Department of Physics and Astronomy, University of Calgary, Calgary AB, Canada, T2N 1N4 (Canada); Fujiwara, M.C.; Gill, D.R. [TRIUMF, 4004 Wesbrook Mall, Vancouver BC, Canada V6T 2A3 (Canada); Gutierrez, A. [Department of Physics and Astronomy, University of British Columbia, Vancouver BC, Canada V6T 1Z4 (Canada); and others


    The ALPHA collaboration, based at CERN, has recently succeeded in confining cold antihydrogen atoms in a magnetic minimum neutral atom trap and has performed the first study of a resonant transition of the anti-atoms. The ALPHA apparatus will be described herein, with emphasis on the structural aspects, diagnostic methods and techniques that have enabled antihydrogen trapping and experimentation to be achieved.

  9. Alpha particle emitters in medicine

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, D.R.


    Radiation-induced cancer of bone, liver and lung has been a prominent harmful side-effect of medical applications of alpha emitters. In recent years, however, the potential use of antibodies labeled with alpha emitting radionuclides against cancer has seemed promising because alpha particles are highly effective in cell killing. High dose rates at high LET, effectiveness under hypoxic conditions, and minimal expectancy of repair are additional advantages of alpha emitters over antibodies labeled with beta emitting radionuclides for cancer therapy. Cyclotron-produced astatine-211 ({sup 211}At) and natural bismuth-212 ({sup 212}Bi) have been proposed and are under extensive study in the United States and Europe. Radium-223 ({sup 223}Ra) also has favorable properties as a potential alpha emitting label, including a short-lived daughter chain with four alpha emissions. The radiation dosimetry of internal alpha emitters is complex due to nonuniformly distributed sources, short particle tracks, and high relative specific ionization. The variations in dose at the cellular level may be extreme. Alpha-particle radiation dosimetry, therefore, must involve analysis of statistical energy deposition probabilities for cellular level targets. It must also account fully for nonuniform distributions of sources in tissues, source-target geometries, and particle-track physics. 18 refs., 4 figs.

  10. Developmental and mutational changes of glycoproteins in the mouse neuronal retina: studies with bovine galactosyltransferase. (United States)

    Wallenfels, B


    Bovine galactosyltransferase (lactose synthase; EC which catalyzes the transfer of galactose from UDPgalactose to glycoproteins with N-acetylglucosamine as the terminal residue of their oligosaccharide side chains was used to label glycoproteins of mouse retina with [14C]galactose. The glycoproteins were separated by isoelectric focusing in the first dimension and by sodium dodecyl sulfate gel electrophoresis in the second dimension. Their position on the gel was determined by autofluorography. With this method, quantitative as well as qualitative changes in the glycoprotein composition of the neuronal mouse retina during postnatal development were observed. Furthermore, it was found that the photoreceptor loss in mice with retinal degeneration was paralleled by the disappearance of certain glycoprotein bands.

  11. Demethoxycurcumin modulates human P-glycoprotein function via uncompetitive inhibition of ATPase hydrolysis activity. (United States)

    Teng, Yu-Ning; Hsieh, Yow-Wen; Hung, Chin-Chuan; Lin, Hui-Yi


    Curcuminoids are major components of Curcuma longa L., which is widely used as spice in food. This study aimed at identifying whether curcumin, demethoxycurcumin, and bisdemethoxycurcumin could modulate efflux function of human P-glycoprotein and be used as chemosensitizers in cancer treatments. Without altering P-glycoprotein expression levels and conformation, the purified curcuminoids significantly inhibited P-glycoprotein efflux function. In rhodamine 123 efflux and calcein-AM accumulation assays, demethoxycurcumin demonstrated the highest inhibition potency (inhibitory IC50 = 1.56 ± 0.13 μM) among the purified curcuminoids, as well as in the fold of reversal assays. Demethoxycurcumin inhibited P-glycoprotein-mediated ATP hydrolysis under concentrations of P-glycoprotein. These results suggested that demethoxycurcumin may be a potential additive natural product in combination with chemotherapeutic agents in drug-resistant cancers.

  12. Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity

    DEFF Research Database (Denmark)

    Horvat, B; Multhaupt, H A; Damjanov, I


    We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had...... a unique binding pattern different from the patterns revealed by other lectins. However, several estrous cycle phase-specific glycoproteins reacted with more than one lectin. The most prominent of these glycoproteins (M(r) 92-95 KD) was weakly expressed in late diestrus and fully expressed only...... in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate...

  13. Loop-acting diuretics do not bind to Tamm-Horsfall urinary glycoprotein. (United States)

    Brunisholz, M C; Lynn, K L; Hunt, J S


    1. Binding between the radiolabelled loop-acting diuretics ([14C]frusemide, [14C]ethacrynic acid and [3H]bumetanide) and human Tamm-Horsfall glycoprotein or human serum albumin in vitro was evaluated by equilibrium dialysis. 2. The diuretic action and binding to urinary Tamm-Horsfall glycoprotein of the radiolabelled diuretics in vivo, after intravenous administration, were examined in rabbits. 3. In vitro, all three radiolabelled diuretics bound strongly to human serum albumin, but not to Tamm-Horsfall glycoprotein. 4. Radiolabelled frusemide and bumetanide, but not ethacrynic acid, caused a diuresis in rabbits, but no binding between the drugs and Tamm-Horsfall glycoprotein was seen in vivo. 5. Binding to Tamm-Horsfall glycoprotein does not appear to be an important mechanism in the action of loop diuretics.

  14. Protective effect of Cardiospermum halicacabum leaf extract on glycoprotein components on STZ-induced hyperglycemic rats

    Institute of Scientific and Technical Information of China (English)

    Chinnadurai Veeramani; Khalid S Al-Numair; Mohammed A Alsaif; Govindasamy Chandramohan; Nouf S Al-Numair; Kodukkur Viswanathan Pugalendi


    Objective: To investigate the protective role of Cardiospermum halicacabum (C. halicacabum) leaf extract on glycoprotein metabolism in streptozotocin (STZ)-induced diabetic rats. Methods:Diabetes was induced in male albino Wistar rats by intraperitonial administration of STZ. TheC. halicacabum leaf extract (CHE) was administered orally to normal and STZ-diabetic rats for 45 days. The effects of C. halicacabum leaf extract (CHE) on plasma and tissue glycoproteins (hexose, hexosamine, fucose and sialic acid) were determined. Results: The levels of plasma and tissues glycoproteins containing hexose, hexosamine and fucose were significantly increased in STZ-induced diabetic rats. In addition, the level of sialic acid significantly increased in plasma and liver while decreased in kidney of STZ-induced diabetic rats. After administration of CHE to diabetic rats, the metabolic alteration of glycoprotein reverted towards normal levels.Conclusions:The present study indicates that the CHE possesses a protective effect on abnormal glycoprotein metabolism in addition to its antihyperglycemic activity.

  15. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation. (United States)

    Arnold, Lindsay; Chen, Rachel


    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

  16. Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants. (United States)

    Goh, John S Y; Chan, Kah Fai; Song, Zhiwei


    The degree of sialylation of therapeutic glycoproteins affects its circulatory half-life and efficacy because incompletely sialylated glycoproteins are cleared from circulation by asialoglycoprotein receptors present in the liver cells. Mammalian expression systems, often employed in the production of these glycoprotein drugs, produce heterogeneously sialylated products. Here, we describe how to produce highly sialylated glycoproteins using a Chinese hamster ovary (CHO) cell glycosylation mutant called CHO-gmt4 with human erythropoietin (EPO) as a model glycoprotein. The protocol describes how to isolate and characterize the CHO glycosylation mutants and how to assess the sialylation of the recombinant protein using isoelectric focusing (IEF). It further describes how to inactivate the dihydrofolate reductase (DHFR) gene in these cells using zinc finger nuclease (ZFN) technology to enable gene amplification and the generation of stable cell lines producing highly sialylated EPO.

  17. Binding of soluble glycoproteins from sugarcane juice to cells of Acetobacter diazotrophicus. (United States)

    Legaz, M E; de Armas, R; Barriguete, E; Vicente, C


    Sugarcane produces two different pools of glycoproteins containing a heterofructan as glycidic moiety, tentatively defined as high-molecular mass (HMMG) and mid-molecular mass (MMMG) glycoproteins. Both kinds of glycoproteins can be recovered in sugarcane juice. Fluorescein-labelled glycoproteins are able to bind to Acetobacter diazotrophicus cells, a natural endophyte of sugarcane. This property implies the aggregation of bacterial cells in liquid culture after addition of HMMG or MMMG. Anionic glycoproteins seem to be responsible for the binding activity whereas cationic fraction is not retained on the surface ofA. diazotrophicus. Bound HMMG is competitively desorbed by sucrose whereas MMMG is desorbed by glucosamine or fructose. On this basis, a hypothesis about the discriminatory ability of sugarcane to choose the compatible endophyte from several possible ones is proposed.

  18. Proteinaceous alpha-araylase inhibitors

    DEFF Research Database (Denmark)

    Svensson, Birte; Fukuda, Kenji; Nielsen, P.K.;


    Proteins that inhibit alpha-amylases have been isolated from plants and microorganisms. These inhibitors can have natural roles in the control of endogenous a-amylase activity or in defence against pathogens and pests; certain inhibitors are reported to be antinutritional factors. The alpha-amylase...... inhibitors belong to seven different protein structural families, most of which also contain evolutionary related proteins without inhibitory activity. Two families include bifunctional inhibitors acting both on alpha-amylases and proteases. High-resolution structures are available of target alpha-amylases...... in complex with inhibitors from five families. These structures indicate major diversity but also some similarity in the structural basis of alpha-amylase inhibition. Mutational analysis of the mechanism of inhibition was performed in a few cases and various protein engineering and biotechnological...

  19. The Mechanism of Henipavirus Fusion: Examining the Relationships between the Attachment and Fusion Glycoproteins

    Institute of Scientific and Technical Information of China (English)

    Andrew C. Hickey; Christopher C. Broder


    The henipaviruses, represented by Nipah virus and Hendra virus, are emerging zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These viruses enter host cells via a class I viral fusion mechanism mediated by their attachment and fusion envelope glycoproteins; efficient membrane fusion requires both these glycoproteins in conjunction with specific virus receptors present on susceptible host cells. The henipavirus attachment glycoprotein interacts with a cellular B class ephrin protein receptor triggering conformational alterations leading to the activation of the viral fusion (F) glycoprotein. The analysis of monoclonal antibody (mAb) reactivity with G has revealed measurable alterations in the antigenic structure of the glycoprotein following its binding interaction with receptor. These observations only appear to occur with full-length native G glycoprotein, which is a tetrameric oligomer, and not with soluble forms of G (sG), which are disulfide-linked dimers. Single amino acid mutations in a heptad repeat-like structure within the stalk domain of G can disrupt its association with F and subsequent membrane fusion promotion activity. Notably, these mutants of G also appear to confer a postreceptor bound conformation implicating the stalk domain as an important element in the G glycoprotein's structure and functional relationship with F. Together, these observations suggest fusion is dependent on a specific interaction between the F and G glycoproteins of the henipaviruses. Further, receptor binding induces measurable changes in the G glycoprotein that appear to be greatest in respect to the interactions between the pairs of dimers comprising its native tetrameric structure. These receptor-induced conformational changes may be associated with the G glycoprotein's promotion of the fusion activity of F.

  20. Effect of reduced renal mass on renal ammonia transporter family, Rh C glycoprotein and Rh B glycoprotein, expression. (United States)

    Kim, Hye-Young; Baylis, Chris; Verlander, Jill W; Han, Ki-Hwan; Reungjui, Sirirat; Handlogten, Mary E; Weiner, I David


    Kidneys can maintain acid-base homeostasis, despite reduced renal mass, through adaptive changes in net acid excretion, of which ammonia excretion is the predominant component. The present study examines whether these adaptations are associated with changes in the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). We used normal Sprague-Dawley rats and a 5/6 ablation-infarction model of reduced renal mass; control rats underwent sham operation. After 1 wk, glomerular filtration rate, assessed as creatinine clearance, was decreased, serum bicarbonate was slightly increased, and Na(+) and K(+) were unchanged. Total urinary ammonia excretion was unchanged, but urinary ammonia adjusted for creatinine clearance, an index of per nephron ammonia metabolism, increased significantly. Although reduced renal mass did not alter total Rhcg protein expression, both light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated hypertrophy of both intercalated cells and principal cells in the cortical and outer medullary collecting duct that was associated with increased apical and basolateral Rhcg polarization. Rhbg expression, analyzed using immunoblot analysis, immunohistochemistry, and measurement of cell-specific expression, was unchanged. We conclude that altered subcellular localization of Rhcg contributes to adaptive changes in single-nephron ammonia metabolism and maintenance of acid-base homeostasis in response to reduced renal mass.

  1. Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum. (United States)

    Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E; Marx, Christine E; Shampine, Lawrence J; Girdler, Susan S; Morrow, A Leslie


    The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, psteroid was detected only in 3/16 control subjects. Levels of 3alpha,5alpha-A, 3alpha,5beta-A and pregnenolone were not altered. This method can be used to investigate the physiological and pathological role of neuroactive steroids and to develop biomarkers and new therapeutics for neurological and psychiatric disorders.

  2. alpha-Smooth muscle actin-expressing cells and lubricin in periprosthetic tissue. (United States)

    Funakoshi, Tadanao; Martin, Scott D; Wolf, Bryce T; Schmid, Thomas M; Thornhill, Thomas S; Spector, Myron


    The objective of the study was to evaluate the distributions of (1) cells expressing the contractile actin isoform, alpha-smooth muscle actin (alpha-SMA) and (2) a lubricating and antiadhesion glycoprotein, lubricin, in the tissue around loose joint replacement prostheses in human subjects. Periprostehtic tissue resected at revision arthroplasty of noncemented glenoid components of total shoulder arthroplasties was obtained from 10 patients. Samples of periprosthetic tissue were stained with monoclonal antibodies to alpha-SMA and lubricin. alpha-SMA was found in cells, principally of fibroblast morphology, in many of the fields of view (FOVs) in samples from all patients. Moderate correlations were observed between the percentage of FOVs containing alpha-SMA-expressing cells and the percentages of FOVs containing polyethylene (R(2) = 0.79) and metallic (R(2) = 0.75) particles. Lubricin was identified (1) as a discrete layer on the surface, (2) within the extracellular matrix, and (3) intracellularly. These lubricin-positive features were found in samples from all patients. Strong correlations were noted between the percentages of FOVs with matrix and intracellular lubricin staining (R(2) = 0.97) and between the percentages of FOVs with surface and matrix staining for lubricin (R(2) = 0.96). Having established the presence of alpha-SMA and lubricin in periprosthetic tissue, hypotheses regarding their role in the development and persistence of periprosthetic tissue can be synthesized for future study: for example, alpha-SMA-enabled contracture of the fibrous periprosthetic tissue results in its densification, and lubricin-coated surfaces interfere with integrative repair processes necessary for resorption and remodeling.

  3. Radiative Corrections to the Muonium Hyperfine Structure; 2, The $\\alpha (Z\\alpha)^2$ Correction

    CERN Document Server

    Nio, M


    This is the second of a series of papers on the radiative corrections of order $\\alpha^2 (Z\\alpha)$, $\\alpha (Z\\alpha )^2$, and various logarithmic terms of order $\\alpha^4$, to the hyperfine structure of the muonium ground state. This paper deals with the $\\alpha (Z\\alpha)^2$ correction. Based on the NRQED bound state theory, we isolated the term of order $\\alpha(Z\\alpha)^2$ exactly. Our result $+16.904~2~(11) \\alpha(Z\\alpha)^2 E_F / \\pi$ for the non-logarithmic part is consistent with the $\\alpha (Z\\alpha)^2$ part of Sapirstein's calculation and the recent result of Pachucki, and reduces the numerical uncertainty in the $\\alpha (Z\\alpha)^2$ term by two orders of magnitude.

  4. Myelin oligodendrocyte glycoprotein (MOG35-55)-induced experimental autoimmune encephalomyelitis is ameliorated in interleukin-32 alpha transgenic mice. (United States)

    Yun, Jaesuk; Gu, Sun Mi; Yun, Hyung Mun; Son, Dong Ju; Park, Mi Hee; Lee, Moon Soon; Hong, Jin Tae


    Multiple sclerosis (MS), also known as disseminated sclerosis or encephalomyelitis disseminate, is an inflammatory disease in which myelin in the spinal cord and brain are damaged. IL-32α is known as a critical molecule in the pathophysiology of immune-mediated chronic inflammatory disease such as rheumatoid arthritis, chronic pulmonary disease, and cancers. However, the role of IL-32α on spinal cord injuries and demyelination is poorly understood. Recently, we reported that the release of proinflammatory cytokines were reduced in IL-32α-overexpressing transgenic mice. In this study, we investigated whether IL-32α plays a role on MS using experimental autoimmune encephalomyelitis (EAE), an experimental mouse model of MS, in human IL-32α Tg mice. The Tg mice were immunized with MOG35-55 suspended in CFA emulsion followed by pertussis toxin, and then EAE paralysis of mice was scored. We observed that the paralytic severity and neuropathology of EAE in IL-32α Tg mice were significantly decreased compared with that of non-Tg mice. The immune cells infiltration, astrocytes/microglials activation, and pro-inflammatory cytokines (IL-1β and IL-6) levels in spinal cord were suppressed in IL-32α Tg mice. Furthermore, NG2 and O4 were decreased in IL-32α Tg mice, indicating that spinal cord damaging was suppressed. In addition, in vitro assay also revealed that IL-32α has a preventive role against Con A stimulation which is evidenced by decrease in T cell proliferation and inflammatory cytokine levels in IL-32α overexpressed Jurkat cell. Taken together, our findings suggested that IL-32α may play a protective role in EAE by suppressing neuroinflammation in spinal cord.

  5. Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

    DEFF Research Database (Denmark)

    Udby, Lene; Sørensen, Ole E; Pass, Jesper


    Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin...

  6. ALPHA freezes antiprotons

    CERN Multimedia

    CERN Bulletin


    Laboratories like CERN can routinely produce many different types of antiparticles. In 1995, the PS210 experiment formed the first antihydrogen atoms and a few years later, in 2002, ATRAP and ATHENA were already able to produce several thousand of them. However, no experiment in the world has succeeded in ‘trapping’ these anti-atoms in order to study them. This is the goal of the ALPHA experiment, which has recently managed to cool down the antiprotons to just a few Kelvin. This represents a major step towards trapping the anti-atom, thus opening a new avenue into the investigation of antimatter properties.   Members of the ALPHA collaboration working on the apparatus in the Antiproton Decelerator experimental hall at CERN. Just like the atom, the anti-atom is neutral. Unlike the atom, the anti-atom is made up of antiprotons (as opposed to protons in the atom) and positrons (as opposed to electrons). In order to thoroughly study the properties of the anti-atoms, scien...

  7. Venus - Alpha Regio (United States)


    The eastern edge of Alpha Regio is shown in this image centered at 30 degrees south latitude and 11.8 degrees east longitude (longitude on Venus is measured from 0 degrees to 360 degrees east). Seven circular, dome-like hills, averaging 25 kilometers (15 miles) in diameter with maximum heights of 750 meters (2,475 feet) dominate the scene. These features are interpreted as very thick lava flows that came from an opening on the relatively level ground, which allowed the lava to flow in an even pattern outward from the opening. The complex fractures on top of the domes suggest that if the domes were created by lava flows, a cooled outer layer formed and then further lava flowing in the interior stretched the surface. The domes may be similar to volcanic domes on Earth. Another interpretation is that the domes are the result of molten rock or magma in the interior that pushed the surface layer upward. The near-surface magma then withdrew to deeper levels, causing the collapse and fracturing of the dome surface. The bright margins possibly indicate the presence of rock debris on the slopes of the domes. Some of the fractures on the plains cut through the domes, while others appear to be covered by the domes. This indicates that active processes pre date and post date the dome-like hills. The prominent black area in the northeast corner of the image is a data gap. North is at the top of the image.

  8. Partitioning $\\alpha$-large sets for $\\alpha<\\varepsilon_{\\omega}$

    CERN Document Server

    De Smet, Michiel


    We generalise the results by Bigorajska and Kotlarski about partitioning $\\alpha$-large sets, by extending the domain up to ordinals below $\\varepsilon_{\\omega}$. These results will be very useful to give a miniaturisation of the infinite Ramsey Theorem.

  9. Two alpha, three alpha and multiple heavy-ion radioactivities

    Energy Technology Data Exchange (ETDEWEB)

    Poenaru, D.N.; Ivascu, M. (Institute for Physics and Nuclear Engineering, Bucharest (Romania))


    New decay modes by spontaneous emission of two and three ..cap alpha.. particles and two identical or different heavy ions, are predicted. The analytical variant of the superasymmetric fission model is used to estimate the half lives.

  10. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun


    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 structures 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.

  11. Structure of the Epstein-Barr virus major envelope glycoprotein. (United States)

    Szakonyi, Gerda; Klein, Michael G; Hannan, Jonathan P; Young, Kendra A; Ma, Runlin Z; Asokan, Rengasamy; Holers, V Michael; Chen, Xiaojiang S


    Epstein-Barr virus (EBV) infection of B cells is associated with lymphoma and other human cancers. EBV infection is initiated by the binding of the viral envelope glycoprotein (gp350) to the cell surface receptor CR2. We determined the X-ray structure of the highly glycosylated gp350 and defined the CR2 binding site on gp350. Polyglycans shield all but one surface of the gp350 polypeptide, and we demonstrate that this glycan-free surface is the receptor-binding site. Deglycosylated gp350 bound CR2 similarly to the glycosylated form, suggesting that glycosylation is not important for receptor binding. Structure-guided mutagenesis of the glycan-free surface disrupted receptor binding as well as binding by a gp350 monoclonal antibody, a known inhibitor of virus-receptor interactions. These results provide structural information for developing drugs and vaccines to prevent infection by EBV and related viruses.

  12. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase (United States)

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


    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.

  13. Stereoselective Modulation of P-Glycoprotein by Chiral Small Molecules. (United States)

    Carocci, Alessia; Catalano, Alessia; Turi, Francesco; Lovece, Angelo; Cavalluzzi, Maria M; Bruno, Claudio; Colabufo, Nicola A; Contino, Marialessandra; Perrone, Maria G; Franchini, Carlo; Lentini, Giovanni


    Inhibition of drug efflux pumps such as P-glycoprotein (P-gp) is an approach toward combating multidrug resistance, which is a significant hurdle in current cancer treatments. To address this, N-substituted aryloxymethyl pyrrolidines were designed and synthesized in their homochiral forms in order to investigate the stereochemical requirements for the binding site of P-gp. Our study provides evidence that the chiral property of molecules could be a strategy for improving the capacity for interacting with P-gp, as the most active compounds of the series stereoselectively modulated this efflux pump. The naphthalene-1-yl analogue (R)-2-[(2,3-dichlorophenoxy)methyl]-1-(naphthalen-1-ylmethyl)pyrrolidine) [(R)-7 a] emerged foremost for its potency and stereoselectivity toward P-gp, with the S enantiomer being nearly inactive. The modulation of P-gp by (R)-7 a involved consumption of ATP, thus demonstrating that the compound behaves as a P-gp substrate.

  14. An analysis of amino acid sequences surrounding archaeal glycoprotein sequons. (United States)

    Abu-Qarn, Mehtap; Eichler, Jerry


    Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N-glycosylation made by sequon-bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N-glycosylation.

  15. An analysis of amino acid sequences surrounding archaeal glycoprotein sequons

    Directory of Open Access Journals (Sweden)

    Mehtap Abu-Qarn


    Full Text Available Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N-glycosylation made by sequon-bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N-glycosylation.


    Directory of Open Access Journals (Sweden)

    Davis F. Ferreira


    Full Text Available We have previously observed that interferon (recIFNa2b blocks the process of morphogenesis of Mayaro virus in TC7 cells (monkey kidney. In this work we show that IFNa inhibits preferentially virus glycoproteins and their precursors, and this effect is probably correlated to the alterations in the morphogenesis process previously observed.Observamos anteriormente que o Interferon (IFN recombinante a2b bloqueia o processo de morfogênese do vírus Mayaro em células TC7 (rim de macaco. Neste trabalho demonstramos que o IFNa inibe preferencialmente as glicoproteínas virais e seus precursores e que este efeito está, provavelmente, correlacionado com as alterações no processo de morfogênese previamente observadas.

  17. Myelin-associated glycoprotein and its axonal receptors. (United States)

    Schnaar, Ronald L; Lopez, Pablo H H


    Myelin-associated glycoprotein (MAG) is expressed on the innermost myelin membrane wrap, directly apposed to the axon surface. Although it is not required for myelination, MAG enhances long-term axon-myelin stability, helps to structure nodes of Ranvier, and regulates the axon cytoskeleton. In addition to its role in axon-myelin stabilization, MAG inhibits axon regeneration after injury; MAG and a discrete set of other molecules on residual myelin membranes at injury sites actively signal axons to halt elongation. Both the stabilizing and the axon outgrowth inhibitory effects of MAG are mediated by complementary MAG receptors on the axon surface. Two MAG receptor families have been described, sialoglycans (specifically gangliosides GD1a and GT1b) and Nogo receptors (NgRs). Controversies remain about which receptor(s) mediates which of MAG's biological effects. Here we review the findings and challenges in associating MAG's biological effects with specific receptors.

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

    Energy Technology Data Exchange (ETDEWEB)

    Schmidt, A. E., E-mail:; Shvetsov, A. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation); Kuklin, A. I. [Joint Institute for Nuclear Research (Russian Federation); Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation)


    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.

  19. Immunoglobulin-E reactivity to wine glycoproteins in heavy drinkers

    DEFF Research Database (Denmark)

    Gonzalez-Quintela, Arturo; Gomez-Rial, Jose; Valcarcel, Catalina;


    and biological significance of IgE antibodies to N-glycans from wine glycoproteins in heavy drinkers. A structured questionnaire, skin prick tests, serum IgE levels, IgE-immunoblotting to wine extracts, and basophil activation tests were used to characterize 20 heavy drinkers and 10 control subjects. Eleven...... heavy drinkers (55%) showed IgE binding to proteins in wine extracts. The proteins were identified by mass spectrometry as grape-derived vacuolar invertase and thaumatin-like protein. Immunoblot reactivity was closely associated with the presence of IgE to CCDs and was inhibited by preincubation...... with a glycoconjugate containing bromelain-type N-glycans. The same conjugate, CCD-bearing allergens, and wine extracts activated basophils in patients with high-titer CCD-specific IgE but not in healthy controls. There was no relationship between immunoblot reactivity and consumption of any specific type of wine...

  20. Bioskin as an affinity matrix for the separation of glycoproteins. (United States)

    Vicente, C; Sebastián, B; Fontaniella, B; Márquez, A; Xavier Filho, L; Legaz, M E


    Bioskin is a natural product produced by a mixed culture of Acetobacter xylinum, Saccharomyces cerevisiae and S. pombe cultured on media containing sucrose. It is of fibrillar nature able to retain some proteins, such as cytochrome c, by adsorption, and mainly composed of glucosamine and N-acetyl-D-glucosamine. This makes it possible that, at an adequate pH value, proteins charged as polyanionic molecules, such as catalase, can be retained by ionic adsorption using the positively charged amino groups of the matrix. In addition, bioskin can also be used as an affinity matrix to retain glycoproteins able to perform specific affinity reactions with the amino sugars of the matrix, such as invertase, fetuin or ovalbumin. Its possible use as a chromatographic support is discussed.

  1. Alpha heating in toroidal devices

    Energy Technology Data Exchange (ETDEWEB)

    Miley, G.H.


    Ignition (or near-ignition) by alpha heating is a key objective for the achievement of economic fusion reactors. While good confinement of high-energy alphas appears possible in larger reactors, near-term tokamak-type ignition experiments as well as some concepts for small reactors (e.g., the Field-Reversed Mirror or FRM) potentially face marginal situations. Consequently, there is a strong motivation to develop methods to evaluate alpha losses and heating profiles in some detail. Such studies for a TFTR-size tokamak and for a small FRM are described here.

  2. Crystal Structure of the Human Cytomegalovirus Glycoprotein B.

    Directory of Open Access Journals (Sweden)

    Heidi G Burke


    Full Text Available 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.

  3. In silico-based vaccine design against Ebola virus glycoprotein

    Directory of Open Access Journals (Sweden)

    Dash R


    Full Text Available Raju Dash,1 Rasel Das,2 Md Junaid,3 Md Forhad Chowdhury Akash,4 Ashekul Islam,5 SM Zahid Hosen1 1Molecular Modeling and Drug Design Laboratory (MMDDL, Pharmacology Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR, Chittagong, Bangladesh; 2Nanotechnology and Catalysis Research Center, University of Malaya, Kuala Lumpur, Malaysia; 3Department of Pharmaceutical Sciences, North South University, Dhaka, Bangladesh; 4Department of Pharmacy, BGC Trust University Bangladesh, Chittagong, Bangladesh; 5Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, Bangladesh Abstract: Ebola virus (EBOV is one of the lethal viruses, causing more than 24 epidemic outbreaks to date. Despite having available molecular knowledge of this virus, no definite vaccine or other remedial agents have been developed yet for the management and avoidance of EBOV infections in humans. Disclosing this, the present study described an epitope-based peptide vaccine against EBOV, using a combination of B-cell and T-cell epitope predictions, followed by molecular docking and molecular dynamics simulation approach. Here, protein sequences of all glycoproteins of EBOV were collected and examined via in silico methods to determine the most immunogenic protein. From the identified antigenic protein, the peptide region ranging from 186 to 220 and the sequence HKEGAFFLY from the positions of 154–162 were considered the most potential B-cell and T-cell epitopes, correspondingly. Moreover, this peptide (HKEGAFFLY interacted with HLA-A*32:15 with the highest binding energy and stability, and also a good conservancy of 83.85% with maximum population coverage. The results imply that the designed epitopes could manifest vigorous enduring defensive immunity against EBOV. Keywords: Ebola virus, epitope, glycoprotein, vaccine design

  4. Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays. (United States)

    Caragata, Michael; Shah, Alok K; Schulz, Benjamin L; Hill, Michelle M; Punyadeera, Chamindie


    Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the sample collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.

  5. Preparation of Concanavalin A-Chelating Magnetic Nanoparticles for Selective Enrichment of Glycoproteins. (United States)

    Dong, Liping; Feng, Shun; Li, Shanshan; Song, Peipei; Wang, Jide


    In this work, a soft and nondestructive approach was developed to prepare concanavalin A-chelating magnetic nanoparticles (Con A-MNPs) for selective enrichment of glycoproteins. Ethylenediamine tetraacetic acid-modified-MNPs (EDTA-MNPs) were prepared by a one-pot chemical coprecipitation method first, and then, Cu(II) cations were used as bridge groups to immobilize Con A on EDTA-MNPs. The as-prepared absorbents with a mean diameter of 15 nm showed a strong magnetic response to an externally applied magnetic field. The results of thermogravimetric analysis showed the content of immobilized Con A was up to 28 wt %. For glycoprotein ovalbumin, the maximum capacity and equilibrium constant were 72.41 mg/g and 0.6035 L/mg, respectively. The as-prepared nanocomposites exhibited a remarkable selectivity for glycoproteins and can enrich glycoproteins specifically from a mixture of glycoprotein and nonglycoprotein even at a molar ratio of 1:600. It was also successfully applied for the enrichment of glycoproteins from real egg white samples. We expect that our finding will serve as a helpful template for others to design new adsorbents for enriching glycoproteins.

  6. Protective Role of α2HS-Glycoprotein in HBV-Associated Liver Failure

    Directory of Open Access Journals (Sweden)

    Xue-Gong Fan


    Full Text Available n this study, levels of plasma α2-Heremans-Schmid glycoprotein, serum tumor necrosis factor-α, serum liver function parameters and short-term mortality were measured in 100 hepatitis B patients. Release of interleukin-6 and tumor necrosis factor-α from the lipopolysaccharide-stimulated peripheral blood mononuclear cells in the presence/absence of spermine and α2-Heremans-Schmid glycoprotein were analyzed by enzyme-linked immunosorbent assay to determine the significance and potential mechanism of α2-Heremans-Schmid glycoprotein in hepatitis B virus-associated liver damage. Results showed that serum α2-Heremans-Schmid glycoprotein levels in acute-on-chronic liver failure patients were significantly lower than that in chronic hepatitis B patients or healthy controls (p < 0.05. A negative dependence between serum human α2-Heremans-Schmid glycoprotein and tumor necrosis factor-α levels was observed. Interleukin-6 and tumor necrosis factor-α levels in the lipopolysaccharide-induced peripheral blood mononuclear cell supernates were significantly reduced by spermine and/or α2-Heremans-Schmid glycoprotein. The latter two proteins jointly inhibited cytokine release. These observations suggest that plasma α2-Heremans-Schmid glycoprotein is an independent marker of liver damage and a prognostic indicator of hepatitis B virus chronicity. It may reduce liver inflammation by partially inhibiting release of inflammatory factors from activated peripheral blood mononuclear cells.

  7. Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain. (United States)

    Quarles, R H


    Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

  8. Bioactivity of proteins isolated from Lactobacillus plantarum L67 treated with Zanthoxylum piperitum DC glycoprotein. (United States)

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


    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

  9. The Lyman alpha reference sample

    DEFF Research Database (Denmark)

    Hayes, M.; Östlin, G.; Schaerer, D.


    We report on new imaging observations of the Lyman alpha emission line (Lyα), performed with the Hubble Space Telescope, that comprise the backbone of the Lyman alpha Reference Sample. We present images of 14 starburst galaxies at redshifts 0.028 alpha emission line (Lyα), performed with the Hubble Space Telescope, that comprise the backbone of the Lyman alpha Reference Sample. We present images of 14 starburst galaxies at redshifts 0.028 ... with quantities that scale with dust content, in the sense that a low dust abundance is a necessary requirement (although not the only one) in order to spread Lyα photons throughout the interstellar medium and drive a large extended Lyα halo. © 2013. The American Astronomical Society. All rights reserved....


    Directory of Open Access Journals (Sweden)

    A. V. Shulkin


    Full Text Available On the basis of the analysis of published data the role of P-glycoprotein, carrier protein, in rational pharmacotherapy in cardiology was shown on the example of its substrates – digoxin, antiplatelet agents and anticoagulants. Determination of C3435T polymorphism of multidrug resistance gene (MDR1, encoding P-glycoprotein, in pharmacotherapy with digoxin, antiplatelet drugs (clopidogrel tikagrelol, prasugrel and anticoagulants (dabigatran etexilate, rivaroxaban, edoxaban is not feasible in routine practice. Drug in- teractions have clinical implications for the efficacy and safety of pharmacotherapy in coadministration of these drugs with P-glycoprotein substrates, inducers and inhibitors.


    Directory of Open Access Journals (Sweden)

    A. V. Shulkin


    Full Text Available On the basis of the analysis of published data the role of P-glycoprotein, carrier protein, in rational pharmacotherapy in cardiology was shown on the example of its substrates – digoxin, antiplatelet agents and anticoagulants. Determination of C3435T polymorphism of multidrug resistance gene (MDR1, encoding P-glycoprotein, in pharmacotherapy with digoxin, antiplatelet drugs (clopidogrel tikagrelol, prasugrel and anticoagulants (dabigatran etexilate, rivaroxaban, edoxaban is not feasible in routine practice. Drug in- teractions have clinical implications for the efficacy and safety of pharmacotherapy in coadministration of these drugs with P-glycoprotein substrates, inducers and inhibitors.

  12. Varicella-zoster virus glycoprotein I is essential for growth of virus in Vero cells.


    Cohen, J I; Nguyen, H.


    Varicella-zoster virus (VZV) encodes at least six glycoproteins. Glycoprotein I (gI), the product of open reading frame 67, is a 58- to 62-kDa glycoprotein found in VZV-infected cells. We constructed two VZV gI deletion mutants. Immunoprecipitation of VZV gE from infected cells indicated that cells infected with VZV deleted for gI expressed a gE that was larger (100 kDa) than that expressed in cells infected with the parental virus (98 kDa). Cell-associated or cell-free VZV deleted for gI gre...

  13. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins (United States)

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Raj, Isha; Singh, Sarman; Singh, Tej P.; Yadav, Savita


    Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma. PMID:22182811

  14. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins

    Directory of Open Access Journals (Sweden)

    Anil Kumar Tomar


    Full Text Available Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma.

  15. Amaranthus leucocarpus lectin recognizes a moesin-like O-glycoprotein and costimulates murine CD3-activated CD4+ T cells (United States)

    Arenas-Del Ángel, Maria; Legorreta-Herrera, Martha; Mendoza-Hernández, Guillermo; Garfias, Yonathan; Chávez, Raul; Zenteno, Edgar; Lascurain, Ricardo


    The Galβ1,3GalNAcα1,O-Ser/Thr specific lectin from Amaranthus leucocarpus (ALL) binds a ∼70 kDa glycoprotein on murine T cell surface. We show that in the absence of antigen presenting cells, murine CD4+ T cells activated by an anti-CD3 antibody plus ALL enhanced cell proliferation similar to those cells activated via CD3/CD28 at 48 h of culture. Moreover, ALL induced the production of IL-4, IL-10, TNF-alpha, and TGF-beta in CD3-activated cells. Proteomic assay using two-dimensional electrophoresis and far-Western blotting, ALL recognized two prominent proteins associated to the lipid raft microdomains in CD3/CD28-activated CD4+ T cells. By mass spectrometry, the peptide fragments from ALL-recognized proteins showed sequences with 33% homology to matricin (gi|347839 NCBInr) and 41% identity to an unnamed protein related to moesin (gi|74186081 NCBInr). Confocal microscopy analysis of CD3/CD28-activated CD4+ T cells confirmed that staining by ALL colocalized with anti-moesin FERM domain antibody along the plasma membrane and in the intercellular contact sites. Our findings suggest that a moesin-like O-glycoprotein is the ALL-recognized molecule in lipid rats, which induces costimulatory signals on CD4+ T cells. PMID:26417436

  16. Mapping sites of herpes simplex virus type 1 glycoprotein D that permit insertions and impact gD and gB receptors usage (United States)

    Fan, Qing; Kopp, Sarah; Connolly, Sarah A.; Muller, William J.; Longnecker, Richard


    Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) is one of four glycoproteins essential for HSV entry and cell fusion. The purpose of this study was to determine the plasticity of gD to tolerate insertion or deletion mutations and to construct an oncolytic HSV-1 that utilizes the disialoganglioside GD2 as a HSV-1 entry receptor. We found that the N-terminus of gD tolerates long insertions, whereas residues adjacent to the gD Ig-like V-type core tolerated shorter insertions (up to 15 amino acids), but not greater than 60 amino acids. Recombinant HSV-1 containing the ch14.18 single chain variable fragment (scFv) at the N-terminus of gD failed to mediate entry, even though the ch14.18 scFv-gD chimera Fc bound to neuroblastoma cells expressing GD2. Finally, we found that hyperfusogenic gB mutants enhanced fusion to a greater degree with the gB receptor the paired immunoglobulin-like type 2 receptor alpha (PILRα) than with gD receptors HVEM and nectin-1. Hyperfusogenic gB could restore the fusion function with PILRα when a gD constructed contained only the “profusion domain” (PFD), suggesting the hyperfusogenic form of gB may regulate fusion of PILRα via a novel mechanism through gH/gL and the gD PFD. PMID:28255168

  17. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion. (United States)

    AlHajri, Salim M; Cunha, Cristina W; Nicola, Anthony V; Aguilar, Hector C; Li, Hong; Taus, Naomi S


    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 cellular

  18. Almost Redundant Components in the 3 alpha Faddeev Equation for the Buck, Friedlich and Wheatly alpha alpha Potential

    CERN Document Server

    Fujiwara, Y; Kohno, M


    The 3 alpha orthogonality condition model using the Pauli-forbidden bound states of the Buck, Friedlich and Wheatly alpha alpha potential can yield a compact 3 alpha ground state with a large binding energy, in which a small admixture of the redundant components can never be eliminated.

  19. Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response. (United States)

    Hidvegi, Tunda; Schmidt, Bela Z; Hale, Pamela; Perlmutter, David H


    In alpha(1)-antitrypsin (alpha1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein alpha1AT, alpha1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells. It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease. One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses. Here we examined the effect of mutant alpha1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant alpha1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant alpha1AT. The results show that ER retention of polymerogenic mutant alpha1ATZ does not result in an unfolded protein response (UPR). The UPR can be induced in the presence of alpha1ATZ by tunicamycin excluding the possibility that the pathway has been disabled. In striking contrast, ER retention of nonpolymerogenic alpha1AT mutants does induce the UPR. These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures. Accumulation of mutant alpha1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFkappaB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic alpha1AT mutants.

  20. Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex. (United States)

    Matsuura, Hisae; Kirschner, Austin N; Longnecker, Richard; Jardetzky, Theodore S


    The Epstein-Barr virus (EBV) is a γ-herpesvirus that infects B cells and epithelial cells and that has been linked to malignancies in both cell types in vivo. EBV, like other herpesviruses, has three glycoproteins, glycoprotein B (gB), gH, and gL, that form the core membrane fusion machinery mediating viral penetration into the cell. The gH and gL proteins associate to form a heterodimeric complex, which is necessary for efficient membrane fusion and also implicated in direct binding to epithelial cell receptors required for viral entry. To gain insight into the mechanistic role of gH/gL, we determined the crystal structure of the EBV gH/gL complex. The structure is comprised of four domains organized along the longest axis of the molecule. Comparisons with homologous HSV-2 gH/gL and partial pseudorabies virus gH structures support the domain boundaries determined for the EBV gH/gL structure and illustrate significant differences in interdomain packing angles. The gL subunit and N-terminal residues of gH form a globular domain at one end of the structure, implicated in interactions with gB and activation of membrane fusion. The C-terminal domain of gH, proximal to the viral membrane, is also implicated in membrane fusion. The gH/gL structure locates an integrin binding motif, implicated in epithelial cell entry, on a prominent loop in the central region of the structure. Multiple regions of gH/gL, including its two extreme ends, are functionally important, consistent with the multiple roles of gH/gL in EBV entry.

  1. DeltaNp73alpha regulates MDR1 expression by inhibiting p53 function. (United States)

    Vilgelm, A; Wei, J X; Piazuelo, M B; Washington, M K; Prassolov, V; El-Rifai, W; Zaika, A


    The p73 protein is a transcription factor and member of the p53 protein family that expresses as a complex variety of isoforms. DeltaNp73alpha is an N-terminally truncated isoform of p73. We found that DeltaNp73 protein is upregulated in human gastric carcinoma suggesting that DeltaNp73 may play an oncogenic role in these tumors. Although it has been shown that DeltaNp73alpha inhibits apoptosis and counteracts the effect of chemotherapeutic drugs, the underlying mechanism by which this p73 isoform contributes to chemotherapeutic drug response remains to be explored. We found that DeltaNp73alpha upregulates MDR1 mRNA and p-glycoprotein (p-gp), which is involved in chemotherapeutic drug transport. This p-gp upregulation was accompanied by increased p-gp functional activity in gastric cancer cells. Our data suggest that upregulation of MDR1 by DeltaNp73alpha is mediated by interaction with p53 at the MDR1 promoter.

  2. Hereditary Persistence of Alpha-Fetoprotein Is Associated with the -119G>A Polymorphism in AFP Gene. (United States)

    Deshpande, Neha; Chavan, Radhika; Bale, Govardhan; Avanthi, Urmila Steffie; Aslam, Mohsin; Ramchandani, Mohan; Reddy, D Nageshwar; Ravikanth, V V


    Alpha-fetoprotein (AFP) is a glycoprotein that is produced by the liver and yolk sac during fetal development. Its levels are usually raised in malignant conditions. Hereditary persistence of AFP (HPAFP) is a rare benign condition with elevated levels of AFP. It is inherited in a dominant mode with complete penetrance and is usually not associated with any clinical disability. We report two individuals with elevated levels of AFP harboring the -119G>A polymorphism in the AFP gene. A genetic screening to rule out variants in the AFP gene is advised in cases with unexplained persistent AFP levels to avoid inappropriate treatment and surgical options.

  3. Hereditary Persistence of Alpha-Fetoprotein Is Associated with the −119G>A Polymorphism in AFP Gene (United States)

    Deshpande, Neha; Chavan, Radhika; Bale, Govardhan; Avanthi, Urmila Steffie; Aslam, Mohsin; Ramchandani, Mohan; Reddy, D. Nageshwar


    Alpha-fetoprotein (AFP) is a glycoprotein that is produced by the liver and yolk sac during fetal development. Its levels are usually raised in malignant conditions. Hereditary persistence of AFP (HPAFP) is a rare benign condition with elevated levels of AFP. It is inherited in a dominant mode with complete penetrance and is usually not associated with any clinical disability. We report two individuals with elevated levels of AFP harboring the −119G>A polymorphism in the AFP gene. A genetic screening to rule out variants in the AFP gene is advised in cases with unexplained persistent AFP levels to avoid inappropriate treatment and surgical options. PMID:28286798

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

    Directory of Open Access Journals (Sweden)

    Yang Gao


    Full Text Available 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.

  5. Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein

    Directory of Open Access Journals (Sweden)

    Shukra M. Aavula


    Full Text Available Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

  6. Prostate Cancer Progression and Serum Sibling (Small Integrin Binding N-Linked Glycoprotein) Levels (United States)


    integrin-binding ligand N-linked glycoproteins (SIBLINGs): multifunctional proteins in cancer Akeila Bellahcène*, Vincent Castronovo*, Kalu U. E...hypertension, angina , myocardial infarction, percutaneous transluminal coronary angioplasty, coronary artery bypass surgery, congestive heart failure, and

  7. Purification of the envelope glycoproteins of western equine encephalitis virus by glass wool column chromatography.


    Yamamoto, K.; Simizu, B


    Glass wool column chromatography was used for separation of the two glycoproteins of western equine encephalitis virus. Cross-contamination of each protein separated was confirmed to be negligible by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  8. Evaluation of the expression of P-glycoprotein in propoxur-resistant Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Shabnam Yazdian


    Full Text Available There is a great concern about the effect of propoxur, as one of the more common N-methyl carbamate pesticides, on human health due to its extensive use in agricultural and non-agricultural applications. Caco-2 cells became resistant to propoxur, and the resistance was confirmed through MTT assay. Then the cell membrane integrity and P-glycoprotein expression were measured by LDH assay and western blot analysis, respectively and compared to the parent cells.  Contrary to what was expected, the expression of P-glycoprotein in propoxur resistant cells was lower than parent cells.This study indicates that the resistance to propoxur may not be related to P-glycoprotein expression directly, since P-glycoprotein expression has decreased in these cells.

  9. Serum sialic acid and glycoprotein levels in some Libyan cancer patients. (United States)

    Balo, N N; Ishaq, M


    Sialic acid is a common conjugate of some serum glycoproteins and glycolipids. Elevated levels of serum sialic acid and alterations in serum glycoproteins have been observed in certain types of cancer. In this study sialic acid concentration in the sera of patients with various types of cancer was determined. In addition to this, serum glycoproteins were also analysed by electrophoretic method. Our results indicate that serum sialic acid levels are generally raised in all types of cancer studied. This increase was more pronounced in case of lung, bronchogenic, intestinal and breast cancer. Some alterations in the serum glycoprotein profiles were also observed, particularly in bronchogenic and gall bladder cancer where an additional band in the low molecular weight region was present and in lung, breast and lymphoma where a band in the middle molecular weight region was found missing when compared with normals.

  10. Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein. (United States)

    Hummel, K B; Martin, M L; Auperin, D D


    A recombinant baculovirus was constructed that expresses the glycoprotein gene of Lassa virus (Josiah strain) under the transcriptional control of the polyhedrin promoter. The expressed protein (B-LSGPC) comigrated with the authentic viral glycoprotein as observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), was reactive with monoclonal antibodies (MAbs) in Western blots, and was glycosylated. Although the recombinant protein was not processed into the mature glycoproteins, G1 and G2, it demonstrated reactivity with all known epitopes as measured by indirect immunofluorescence (IFA), and it was immunogenic, eliciting antisera in rabbits that recognized whole virus in IFAs. Regarding future applications to diagnostic assays, the recombinant glycoprotein proved to be an effective substitute for Lassa virus-infected mammalian cells in IFAs and it was able to distinguish sera from several human cases of Lassa fever, against a panel of known negative sera of African origin, in an enzyme immunoassay (EIA).

  11. Distribution of surface glycoproteins on influenza A virus determined by electron cryotomography. (United States)

    Wasilewski, Sebastian; Calder, Lesley J; Grant, Tim; Rosenthal, Peter B


    We use electron cryotomography to reconstruct virions of two influenza A H3N2 virus strains. The maps reveal the structure of the viral envelope containing hemagglutinin (HA) and neuraminidase (NA) glycoproteins and the virus interior containing a matrix layer and an assembly of ribonucleoprotein particles (RNPs) that package the genome. We build a structural model for the viral surface by locating copies of the X-ray structure of the HA ectodomain into density peaks on the virus surface. We calculate inter-glycoprotein distances and the fractional volume occupied by glycoproteins. The models suggest that for typical HA densities on virus, Fabs can bind to epitopes on the HA stem domain. The models also show how membrane curvature may influence the number of glycoproteins that can simultaneously interact with a target surface of receptors.

  12. Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36

    Directory of Open Access Journals (Sweden)

    Roger S. Holmes


    Full Text Available Platelet glycoprotein 4 (CD36 (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3] is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, malaria, diabetes, steatosis, dementia and obesity. Genetic deficiency of this protein results in significant changes in fatty acid and oxidized lipid uptake. Comparative CD36 amino acid sequences and structures and CD36 gene locations were examined using data from several vertebrate genome projects. Vertebrate CD36 sequences shared 53–100% identity as compared with 29–32% sequence identities with other CD36-like superfamily members, SCARB1 and SCARB2. At least eight vertebrate CD36 N-glycosylation sites were conserved which are required for membrane integration. Sequence alignments, key amino acid residues and predicted secondary structures were also studied. Three CD36 domains were identified including cytoplasmic, transmembrane and exoplasmic sequences. Conserved sequences included N- and C-terminal transmembrane glycines; and exoplasmic cysteine disulphide residues; TSP-1 and PE binding sites, Thr92 and His242, respectively; 17 conserved proline and 14 glycine residues, which may participate in forming CD36 ‘short loops’; and basic amino acid residues, and may contribute to fatty acid and thrombospondin binding. Vertebrate CD36 genes usually contained 12 coding exons. The human CD36 gene contained transcription factor binding sites (including PPARG and PPARA contributing to a high gene expression level (6.6 times average. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate CD36 gene with vertebrate

  13. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Filipski, Elisabeth; Berland, Elodie [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Ozturk, Narin [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Istanbul University Faculty of Pharmacy, Department of Pharmacology, Beyazit TR-34116, Istanbul (Turkey); Guettier, Catherine [Assistance Publique-Hôpitaux de Paris, Unité de Chronothérapie, Département de Cancérologie, Hôpital Paul Brousse, Villejuif F-94807 (France); Horst, Gijsbertus T.J. van der [Department of Genetics, Erasmus University Medical Center, 3000 CA Rotterdam (Netherlands); Lévi, Francis [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Assistance Publique-Hôpitaux de Paris, Unité de Chronothérapie, Département de Cancérologie, Hôpital Paul Brousse, Villejuif F-94807 (France); and others


    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 to 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.

  14. Recognition of helper T cell epitopes in envelope (E) glycoprotein of Japanese encephalitis, west Nile and Dengue viruses. (United States)

    Kutubuddin, M; Kolaskar, A S; Galande, S; Gore, M M; Ghosh, S N; Banerjee, K


    Helper T (Th) cell antigenic sites were predicted from the primary amino acid sequence (approximately 500 in length) of the envelope (E) glycoprotein (gp) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV flaviviruses. Prediction of Th epitopes was done by analyzing the occurrence of amphipathic segments, Rothbard-Taylor tetra/pentamer motifs and presence of alpha helix-preferring amino acids. The simultaneous occurrence of all these parameters in segments of E gp were used as criteria for prediction as Th epitopes. Only one cross reactive epitope was predicted in the C-terminal region of the E gp predicted segments of all flaviviruses analyzed. This region is one of the longest amphipathic stretch (approximately from 420 to 455) and also has a fairly large amphipathic score. Based on the predicted findings three selected peptides were synthesized and analyzed for their ability to induce in vitro T cell proliferative response in different inbred strains of mice (Balb/c, C57BL6, C3H/HeJ). Synthetic peptide I and II prepared from C-terminal region gave a cross reactive response to JE, WN and Den-II in Balb/c and C3H/HeJ mice. Synthetic peptide III prepared from N-terminal region gave a proliferative response to DEN-II in Balb/c strain only, indicating differential antigen presentation.

  15. Investigation of the Pygmy Dipole Resonance in (alpha, alpha 'gamma) coincidence experiments

    NARCIS (Netherlands)

    Savran, D.; Babilon, M.; van den Berg, A. M.; Harakeh, M. N.; Hasper, J.; Wortche, H. J.; Zilges, A.


    We report on first results from experiments using the (alpha, alpha'gamma) reaction at E alpha = 136 MeV to investigate bound electric dipole (El) excitations building the so-called Pygmy Dipole Resonance (PDR) in the semi-magic nucleus Ce-140. The method of (alpha, alpha'gamma) allows the separatio

  16. Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.


    Sturman, L S; Holmes, K V; Behnke, J.


    The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus a...

  17. Spatial distribution of Reissner's fiber glycoproteins in the filum terminale of the rat and rabbit. (United States)

    Molina, B; Rodríguez, E M; Peruzzo, B; Caprile, T; Nualart, F


    The subcommissural organ secretes into the third ventricle glycoproteins that condense to form the Reissner's fiber (RF). At the distal end of the central canal of the spinal cord, the RF-glycoproteins accumulate in the form of an irregular mass known as massa caudalis. Antibodies against RF-glycoproteins and a set of lectins were used at the light and electron microscopic level to investigate the spatial distribution of the massa caudalis material in the rat and rabbit filum terminale. In the sacral region of the rat, the central canal presents gaps between the ependymal cells through which RF-glycoproteins spread out. The bulk of massa caudalis material, however, escapes through openings in the dorsal wall of the terminal ventricle. In the rabbit, the massa caudalis is formed within the ependymal canal, at the level of the second coccygeal vertebra, it accumulates within preterminal and terminal dilatations of the central canal, and it escapes out through gaps in the dorsal ependymal wall of the terminal ventricle. The existence of wide intercellular spaces and a large orifice (neuroporous) in the dorsal ependymal wall of the terminal ventricle, and the passage of RF-material through them, appear to be conserved evolutionary features. After leaving the terminal ventricle of the rat and rabbit, RF-glycoproteins establish a close spatial association with the numerous blood vessels irrigating the filum terminale, suggesting that in these species the blood vessels are the site of destination of the RF-glycoproteins escaping from the central canal, thus resembling the situation found in lower vertebrates. When passing from the RF stage to the massa caudalis stage, the rabbit RF-glycoproteins lose their sialic acid residues, exposing galactose as the terminal residue. Since this sialic acid-galactose modification of RF-glycoproteins had also been described in lamprey larvae, it may be regarded as a conserved evolutionary feature associated with the formation of the

  18. The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

    Directory of Open Access Journals (Sweden)

    A.J. Parodi


    Full Text Available The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

  19. Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry (United States)


    and form enveloped virions [1]. Seven arenaviruses cause viral hemorrhagic fever in humans: the Old World arenaviruses Lassa and ‘Lujo,’ and the New...hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to... fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular

  20. Enantiomeric separation of ibuprofen by HPLC using α1-acid glycoprotein chiral stationary phase%α1-酸性糖蛋白柱拆分布洛芬对映体

    Institute of Scientific and Technical Information of China (English)

    杨凌; 冉桂梅; 张才华; 范岩; 郭兴杰


    目的使用α1-酸性糖蛋白手性固定相(α1-AGP)拆分布洛芬对映体.方法分别以异丙醇、甲醇、乙腈为有机改性剂,考察了有机改性剂的种类、浓度、磷酸盐缓冲液的pH值及流速对对映体拆分的影响.结果布洛芬对映体在AGP柱上的最佳分离条件是:流动相为甲醇-10 ol·L-1磷酸盐缓冲液(V∶V=1∶99,pH7.0),流速为0.3 mL·min-1,柱温30 ℃.结论布洛芬对映体可以在AGP固定相上得到完全分离.

  1. Workshop on Precision Measurements of $\\alpha_s$

    Energy Technology Data Exchange (ETDEWEB)

    Bethke, Siegfried; /Munich, Max Planck Inst.; Hoang, Andre H.; /Vienna U.; Kluth, Stefan; /Munich, Max Planck Inst.; Schieck, Jochen; /Munich U.; Stewart, Iain W.; Aoki, S.; Beneke, M.; Bethke, S.; Blumlein, J.; Brambilla, N.; Brodsky, S.; /MIT, LNS


    These are the proceedings of the Workshop on Precision Measurements of {alpha}{sub s} held at the Max-Planck-Institute for Physics, Munich, February 9-11, 2011. The workshop explored in depth the determination of {alpha}{sub s}(m{sub Z}) in the {ovr MS} scheme from the key categories where high precision measurements are currently being made, including DIS and global PDF fits, {tau}-decays, electro-weak precision observables and Z-decays, event-shapes, and lattice QCD. These proceedings contain a short summary contribution from the speakers, as well as the lists of authors, conveners, participants, and talks.

  2. Scale Setting for $\\alpha_{s}$ Beyond Leading Order

    CERN Document Server

    Hornbostel, K; Morningstar, C J


    We present a general procedure for applying the scale-setting prescription of Brodsky, Lepage and Mackenzie to higher orders in the strong coupling constant $\\alphas$. In particular, we show how to apply this prescription when the leading coefficient or coefficients in a series in $\\alphas$ are anomalously small. We give a general method for computing an optimum scale numerically, within dimensional regularization, and in cases when the coefficients of a series are known. We find significant corrections to the scales for $R_{e^+ e^-}$, $\\Gamma(B \\to X_u e \\bar{\

  3. Proxima's orbit around Alpha Centauri

    CERN Document Server

    Kervella, P


    Proxima and Alpha Centauri AB have almost identical distances and proper motions with respect to the Sun. Although the probability of such similar parameters is in principle very low, the question whether they actually form a single gravitationally bound triple system has been open since the discovery of Proxima one century ago. Owing to recent high precision radial velocity measurements and the revision of the parameters of the Alpha Cen pair, we show that Proxima and Alpha Cen are gravitationally bound with a high degree of confidence. The orbital period of Proxima is approximately 600 000 years, with a moderate excentricity of 0.42 +0.07 -0.08. Proxima comes within 5.3 -0.9 +1.2 kAU of Alpha Cen at periastron, and the apastron occurs at 12.9 +0.3 -0.1 kAU. This orbital motion may have influenced the formation or evolution of the recently discovered planet orbiting Proxima as well as circumbinary planet formation around Alpha Cen.

  4. Surface plasmon resonance for real-time study of lectin-carbohydrate interactions for the differentiation and identification of glycoproteins. (United States)

    Safina, Gulnara; Duran, Iu Benet; Alasel, Mohammed; Danielsson, Bengt


    A study of specific interactions between lectins and glycoproteins has been carried out using surface plasmon resonance (SPR) in a flow-injection mode. Lectins were covalently immobilised on the surfaces of the microfluidic sensor chip via amine coupling and serum glycoproteins were injected into the flow channels. Specific lectin-glycoprotein interactions caused the shift of refractive index proportional to the mass concentration accumulated on the channel surface. Lectins showed different affinity to the tested glycoproteins and each glycoprotein displayed its own lectin-binding pattern. It is possible to distinguish and identify even glycoproteins with similar sugar structures by simple and quick screening. The working conditions of the assay were optimised. The lectin-based SPR made it possible to carry out the label-free detection of glycoproteins within a broad concentration range with a good linearity. Regeneration conditions for the surface of the sensor chip were found and optimised. Combination of 10mM HCl and 10mM glycine-HCl (pH 2.5) removes the bound glycoproteins from the lectin surface without damaging it. The kinetic and affinity parameters of lectin-glycoprotein binding were evaluated. The proposed method was tested on human glycosylated serum. Combination of the lectin panel with SPR is suitable both for specific screening and for sensitive assay of serum glycoproteins.

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

    Directory of Open Access Journals (Sweden)

    Famida G. Hoosain


    Full Text Available 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.

  6. Lipid modification gives rise to two distinct Haloferax volcanii S-layer glycoprotein populations. (United States)

    Kandiba, Lina; Guan, Ziqiang; Eichler, Jerry


    The S-layer glycoprotein is the sole component of the protein shell surrounding Haloferax volcanii cells. The deduced amino acid sequence of the S-layer glycoprotein predicts the presence of a C-terminal membrane-spanning domain. However, several earlier observations, including the ability of EDTA to selectively solubilize the protein, are inconsistent with the presence of a trans-membrane sequence. In the present report, sequential solubilization of the S-layer glycoprotein by EDTA and then with detergent revealed the existence of two distinct populations of the S-layer glycoprotein. Whereas both S-layer glycoprotein populations underwent signal peptide cleavage and N-glycosylation, base hydrolysis followed by mass spectrometry revealed that a lipid, likely archaetidic acid, modified only the EDTA-solubilized version of the protein. These observations are consistent with the S-layer glycoprotein being initially synthesized as an integral membrane protein and subsequently undergoing a processing event in which the extracellular portion of the protein is separated from the membrane-spanning domain and transferred to a waiting lipid moiety.

  7. The importance of drug-transporting P-glycoproteins in toxicology. (United States)

    van Tellingen, O


    The importance of specific transport in toxicology is becoming increasingly clear and the work on P-glycoprotein has certainly been a major contribution to these growing insights. P-Glycoproteins were discovered by their ability to confer multidrug resistance in mammalian tumour cells. They are localised in the cell membrane where they actively extrude a wide range of compounds including many anti-cancer drugs from the cell. Besides in tumour cells, drug-transporting P-glycoproteins are also expressed in a polarised fashion in normal tissues that perform an excretory or barrier function, such as the liver, kidneys, intestines, brain endothelial cells. Based on this expression profile, it has been proposed that P-glycoproteins are important in protecting the host by reducing exposure to xenobiotics. Further studies with P-glycoprotein knockout mice have clearly established this protective function. In general, the clearance of substrate drugs is lower in knockout mice due to a diminished hepatobiliary excretion, direct intestinal excretion and/or increased enterohepatic cycling. Moreover, their uptake in sanctuary sites, such as the brain or the foetus, was profoundly higher in P-glycoprotein knockout mice, as was the uptake of drugs from the gastro-intestinal tract into the systemic circulation following oral ingestion. These results clearly highlight the impact that transport proteins can play in toxicology.

  8. Bile canalicular cationic dye secretion as a model for P-glycoprotein mediated transport. (United States)

    Thalhammer, T; Stapf, V; Gajdzik, L; Graf, J


    This study explores properties of P-glycoprotein dependent membrane transport in rat liver with the use of acridine orange as the substrate. We studied the biliary secretion of the dye, its binding to canalicular membrane P-glycoprotein, and effects of the inhibitor cyclosporin A: acridine orange is excreted into bile together with less hydrophobic and glucuronidated metabolites. Cyclosporin A inhibited both the secretion of acridine orange and of its metabolites. In TR- animals, a rat strain that is deficient of the canalicular multi-specific organic anion transport system, non-metabolized acridine orange is the predominant species in bile and its secretion is also inhibited by cyclosporin A. Binding of acridine orange to liver P-glycoprotein was analyzed by photoaffinity labeling with azidopine, a substrate of P-glycoprotein dependent transport in multi-drug resistant tumor cells. Labeling of the immunoprecipitated P-glycoprotein was inhibited by acridine orange, verapamil, and by cyclosporin A. The results show that biliary secretion of acridine orange is highly analogous to P-glycoprotein mediated membrane drug transport in tumor cells that exhibit multi-drug resistance.

  9. Molecular docking studies with rabies virus glycoprotein to design viral therapeutics

    Directory of Open Access Journals (Sweden)

    Tomar N


    Full Text Available The genome of rabies virus encodes five proteins; the nucleoprotein, the phosphoprotein, the matrix protein, the glycoprotein, and the RNA-dependent RNA polymerase. Among these, the glycoprotein is the most important as it is the major contributor to pathogenicity and virus neutralizing antibody response. Keeping in mind that glycoprotein is the only protein exposed on the surface of virus and is thought to be responsible for the interaction with the cell membrane, it was attempted to target glycoprotein by a ligand polyethylene glycol 4000, which blocks its active site, as seen by molecular operating environment software, so that it may be possible to prevent the spread of virus into the host. The ligand polyethylene glycol 4000 was retrieved from Research Collaboratory for Structural Bioinformatics protein data bank by providing the glycoprotein sequence to the databank. In this study it was observed that the ligand was successfully docked on a major portion of antigenic site II of glycoprotein by mimicking the virus neutralizing antibodies. This knowledge may be important for the development of novel therapies for the treatment of rabies and other viral diseases in the future.

  10. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Luis Mario Rodríguez-Martínez

    Full Text Available Current Ebola virus (EBOV detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV proteins. In particular, several monoclonal antibodies (mAbs have been described that bind the capsid glycoprotein (GP of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV.We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude and they are easily and economically produced in bacterial cultures.Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.

  11. P-Glycoprotein and Drug Resistance in Systemic Autoimmune Diseases

    Directory of Open Access Journals (Sweden)

    Andrea Picchianti-Diamanti


    Full Text Available Autoimmune diseases such as systemic lupus erythematosus (SLE, rheumatoid arthritis (RA and psoriatic arthritis (PsA are chronic inflammatory disorders of unknown etiology characterized by a wide range of abnormalities of the immune system that may compromise the function of several organs, such as kidney, heart, joints, brain and skin. Corticosteroids (CCS, synthetic and biologic immunosuppressive agents have demonstrated the capacity to improve the course of autoimmune diseases. However, a significant number of patients do not respond or develop resistance to these therapies over time. P-glycoprotein (P-gp is a transmembrane protein that pumps several drugs out of the cell, including CCS and immunosuppressants; thus, its over-expression or hyper-function has been proposed as a possible mechanism of drug resistance in patients with autoimmune disorders. Recently, different authors have demonstrated that P-gp inhibitors, such as cyclosporine A (CsA and its analogue Tacrolimus, are able to reduce P-gp expression and or function in SLE, RA and PsA patients. These observations suggest that P-gp antagonists could be adopted to revert drug resistance and improve disease outcome. The complex inter-relationship among drug resistance, P-gp expression and autoimmunity still remains elusive.

  12. [Classification models of structure - P-glycoprotein activity of drugs]. (United States)

    Grigorev, V Yu; Solodova, S L; Polianczyk, D E; Raevsky, O A


    Thirty three classification models of substrate specificity of 177 drugs to P-glycoprotein have been created using of the linear discriminant analysis, random forest and support vector machine methods. QSAR modeling was carried out using 2 strategies. The first strategy consisted in search of all possible combinations from 1÷5 descriptors on the basis of 7 most significant molecular descriptors with clear physico-chemical interpretation. In the second case forward selection procedure up to 5 descriptors, starting from the best single descriptor was used. This strategy was applied to a set of 387 DRAGON descriptors. It was found that only one of 33 models has necessary statistical parameters. This model was designed by means of the linear discriminant analysis on the basis of a single descriptor of H-bond (ΣC(ad)). The model has good statistical characteristics as evidenced by results to both internal cross-validation, and external validation with application of 44 new chemicals. This confirms an important role of hydrogen bond in the processes connected with penetration of chemical compounds through a blood-brain barrier.

  13. Effect of Urea on Activity and Conformation of a Glycoprotein

    Institute of Scientific and Technical Information of China (English)

    WEI Xiang; WANG Xiaoyun; ZHOU Bo; ZHOU Haimeng


    The changes of the activity and conformation of Aspergillus niger phytase in urea were detected by farultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays. The results show that no enzyme activity can be detected after phytase is incubated for 10 h in 3.0 mol/L urea, even though at this urea concentration, less than 20% of the tertiary and secondary structures in the native enzyme changed. The inactivation reaction kinetics is found to be a monophasic first-order reaction, but the unfolding is a biphasic process consisting of two first-order reactions. The inactivation rates of the free enzyme and the substrate-enzyme complex are much faster than the conformational changes during urea denaturation. All of the results indicate that, as a glycoprotein, phytase's activity is strongly dependent on its conformational integrity. The phytase active sites seem to be located in a limited region in the molecule and display more conformational fragility and flexibility to denaturants than enzyme molecular structure as a whole.

  14. An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids

    Directory of Open Access Journals (Sweden)

    Harriet Allison


    Full Text Available African trypanosomes are evolutionarily highly divergent parasitic protozoa, and as a consequence the vast majority of trypanosome membrane proteins remain uncharacterised in terms of location, trafficking or function. Here we describe a novel family of type I membrane proteins which we designate ‘invariant glycoproteins’ (IGPs. IGPs are trypanosome-restricted, with extensive, lineage-specific paralogous expansions in related taxa. In T. brucei three IGP subfamilies, IGP34, IGP40 and IGP48 are recognised; all possess a putative C-type lectin ectodomain and are ER-localised, despite lacking a classical ER-retention motif. IGPs exhibit highest expression in stumpy stage cells, suggesting roles in developmental progression, but gene silencing in mammalian infective forms suggests that each IGP subfamily is also required for normal proliferation. Detailed analysis of the IGP48 subfamily indicates a role in maintaining ER morphology, while the ER lumenal domain is necessary and sufficient for formation of both oligomeric complexes and ER retention. IGP48 is detected by antibodies from T. b. rhodesiense infected humans. We propose that the IGPs represent a trypanosomatid-specific family of ER-localised glycoproteins, with potential contributions to life cycle progression and immunity, and utilise oligomerisation as an ER retention mechanism.

  15. HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies. (United States)

    Sliepen, Kwinten; Sanders, Rogier W


    The long pursuit for a vaccine against human immunodeficiency virus 1 (HIV-1) has recently been boosted by a number of exciting developments. An HIV-1 subunit vaccine ideally should elicit potent broadly neutralizing antibodies (bNAbs), but raising bNAbs by vaccination has proved extremely difficult because of the characteristics of the HIV-1 envelope glycoprotein complex (Env). However, the isolation of bNAbs from HIV-1-infected patients demonstrates that the human humoral immune system is capable of making such antibodies. Therefore, a focus of HIV-1 vaccinology is the elicitation of bNAbs by engineered immunogens and by using vaccination strategies aimed at mimicking the bNAb maturation pathways in HIV-infected patients. Important clues can also be taken from the successful subunit vaccines against hepatitis B virus and human papillomavirus. Here, we review the different types of HIV-1 immunogens and vaccination strategies that are being explored in the search for an HIV-1 vaccine that induces bNAbs.

  16. Analysis of Determinants in Filovirus Glycoproteins Required for Tetherin Antagonism

    Directory of Open Access Journals (Sweden)

    Kerstin Gnirß


    Full Text Available The host cell protein tetherin can restrict the release of enveloped viruses from infected cells. The HIV-1 protein Vpu counteracts tetherin by removing it from the site of viral budding, the plasma membrane, and this process depends on specific interactions between the transmembrane domains of Vpu and tetherin. In contrast, the glycoproteins (GPs of two filoviruses, Ebola and Marburg virus, antagonize tetherin without reducing surface expression, and the domains in GP required for tetherin counteraction are unknown. Here, we show that filovirus GPs depend on the presence of their authentic transmembrane domains for virus-cell fusion and tetherin antagonism. However, conserved residues within the transmembrane domain were dispensable for membrane fusion and tetherin counteraction. Moreover, the insertion of the transmembrane domain into a heterologous viral GP, Lassa virus GPC, was not sufficient to confer tetherin antagonism to the recipient. Finally, mutation of conserved residues within the fusion peptide of Ebola virus GP inhibited virus-cell fusion but did not ablate tetherin counteraction, indicating that the fusion peptide and the ability of GP to drive host cell entry are not required for tetherin counteraction. These results suggest that the transmembrane domains of filoviral GPs contribute to tetherin antagonism but are not the sole determinants.

  17. Histidine-rich glycoprotein protects from systemic Candida infection.

    Directory of Open Access Journals (Sweden)

    Victoria Rydengård


    Full Text Available Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG, an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg(-/- mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity.

  18. Bioisosteric phentolamine analogs as selective human alpha(2)- versus alpha(1)-adrenoceptor ligands. (United States)

    Bavadekar, Supriya A; Hong, Seoung-Soo; Lee, Sang-Ii; Miller, Duane D; Feller, Dennis R


    Phentolamine is known to act as a competitive, non-subtype-selective alpha-adrenoceptor antagonist. In an attempt to improve alpha(2)- versus alpha(1)-adrenoceptor selectivity and alpha(2)-adrenoceptor subtype-selectivity, two new chemical series of bioisosteric phentolamine analogs were prepared and evaluated. These compounds were evaluated for binding affinities on alpha(1)- (alpha(1A)-, alpha(1B)-, alpha(1D)-) and alpha(2)- (alpha(2A)-, alpha(2B)-, alpha(2C)-) adrenoceptor subtypes that had been stably expressed in human embryonic kidney and Chinese hamster ovary cell lines, respectively. Methylation of the phenolic hydroxy group and replacement of the 4-methyl group of phentolamine with varying lipophilic substituents yielded bioisosteric analogs selective for the alpha(2)- versus alpha(1)-adrenoceptors. Within the alpha(2)-adrenoceptors, these analogs bound with higher affinity at the alpha(2A)- and alpha(2C)-subtypes as compared to the alpha(2B)-subtype. In particular, the t-butyl analog was found to be the most selective, its binding at the alpha(2C)-adrenoceptor (Ki=3.6 nM) being 37- to 173-fold higher than that at the alpha(1)-adrenoceptors, and around 2- and 19-fold higher than at the alpha(2A)- and alpha(2B)-adrenoceptors, respectively. Data from luciferase reporter gene assays confirmed the functional antagonist activities of selected compounds from the bioisosteric series on human alpha(1A)- and alpha(2C)-adrenoceptors. Thus, the results with these bioisosteric analogs of phentolamine provide a lead to the rational design of potent and selective alpha(2)-adrenoceptor ligands that may be useful in improving the therapeutic profile of this drug class for human disorders.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    Highlights: • The method enables relative quantitation of hAGP glycans from pathological samples • Pancreatic cancer samples clearly showed an increase of hAGP fucosylated glycans. • Fucosylated glycans could be potential biomarkers for diagnosing pancreatic cancer. • The established method could be extremely useful to find novel glycoprotein biomarkers - Abstract: In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [{sup 12}C]- and [{sup 13}C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α{sub 1}-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in h

  20. NACA Physicist Studying Alpha Rays (United States)


    NACA Physicits studying Alpha Rays in a continuous cloud chamber. A cloud chamber is used by Lewis scientists to obtain information aimed at minimizing undesirable effects of radiation on nuclear-powered aircraft components. Here, alpha particles from a polonium source emit in a flower-like pattern at the cloud chamber's center. The particles are made visible by means of alcohol vapor diffusing from an area at room temperature to an area at minus -78 deg. Centigrade. Nuclear-powered aircraft were never developed and aircraft nuclear propulsion systems were canceled in the early 1960s.

  1. Simulating the Lyman Alpha Forest

    CERN Document Server

    Machacek, M E; Anninos, P; Meiksin, A; Norman, M L; Machacek, Marie E.; Bryan, Greg L.; Anninos, Peter; Meiksin, Avery; Norman, Michael L.


    In this paper we review the importance of the Lyman alpha forest as a probe of structure formation in the universe. We first discuss the statistics used to describe the Lyman alpha forest and the numerical techniques used to produce simulated spectra of the forest from a given cosmological model. We then discuss the physical picture of the absorbing structures that emerges from these numerical simulations. Finally, we comment on how two of the statistics, the slope of the column density distribution and the b parameter distribution, may be used to constrain competing cosmologies.

  2. Pathogenesis of mucosal injury in the blind loop syndrome. Brush border enzyme activity and glycoprotein degradation. (United States)

    Jonas, A; Flanagan, P R; Forstner, G G


    The effect of intestinal bacterial over-growth on brush border hydrolases and brush border glycoproteins was studied in nonoperated control rats, control rats with surgically introduced jejunal self-emptying blind loops, and rats with surgically introduced jejunal self-filling blind loops. Data were analyzed from blind loop segments, segments above and below the blind loops, and three corresponding segments in the nonoperated controls. Rats with self-filling blind loops had significantly greater fat excretion than controls and exhibited significantly lower conjugated:free bile salt ratios in all three segments. Maltase, sucrase, and lactase activities were significantly reduced in homogenates and isolated brush borders from the self-filling blind loop, but alkaline phosphatase was not affected. The relative degradation rate of homogenate and brush border glycoproteins was assessed by a double-isotope technique involving the injection of d-[6-(3)H]glucosamine 3 h and d-[U-(14)C]glucosamine 19 h before sacrifice, and recorded as a (3)H:(14)C ratio. The relative degradation rate in both homogenate and brush border fractions was significantly greater in most segments from rats with self-filling blind loops. In the upper and blind loop segments from rats with self-filling blind loops, the (3)H:(14)C ratios were higher in the brush border membrane than in the corresponding homogenates, indicating that the increased rates of degradation primarily involve membrane glycoproteins. Incorporation of d-[6-(3)H]glucosamine by brush border glycoproteins was not reduced in rats with self-filling blind loops, suggesting that glycoprotein synthesis was not affected. Polyacrylamide gel electrophoresis of brush border glycoproteins from the contaminated segments indicated that the large molecular weight glycoproteins, which include many of the surface hydrolases, were degraded most rapidly. Brush border maltase, isolated by immunoprecipitation, had (3)H:(14)C ratios characteristic of

  3. Stable isotope utilization for the study of the metabolism nutritional control for glycoproteins neutral oses; Utilisation des isotopes stables pour l`etude de la regulation nutritionnelle du metabolisme des oses neutres des glycoproteines

    Energy Technology Data Exchange (ETDEWEB)

    Rambal, C.; Pachiaudi, C.; Normand, S.; Riou, J.P.; Louisot, P.; Martin, A. [Centre de Recherche en Nutrition Humaine, 69 - Lyon (France)


    The aim of this work is to assess the role of minor oses, constituents of the glycoproteins and present in small quantities, in food, on glycosylation and its control. Stable isotopes are necessary for metabolic studies on man. These studies have never been used for glycoprotein oses, so, application of gaseous phase chromatography coupled to isotopic mass spectrometry is validated here on animal for ose study; exploratory tests on man seric glycoproteins have been also carried out. 6 figs., 7 refs.

  4. Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins. (United States)

    Mislovičová, D; Katrlík, J; Paulovičová, E; Gemeiner, P; Tkac, J


    A procedure for determination of apparent affinity constants K(D)(app) between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10(-7) to 9×10(-6)M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

  5. Inhibition of rhodamine 123 secretion by cyclosporin A as a model of P-glycoprotein mediated transport in liver. (United States)

    Stapf, V; Thalhammer, T; Huber-Huber, R; Felberbauer, F; Gajdzik, L; Graf, J


    The interaction between P-glycoprotein modulators and P-glycoprotein mediated transport was investigated using rhodamine 123 in the isolated perfused rat liver of a mutant (TR-) rat strain. TR- rats, deficient in the canalicular multispecific anion transport system, are unable to extrude organic anions (glucuronides) and therefore excrete solely unconjugated rhodamine 123 via P-glycoprotein. Cyclosporin A, a modulator of multidrug resistance in tumor cells, inhibited the biliary secretion of rhodamine 123 dose dependently in a non-competitive manner. Both cyclosporin A and rhodamine inhibited photoaffinity labeling of immunoprecipitated P-glycoprotein with azidopine, indicating binding to hepatic P-glycoprotein. Our results indicate that monitoring the biliary rhodamine 123 secretion in the isolated perfused liver of TR- rats offers a new system for testing modulators of P-glycoprotein like cyclosporin A.

  6. Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

    Directory of Open Access Journals (Sweden)

    C.G. Machado


    Full Text Available The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

  7. Biogenesis of lysosomal enzymes in the alpha-glucosidase II-deficient modA mutant of Dictyostelium discoideum: retention of alpha-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of alpha-mannosidase or beta-glucosidase. (United States)

    Ebert, D L; Bush, J M; Dimond, R L; Cardelli, J A


    The endoplasmic reticulum-localized enzyme alpha-glucosidase II is responsible for removing the two alpha-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum. Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, alpha-mannosidase and beta-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant alpha-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of alpha-mannosidase and beta-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N-acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.

  8. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujise


    Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

  9. Alpha-nucleus potential for alpha-decay and sub-barrier fusion

    CERN Document Server

    Denisov, V Y


    The set of parameters for alpha-nucleus potential is derived by using the data for both the alpha-decay half-lives and the fusion cross-sections around the barrier for reactions alpha+40Ca, alpha+59Co, alpha+208Pb. The alpha-decay half-lives are obtained in the framework of a cluster model using the WKB approximation. The evaluated alpha-decay half-lives and the fusion cross-sections agreed well with the data. Fusion reactions between alpha-particle and heavy nuclei can be used for both the formation of very heavy nuclei and spectroscopic studies of the formed compound nuclei.

  10. Alpha-nucleus potential for alpha-decay and sub-barrier fusion


    Denisov, V. Yu.; Ikezoe, H.


    The set of parameters for alpha-nucleus potential is derived by using the data for both the alpha-decay half-lives and the fusion cross-sections around the barrier for reactions alpha+40Ca, alpha+59Co, alpha+208Pb. The alpha-decay half-lives are obtained in the framework of a cluster model using the WKB approximation. The evaluated alpha-decay half-lives and the fusion cross-sections agreed well with the data. Fusion reactions between alpha-particle and heavy nuclei can be used for both the f...

  11. Inflaton Decay in an Alpha Vacuum

    CERN Document Server

    Naidu, S; Naidu, Siddartha; Holman, Richard


    We study the alpha vacua of de Sitter space by considering the decay rate of the inflaton field coupled to a scalar field placed in an alpha vacuum. We find an {\\em alpha dependent} Bose enhancement relative to the Bunch-Davies vacuum and, surprisingly, no non-renormalizable divergences. We also consider a modified alpha dependent time ordering prescription for the Feynman propagator and show that it leads to an alpha independent result. This result suggests that it may be possible to calculate in any alpha vacuum if we employ the appropriate causality preserving prescription.

  12. Discovery & development of small molecule allosteric modulators of glycoprotein hormone receptors

    Directory of Open Access Journals (Sweden)

    Selvaraj G Nataraja


    Full Text Available Glycoprotein hormones, follicle-stimulating hormone (FSH, luteinizing hormone (LH, and thyroid stimulating hormone (TSH are heterodimeric proteins with a common subunit and hormone-specific subunit. These hormones are dominant regulators of reproduction and metabolic processes. Receptors for the glycoprotein hormones belong to the family of G-protein coupled receptors (GPCR. FSH receptor (FSHR and LH receptor (LHR are primarily expressed in somatic cells in ovary and testis to promote egg and sperm production in women & men respectively. TSH receptor (TSHR is expressed in thyroid cells and regulates the secretion of T3 & T4. Glycoprotein hormones bind to the large extracellular domain of the receptor and cause a conformational change in the receptor that leads to activation of more than one intracellular signaling pathway. Several small molecules have been described to activate/inhibit glycoprotein hormone receptors through allosteric sites of the receptor. Small molecule allosteric modulators have the potential to be administered orally to patients thus improving the convenience of treatment. It has been a challenge to develop a small molecule allosteric agonist for glycoprotein hormones that can mimic the agonistic effects of the large natural ligand to activate similar signaling pathways. However, in the past few years, there have been several promising reports describing distinct chemical series with improved potency in preclinical models. In parallel, proposal of new structural model for FSH receptor and in silico docking studies of small molecule ligands to glycoprotein hormone receptors provide a giant leap on the understanding of the mechanism of action of the natural ligands and new chemical entities on the receptors. This review will focus on the current status of small molecule allosteric modulators of glycoprotein hormone receptors, their effects on common signaling pathways in cells, their utility for clinical

  13. What Powers Lyman alpha Blobs?

    CERN Document Server

    Ao, Y; Beelen, A; Henkel, C; Cen, R; De Breuck, C; Francis, P; Kovacs, A; Lagache, G; Lehnert, M; Mao, M; Menten, K M; Norris, R; Omont, A; Tatemastu, K; Weiss, A; Zheng, Z


    Lyman alpha blobs (LABs) are spatially extended lyman alpha nebulae seen at high redshift. The origin of Lyman alpha emission in the LABs is still unclear and under debate. To study their heating mechanism(s), we present Australia Telescope Compact Array (ATCA) observations of the 20 cm radio emission and Herschel PACS and SPIRE measurements of the far-infrared (FIR) emission towards the four LABs in the protocluster J2143-4423 at z=2.38. Among the four LABs, B6 and B7 are detected in the radio with fluxes of 67+/-17 microJy and 77+/-16 microJy, respectively, and B5 is marginally detected at 3 sigma (51+/-16 microJy). For all detected sources, their radio positions are consistent with the central positions of the LABs. B6 and B7 are obviously also detected in the FIR. By fitting the data with different templates, we obtained redshifts of 2.20$^{+0.30}_{-0.35}$ for B6 and 2.20$^{+0.45}_{-0.30}$ for B7 which are consistent with the redshift of the lyman alpha emission within uncertainties, indicating that both ...

  14. Alpha proton x ray spectrometer (United States)

    Rieder, Rudi; Waeke, H.; Economou, T.


    Mars Pathfinder will carry an alpha-proton x ray spectrometer (APX) for the determination of the elemental chemical composition of Martian rocks and soils. The instrument will measure the concentration of all major and some minor elements, including C, N, and O at levels above typically 1 percent.

  15. Sparse Coding for Alpha Matting. (United States)

    Johnson, Jubin; Varnousfaderani, Ehsan; Cholakkal, Hisham; Rajan, Deepu


    Existing color sampling based alpha matting methods use the compositing equation to estimate alpha at a pixel from pairs of foreground (F) and background (B) samples. The quality of the matte depends on the selected (F,B) pairs. In this paper, the matting problem is reinterpreted as a sparse coding of pixel features, wherein the sum of the codes gives the estimate of the alpha matte from a set of unpaired F and B samples. A non-parametric probabilistic segmentation provides a certainty measure on the pixel belonging to foreground or background, based on which a dictionary is formed for use in sparse coding. By removing the restriction to conform to (F,B) pairs, this method allows for better alpha estimation from multiple F and B samples. The same framework is extended to videos, where the requirement of temporal coherence is handled effectively. Here, the dictionary is formed by samples from multiple frames. A multi-frame graph model, as opposed to a single image as for image matting, is proposed that can be solved efficiently in closed form. Quantitative and qualitative evaluations on a benchmark dataset are provided to show that the proposed method outperforms current state-of-the-art in image and video matting.

  16. Thyroid Hormone and P-Glycoprotein in Tumor Cells

    Directory of Open Access Journals (Sweden)

    Paul J. Davis


    Full Text Available P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1 is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1 and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrin αvβ3 also binds tetraiodothyroacetic acid (tetrac, a derivative of L-thyroxine (T4 that blocks nongenomic actions of T4 and of 3,5,3′-triiodo-L-thyronine (T3 at αvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+ exchanger (NHE1 and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF and osteopontin (OPN, apparently via αvβ3. Intracellular acidification and decreased H+ efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein.

  17. Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Suchita


    Full Text Available Abstract The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1. However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER, Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP, it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.

  18. Structural mechanism of trimeric HIV-1 envelope glycoprotein activation.

    Directory of Open Access Journals (Sweden)

    Erin E H Tran

    Full Text Available HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.

  19. Alternative promoter usage of the membrane glycoprotein CD36

    Directory of Open Access Journals (Sweden)

    Whatling Carl


    Full Text Available Abstract Background CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation. Results We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters. Conclusion Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.

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

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    Chang, Shan-Yan [Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Liu, Fu-Feng, E-mail:, E-mail:; Dong, Xiao-Yan; Sun, Yan, E-mail:, E-mail: [Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072 (China)


    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.

  1. Molecular insight into conformational transmission of human P-glycoprotein (United States)

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


    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.

  2. Multiple Drug Transport Pathways through Human P-Glycoprotein. (United States)

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


    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 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, 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 that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

  3. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

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    Oliveira M.C.


    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  4. Membrane topology analysis of HIV-1 envelope glycoprotein gp41

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    Xiao Dan


    Full Text Available Abstract Background The gp41 subunit of the HIV-1 envelope glycoprotein (Env has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD. An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP that is only active in the cytoplasm. The tag protein (HaloTag and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. Results In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells, the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. Conclusions It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

  5. Interaction of the P-Glycoprotein Multidrug Transporter with Sterols. (United States)

    Clay, Adam T; Lu, Peihua; Sharom, Frances J


    The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3-9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains.

  6. Effect of expression of P-glycoprotein on technetium-99m methoxyisobutylisonitrile single photon emission computed tomography of brain tumors

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    Shibata, Yasushi; Matsumura, Akira; Nose, Tadao [Tsukuba Univ., Ibaraki (Japan). Inst. of Clinical Medicine


    The expression of P-glycoprotein was investigated imunohistochemically in 26 brain tumor tissues and compared with the findings of technetium-99m methoxyisobutylisonitrile single photon emission computed tomography ({sup 99m}Tc-MIBI SPECT) to clarify the effect of P-glycoprotein on the diagnostic accuracy. P-glycoprotein labeling index of both tumor cells and vascular endothelial cells showed no clear relationship with the findings of {sup 99m}Tc-MIBI SPECT imaging. Expression of P-glycoprotein has no effect on the diagnostic accuracy of {sup 99m}Tc-MIBI SPECT. (author)

  7. Envelope glycoproteins of HIV-1, HIV-2, and SIV purified with Galanthus nivalis agglutinin induce strong immune responses. (United States)

    Gilljam, G


    Lectin affinity chromatography was used to purify in a single step the envelope glycoproteins of HIV-1, HIV-2, and SIV. Envelope glycoproteins carry the major determinants essential for protection by the humoral immune response. The purification of these proteins has previously been a laborious procedure. The glycoproteins were purified by a one-step procedure to a high level of purity by using Galanthus nivalis agglutinin (GNA). The purified glycoprotein had CD4-binding and antigenic reactivities. Strong immune responses to envelope proteins and peptides were seen in mice and primates after immunization with these preparations.

  8. Isolation from Gluconacetobacter diazotrophicus cell walls of specific receptors for sugarcane glycoproteins, which act as recognition factors. (United States)

    Blanco, Y; Arroyo, M; Legaz, M E; Vicente, C


    Glycoproteins from sugarcane stalks have been isolated from plants field-grown by size-exclusion chromatography. Some of these glycoproteins, previously labelled with fluorescein isothiocyanate, are able to bind to the cell wall of the sugarcane endophyte, N2-fixing Gluconacetobacter diazotrophicus, and largely removed after washing the bacterial cells with sucrose. This implies that sugarcane glycoproteins use beta-(1-->2)-fructofuranosyl fructose domains in their glycosidic moiety to bind to specific receptors in the bacterial cell walls. These receptors have been isolated by affinity chromatography on a sugarcane glycoprotein-agarose matrix, desorbed with sucrose and characterized by sodium dodecyl sulfate polyacrylamide gel electrophresisand capillary electrophoresis (CE).

  9. Inhibition of platelet (/sup 3/H)- imipramine binding by human plasma protein fractions

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    Strijewski, A.; Chudzik, J.; Tang, S.W.


    Inhibition of high-affinity (/sup 3/H)-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha/sub 1/ acid glycoprotein, high density and low density lipoprotein, IgG and ..cap alpha../sub 1/-antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of (/sup 3/H)-imipramine binding site.

  10. Purification and biochemical characterization of an alpha-glucosidase from Xanthophyllomyces dendrorhous. (United States)

    Marín, Dolores; Linde, Dolores; Fernández Lobato, María


    Xanthophyllomyces dendrorhous grown in different media shows amylolytic activity, consisting in an extracellular exo-acting enzyme able to hydrolysed alpha,1-4 glycosidic bonds from soluble starch, which also cleaves maltose and malto-oligosaccharides. The enzyme was purified, using basically a couple of chromatography process on DEAE-Sephacel. It is a glycoprotein with a molecular weight estimated to be 60.2 kDa based on its mobility in SDS-PAGE and 115 kDa based on gel filtration. N-linked carbohydrate accounts for 12% of the total mass. It exhibited optimum activity at pH 5.5 and 45 degrees C. Thermostability analysis indicated that it was stable to thermal treatment up to 50 degrees C; 50% of the activity was maintained after 3 h. The rate parameters measured for the hydrolysis of starch and various chain length malto-oligosaccharides shows high catalytic efficiency, calculated by the relationship V(cat)/K(m), for malto-oligosaccharides, such as maltotriose (873 mM(-1) min(-1)), or maltoheptose (698 mM(-1) min(-1)). The new enzyme hydrolysed soluble starch with nearly 3.5- and 1.4-fold lower efficiency than that for maltotriose and maltose, respectively. No activity was found on heterogeneous substrates, such as sucrose and aryl alpha-glucoside, or on isomalto-oligosaccharides. In accordance to substrate specificity profile, the new enzyme was classified as an alpha-glucosidase.

  11. Expression of herpes simplex virus. beta. and. gamma. genes integrated in mammalian cells and their induction by an. cap alpha. gene product

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    Sandri-Goldin, R.M.; Goldin, A.L.; Holland, L.E.


    The proteins of herpes simplex virus type 1 (HSV-1) form three kinetic groups termed ..cap alpha..,..beta..,and ..gamma.., whose synthesis is regulated in a cascade fashion, ..cap alpha.. products are synthesized first during infection, and they are required for synthesis of ..beta.. and ..gamma.. proteins. To examine the expression of several HSV-1 ..beta.. and ..gamma.. genes in the absence of ..cap alpha.. functions, we transferred into mammalian cells a plasmid containing a region of the HSV-1 genome that codes for only ..beta.. and ..gamma.. genes (0.315 to 0.421 map units). The authors found stable integration of at least one copy of the intact plasmid in each cell line. Four HSV-1 transcripts of the ..beta.. and ..gamma.. classes were transcribed constitutively in the cells, including the genes for glycoprotein B and DNA-binding protein. No constitutive synthesis of these two proteins could be demonstrated, however. The integrated HSV-1 genes responded to viral regulatory signals in that they could be induced by infection with HSV-1 mutants resulting in a high level of synthesis of both glycoprotein B and DNA-binding protein. The HSV-1 ..cap alpha.. gene product ICP4 was necessary for this induction, and it was found to be most efficient at a low multiplicity of infection. Functional expression of four genes was demonstrated in that the cell lines complemented infecting HSV-1 temperature-sensitive mutants. The same genes were not available for homologous recombination with infecting virus, however, since no recombinant wild-type virus could be detected. These data demonstrate that HSV-1 ..beta.. and ..gamma.. genes can be transcribed in the absence of ..cap alpha.. functions in mammalian cells, but that they still respond to HSV-1 regulatory signals such as the ..cap alpha.. gene product ICP4.

  12. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis. (United States)

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M


    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

  13. Glycoprotein processing in mutants of HSV-1 that induce cell fusion

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    Person, S.; Kousoulas, K.G.; Knowles, R.W.; Read, G.S.; Holland, T.C.; Keller, P.M.; Warner, S.C.


    The synthesis of viral-specified glycoproteins, and their appearance on cell surfaces, were compared for cells infected either with syncytial mutants of HSV-1 or with the parental strains from which the mutants were derived. The mutants MP and tsB5, representatives of two different viral genes that affect fusion, were employed in these studies. Cells infected with either mutant gave rise to reduce surface labeling of major viral-specified glycoproteins throughout infection relative to the parental strains. Putative glycoprotein precursors were synthesized in similar amounts throughout infection in mutant and wild-type infected cells. However, during a chase period after a pulse of labeling, fully glycosylated species of glycoproteins accumulated more slowly in mutant than in parent infected cells. The effect was clearly visible early in MP-infected cells and by 9 hr after infection for tsB5-infected cells, and increased thereafter. Apparently the decrease in appearance of glycoproteins on the cell surface of mutant-infected cells is due to a decrease in their rate of processing. 26 references, 7 figures.

  14. Recognition of glycoprotein peroxidase via Con A-carrying self-assembly layer on gold. (United States)

    Liu, Songqin; Wang, Kewei; Du, Dan; Sun, Yueming; He, Lin


    We have successfully fabricated a self-assembled layer of concanavalin A (Con A) on a gold surface for recognition of glycoproteins. The type IV Con A is covalently bound to 11-mercaptoundecanoic acid (MUA) on gold with a 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) linkage. The binding interaction between glycoproteins and self-assembled Con A is studied using horseradish peroxidase (HRP) as a model glycoprotein. Voltammetric, electrochemical impedance studies, and photometric activity measurements show the presence of both specific and nonspecific bindings of HRP to the Con A interface. The specific binding is attributed to the Con A-sugar interaction where Con A selectively recognizes the glycosylation sites of HRP. The catalytic current of the HRP-loaded electrode, because of catalytic oxidation of thionine in the presence of hydrogen peroxide (H2O2), is found to be proportional to the HRP concentrations in the incubation solution. A linear correlation coefficient of 0.993 was obtained over a wide HRP concentration range of 12.5 microg/mL to 1 mg/mL. The approach described in this study provides a simple yet selective means to immobilize glycoproteins on a solid support. The specific binding achieved is desirable in biosensor fabrication, glycoprotein separation, recognition, and purification as well as in drug-releasing systems.

  15. Haemonchus contortus P-glycoprotein-2: in situ localisation and characterisation of macrocyclic lactone transport. (United States)

    Godoy, Pablo; Lian, Jing; Beech, Robin N; Prichard, Roger K


    Haemonchus contortus is a veterinary nematode that infects small ruminants, causing serious decreases in animal production worldwide. Effective control through anthelmintic treatment has been compromised by the development of resistance to these drugs, including the macrocyclic lactones. The mechanisms of resistance in H. contortus have yet to be established but may involve efflux of the macrocyclic lactones by nematode ATP-binding-cassette transporters such as P-glycoproteins. Here we report the expression and functional activity of H. contortus P-glycoprotein 2 expressed in mammalian cells and characterise its interaction with the macrocyclic lactones, ivermectin, abamectin and moxidectin. The ability of H. contortus P-glycoprotein 2 to transport different fluorophore substrates was markedly inhibited by ivermectin and abamectin in a dose-dependent and saturable way. The profile of transport inhibition by moxidectin was markedly different. H. contortus P-glycoprotein 2 was expressed in the pharynx, the first portion of the worm's intestine and perhaps in adjacent nervous tissue, suggesting a role for this gene in regulating the uptake of avermectins and in protecting nematode tissues from the effects of macrocyclic lactone anthelmintic drugs. H. contortus P-glycoprotein 2 may thus contribute to resistance to these drugs in H. contortus.

  16. Archaeal S-layer glycoproteins: Post-translational modification in the face of extremes

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    Jerry eEichler


    Full Text Available Corresponding to the sole or basic component of the surface (S-layer surrounding the archaeal cell in most known cases, S-layer glycoproteins are in direct contact with the harsh environments that characterize niches where Archaea can thrive. Accordingly, early work examining archaeal S-layer glycoproteins focused on identifying those properties that allow members of this group of proteins to maintain their structural integrity in the face of extremes of temperature, pH and salinity, as well as other physical challenges. However, with expansion of the list of archaeal strains serving as model systems, as well as growth in the number of molecular tools available for the manipulation of these strains, studies on archaeal S-layer glycoproteins are currently more likely to consider the various post-translational modifications these polypeptides undergo. For instance, archaeal S-layer glycoproteins can undergo proteolytic cleavage, both N- and O-glycosylation, lipid-modification and oligomerization. In this mini-review, recent findings related to the post-translational modification of archaeal S-layer glycoproteins are considered.

  17. Ammonia secretion from fish gill depends on a set of Rh glycoproteins. (United States)

    Nakada, Tsutomu; Westhoff, Connie M; Kato, Akira; Hirose, Shigehisa


    Ammonia excretion from the gill in teleost fish is essential for nitrogen elimination. Although numerous physiological studies have measured ammonia excretion, the mechanism of ammonia movement through the membranes of gill epithelial cells is still unknown. Mammalian Rh glycoproteins are members of a family of proteins that mediate ammonia transport in bacteria, yeast, and plants. We identified the Rh glycoprotein homologs, fRhag, fRhbg, fRhcg1, and fRhcg2, of the pufferfish, Takifugu rubripes. Northern blot, in situ hybridization, and immunohistochemistry revealed that the pufferfish erythroid Rh glycoprotein homologue fRhag was present in red blood cells and the hematological organs (spleen and kidney) in fish. All four pufferfish Rh glycoproteins are specifically localized in the gill and line the pillar cells, pavement cells, and the mitochondrion-rich cells. Heterologous expression in Xenopus oocytes showed that they mediate methylammonium (an analog of ammonium) transport. These results suggest that pufferfish Rh glycoproteins are involved in ammonia excretion from the gill. These findings challenge the classic view that ammonia excretion in the fish gill occurs by passive diffusion.

  18. Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

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    Mahsa Mohseni


    Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1 to (15±2.6 nM and for vinblastine from (30±5.9 to (5±5.6 nM (P≤0.05.               P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001. Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05. Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents.  Conclusion:Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression.

  19. Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G. (United States)

    Baquero, Eduard; Buonocore, Linda; Rose, John K; Bressanelli, Stéphane; Gaudin, Yves; Albertini, Aurélie A


    Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV G(th)) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV G(th) obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal.

  20. Biochemical reconstitution of hemorrhagic-fever arenavirus envelope glycoprotein-mediated membrane fusion.

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    Celestine J Thomas

    Full Text Available 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.

  1. Immunological responses to envelope glycoprotein 120 from subtypes of human immunodeficiency virus type 1. (United States)

    Gilljam, G; Svensson, A; Ekström, A; Wahren, B


    The outer envelope glycoprotein (gp120) from subtypes A-E of HIV-1 was purified using a specific high mannose-binding lectin, Galanthus nivalis agglutinin. All isolates were grown in peripheral blood lymphocyte cells in order to avoid selection in cell lines. A comparison of the reactivities of the envelope proteins was made using sera from patients infected with the different subtypes. In this study, the B and C subtype envelope glycoproteins showed the strongest immunological reactivity, when reacted with sera from patients infected with the same subtype of virus. On the other hand, sera of patients infected with subtype A or C virus had the strongest and broadest reactivities, to envelope glycoproteins of many subtypes. The purified gp120 proteins from all five subtypes stimulated mononuclear cells from HIV-1 (subtype B)-infected patients, indicating conserved T cell-activating epitopes. The immunological reactivities indicate that strong antigenicity does not always predict the broadest immunogenicity of an envelope glycoprotein. Glycoprotein 120 from foreign subtypes may serve to induce strong cross-reactive immune responses.

  2. Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells (United States)

    Goh, John SY; Liu, Yingwei; Chan, Kah Fai; Wan, Corrine; Teo, Gavin; Zhang, Peiqing; Zhang, Yuanxing; Song, Zhiwei


    Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors. PMID:24911584

  3. Defence sugarcane glycoproteins disorganize microtubules and prevent nuclear polarization and germination of Sporisorium scitamineum teliospores. (United States)

    Sánchez-Elordi, Elena; Baluška, František; Echevarría, Clara; Vicente, Carlos; Legaz, M Estrella


    Microtubules (MTs) are involved in the germination of Sporisorium scitamineum teliospores. Resistant varieties of sugar cane plants produce defence glycoproteins that prevent the infection of the plants by the filamentous fungi Sporisorium scitamineum. Here, we show that a fraction of these glycoproteins prevents the correct arrangement of MTs and causes nuclear fragmentation defects. As a result, nuclei cannot correctly migrate through the growing hyphae, causing germinative failure. Arginase activity contained in defence glycoproteins is already described for preventing fungal germination. Now, its enzymatically active form is presented as a link between the defensive capacity of glycoproteins and the MT disorganization in fungal cells. Active arginase is produced in healthy and resistant plants; conversely, it is not detected in the juice from susceptible varieties, which explains why MT depolarization, nuclear disorganization as well as germination of teliospores are not significantly affected by glycoproteins from non-resistant plants. Our results also suggest that susceptible plants try to increase their levels of arginase after detecting the presence of the pathogen. However, this signal comes "too late" and such defensive mechanism fails.

  4. Evolution and seismology of alpha Centauri

    CERN Document Server

    Montalban, J; Montalban, Josefina; Miglio, Andrea


    Solar-like oscillations detected in both components of the binary system alpha Centauri provide strong constraints on the fundamental parameters of the stellar system. We model alpha Centauri by means of a Levenberg-Marquardt minimization algorithm including seismic and classical constraints. Computations, that were perfomed decreasing significanly the weight of alpha Cen B seismic data in the calibration procedure, predict small separations in good agreement with new observations of solar-like oscillations in alpha Cen B by Bedding (these proceedings).

  5. Astrophysical reaction rate for $\\alpha(\\alpha n,\\gamma)$

    CERN Document Server

    Sumiyoshi, K; Goko, S; Kajino, T


    We study the astrophysical reaction rate for the formation of $^{9}$Be through the three body reaction $\\alpha(\\alpha n,\\gamma)$. This reaction is one of the key reactions which could bridge the mass gap at A = 8 nuclear systems to produce intermediate-to-heavy mass elements in alpha- and neutron-rich environments such as r-process nucleosynthesis in supernova explosions, s-process nucleosynthesis in asymptotic giant branch (AGB) stars, and primordial nucleosynthesis in baryon inhomogeneous cosmological models. To calculate the thermonuclear reaction rate in a wide range of temperatures, we numerically integrate the thermal average of cross sections assuming a two-steps formation through a metastable $^{8}$Be. Off-resonant and on-resonant contributions from the ground state in $^{8}$Be are taken into account. As input cross section, we adopt the latest experimental data by photodisintegration of $^{9}$Be with laser-electron photon beams, which covers all relevant resonances in $^{9}$Be. We provide the reactio...


    NARCIS (Netherlands)



    We have identified a new protein fold-the alpha/beta-hydrolase fold-that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta-sheet, not barrel, of eight beta-sheets connected by alpha-helices. These

  7. Effectiveness of Alpha Biofeedback Therapy: Negative Results. (United States)

    Watson, Charles G.; Herder, Joseph


    Assessed the utility of alpha biofeedback training in the treatment of patients (N=66). Biofeedback and placebo biofeedback groups were given alpha or mock-alpha training sessions. Improvement on 54 variables was compared to that of no-treatment controls. Only a chance number of significant changes appeared among the groups. (Author)

  8. Enzyme replacement therapy for alpha-mannosidosis

    DEFF Research Database (Denmark)

    Borgwardt, Line Gutte; Dali, Christine I.; Fogh, J


    Alpha-mannosidosis (OMIM 248500) is a rare lysosomal storage disease (LSD) caused by alpha-mannosidase deficiency. Manifestations include intellectual disabilities, facial characteristics and hearing impairment. A recombinant human alpha-mannosidase (rhLAMAN) has been developed for weekly intrave...... intravenous enzyme replacement therapy (ERT). We present the preliminary data after 12 months of treatment....

  9. Refinement of the $n-\\alpha$ and $p-\\alpha$ fish-bone potential

    CERN Document Server

    Smith, E; Papp, Z


    The fishbone potential of composite particles simulates the Pauli effect by nonlocal terms. We determine the $n-\\alpha$ and $p-\\alpha$ fish-bone potential by simultaneously fitting to the experimental phase shifts. We found that with a double Gaussian parametrization of the local potential can describe the $n-\\alpha$ and $p-\\alpha$ phase shifts for all partial waves.

  10. Nature of the pygmy dipole resonance in Ce-140 studied in (alpha, alpha 'gamma) experiments

    NARCIS (Netherlands)

    Savran, D.; Babilon, M.; van den Berg, A. M.; Harakeh, M. N.; Hasper, J.; Matic, A.; Wortche, H. J.; Zilges, A.


    A concentration of electric-dipole excitations below the particle threshold, which is frequently denoted as the pygmy dipole resonance, has been studied in the semimagic nucleus Ce-140 in (alpha, alpha(')gamma) experiments at E-alpha=136 MeV. The technique of alpha-gamma coincidence experiments allo

  11. Alpha-1 antitrypsin is markedly decreased following pulmonary F. tularensis challenge

    Directory of Open Access Journals (Sweden)

    James Patrick Chambers


    Full Text Available Alpha-1 antitrypsin, a small glycoprotein clade A serpine serine protease inhibitor of neutrophil elastase has been shown to increase in humans following bacterial and viral infection. However, we report here significant reduction of this major inhibitor of elastase in plasma of F. tularensis LVS and SCHU S4 (Type A strain following pulmonary challenge. Consistent with an imbalance of protease-antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresistivity. These data are suggestive of targeted tissue destruction via unchecked neutrophhil elastase activity in infected animals.

  12. Separation of the subtypes of type V collagen molecules, [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), by chain composition-dependent affinity for heparin: single alpha 1(V) chain shows intermediate heparin affinity between those of the type V collagen subtypes composed of [alpha 1(V)]2 alpha 2(V) and of alpha 1(V) alpha 2(V) alpha 3(V). (United States)

    Mizuno, K; Hayashi, T


    The heparin affinities of heat-treated type V collagen alpha-chains and the triple-helical molecules were evaluated in terms of the NaCl concentration required for prevention of binding to a heparin-Sepharose column. After heat treatment, alpha 1(V) chain required approximately two-fold higher NaCl concentration to pass through the column than the other two chains, alpha 2(V) and alpha 3(V). Thus, the heparin affinity of alpha 1(V) may be approximately two-fold higher than those of the other alpha (V)-chains. The type V collagen molecules in triple-helical conformation were separated into two fractions at 170 mM NaCl in 20 mM phosphate buffer, pH 7.2, containing 2 M urea; bound and non-bound. The ratio of the three alpha-chains, alpha 1(V): alpha 2(V): alpha 3(V) was 2 : 1 : 0 and 1 : 1 : 1 in the bound and flow-through fractions, respectively, on analysis by SDS-PAGE. The differential affinity of the two fractions could be accounted for by the number of alpha 1(V) chains in the triple-helical molecule, if these fractions contained triple-helical subtypes with the chain compositions of [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), respectively. From the comparison of the NaCl concentration required for prevention of the binding, [alpha 1(V)]2, alpha 2(V) had about two-fold higher affinity than alpha 1(V) alpha 2(V) alpha 3(V), and the separated alpha 1(V) chain showed an intermediate affinity. A possible explanation for difference in heparin affinity among the subtypes of molecules and the separated alpha-chains is that the heparin affinity of type V collagen molecule is governed by the number of alpha 1(V) chains contained in the molecule and that steric restraint in a triple-helical conformation weakens the binding of alpha 1(V) chain to heparin.

  13. Crystalline anhydrous {alpha},{alpha}-trehalose (polymorph {beta}) and crystalline dihydrate {alpha},{alpha}-trehalose: A calorimetric study

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, Susana S. [Centro de Quimica Estrutural, Complexo Interdisciplinar, Instituto Superior Tecnico, 1049-001 Lisbon (Portugal)]. E-mail:; Diogo, Herminio P. [Centro de Quimica Estrutural, Complexo Interdisciplinar, Instituto Superior Tecnico, 1049-001 Lisbon (Portugal)]. E-mail:; Moura-Ramos, Joaquim J. [Centro de Quimica-Fisica Molecular, Complexo Interdisciplinar, Instituto Superior Tecnico, 1049-001 Lisbon (Portugal)]. E-mail:


    The mean values of the standard massic energy of combustion of crystalline anhydrous {alpha},{alpha}-trehalose (C{sub 12}H{sub 22}O{sub 11}, polymorph {beta}) and crystalline dihydrate {alpha},{alpha}-trehalose (C{sub 12}H{sub 26}O{sub 13}) measured by static-bomb combustion calorimetry in oxygen, at the temperature T=298.15K, are {delta}{sub c}u{sup o}=-(16434.05+/-4.50)J.g{sup -1} and {delta}{sub c}u{sup o}=-(14816.05+/-3.52)J.g{sup -1}, respectively. The standard (p{sup o}=0.1MPa) molar enthalpy of formation of these compounds were derived from the corresponding standard molar enthalpies of combustion, respectively, {delta}{sub f}H{sub m}{sup o} (C{sub 12}H{sub 22}O{sub 11},cr)=-(2240.9+/-3.9)kJ.mol{sup -1}, and {delta}{sub f}H{sub m}{sup o} (C{sub 12}H{sub 26}O{sub 13},cr)=-(2832.6+/-3.6)kJ.mol{sup -1}. The values of the standard enthalpies of formation obtained in this work, together with data on enthalpies of solution at infinite dilution ({delta}{sub sol}H{sup {approx}}) for crystalline dihydrate and amorphous anhydrous trehalose, allow a better insight on the thermodynamic description of the trehalose system which can provide, together with the future research on the subject, a contribution for understanding the metabolism in several organisms, as well as the phase transition between the different polymorphs.

  14. Efficient transduction of neurons using Ross River glycoprotein-pseudotyped lentiviral vectors

    DEFF Research Database (Denmark)

    Jakobsson, J; Nielsen, T Tolstrup; Staflin, K;


    Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV-G-pseudotyped le......Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV...... and human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells....

  15. Complement inhibition enables tumor delivery of LCMV glycoprotein pseudotyped viruses in the presence of antiviral antibodies (United States)

    Evgin, Laura; Ilkow, Carolina S; Bourgeois-Daigneault, Marie-Claude; de Souza, Christiano Tanese; Stubbert, Lawton; Huh, Michael S; Jennings, Victoria A; Marguerie, Monique; Acuna, Sergio A; Keller, Brian A; Lefebvre, Charles; Falls, Theresa; Le Boeuf, Fabrice; Auer, Rebecca A; Lambris, John D; McCart, J Andrea; Stojdl, David F; Bell, John C


    The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody. PMID:27909702

  16. Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells. (United States)

    Sheta, Razan; Roux-Dalvai, Florence; Woo, Christina M; Fournier, Frédéric; Bourassa, Sylvie; Bertozzi, Carolyn R; Droit, Arnaud; Bachvarov, Dimcho


    This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets.

  17. Alpha voltaic batteries and methods thereof (United States)

    Raffaelle, Ryne P. (Inventor); Jenkins, Phillip (Inventor); Wilt, David (Inventor); Scheiman, David (Inventor); Chubb, Donald (Inventor); Castro, Stephanie (Inventor)


    An alpha voltaic battery includes at least one layer of a semiconductor material comprising at least one p/n junction, at least one absorption and conversion layer on the at least one layer of semiconductor layer, and at least one alpha particle emitter. The absorption and conversion layer prevents at least a portion of alpha particles from the alpha particle emitter from damaging the p/n junction in the layer of semiconductor material. The absorption and conversion layer also converts at least a portion of energy from the alpha particles into electron-hole pairs for collection by the one p/n junction in the layer of semiconductor material.

  18. Secretion of hepatitis C virus envelope glycoproteins depends on assembly of apolipoprotein B positive lipoproteins.

    Directory of Open Access Journals (Sweden)

    Vinca Icard

    Full Text Available The density of circulating hepatitis C virus (HCV particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB positive and triglyceride rich lipoproteins (TRL likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1-E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.

  19. Regulation of HSV glycoprotein induced cascade of events governing cell-cell fusion. (United States)

    Atanasiu, Doina; Saw, Wan Ting; Eisenberg, Roselyn J; Cohen, Gary H


    Receptor dependent HSV-induced 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 transformation of the pre-fusion 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 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 gH2/gL2, thereby enhancing their activity. We also found that deregulated forms of 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.

  20. Effect of mild-thiol reducing agents and alpha2,3-sialyltransferase expression on secretion and sialylation of recombinant EPO in CHO cells. (United States)

    Chang, Kern Hee; Jeong, Yeon Tae; Kwak, Chan Yeong; Choi, One; Kim, Jung Hoe


    We have previously reported that N-acetylcysteine (NAC) not only delayed apoptosis but also enhanced the production of recombinant erythropoietin (EPO) in Chinese hamster ovary (CHO) cell culture. To investigate the production enhancement mechanism, the effects of similar thiolreducing agents were studied. Intriguingly, all mild reducing agents examined including mercaptoethanesulfonic acid (MESNA), thiolactic acid (TLA), and thioglycolate (TG) were shown to block apoptosis and increase EPO production. A pulse-chase study of EPO secretion revealed that all four thiol-reducing agents increased the EPO secretion rate; among them TLA showed the highest rate. In terms of product quality, the sialic acid content of the glycoprotein is one of the most important factors. It was reported that a number of glycoproteins produced by CHO cells often have incomplete sialylation, particularly under high-producing conditions. Human alpha2,3-sialyltransferase (alpha2,3-ST) was introduced into EPO-producing CHO cells in order to compensate for the reduced sialylation during supplementation with NAC. When alpha2,3-ST was expressed in the presence of NAC, reduced sialylation was restored and an even more sialylated EPO was produced. Thus, our study is significant in that it offers increased EPO production while still allowing the prevention of decreased sialylation of EPO.

  1. Collagen IV alpha 3, alpha 4, and alpha 5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches



    Collagen IV is a major component of vertebrate basal laminae (BLs). Studies in humans have revealed a family of genes encoding alpha 1- alpha 6 collagen IV chains and implicated alpha 3-alpha 6 in disease processes (Goodpasture and Alport syndromes and diffuse leiomyomatosis). To extend studies of these components to an experimentally accessible animal, we cloned cDNAs encoding partial collagen alpha 3, alpha 4, and alpha 5(IV) chains from the mouse. Ribonuclease protection assays showed that...

  2. Development of oligoclonal nanobodies for targeting the tumor-associated glycoprotein 72 antigen

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali;


    The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specific......The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies...

  3. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Institute of Scientific and Technical Information of China (English)

    Beuy Joob; Viroj Wiwanitkit


    The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present. Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus. The in-depth analysis of the viral protein to find the binding site, active pocket is needed. Here, the authors analyzed the envelope glycoprotein GP2 from Ebola virus. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus was done. According to this assessment, 7 active pockets with varied druggability could be identified.

  4. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Institute of Scientific and Technical Information of China (English)

    Beuy; Joob; Viroj; Wiwanitkit


    The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present.Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus.The in-depth analysis of the viral protein to find the binding site,active pocket is needed.Here,the authors analyzed the envelope glycoprotein GP2 from Ebola virus.Identification of active pocket and protein draggability within envelope glycoprotein GP2 from Ebola virus was done.According to this assessment,7 active pockets with varied draggability could be identified.

  5. Electrophoretic demonstration of glycoproteins, lipoproteins, and phosphoproteins in human and bovine enamel

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D; Bøg-Hansen, T C;


    Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human...... enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs....

  6. A Novel Method for Detection of Glycoproteins on Sodium Dodecyl Sulphate Polyacrylamide Gel Using Radio-Iodinated Tyrosine

    DEFF Research Database (Denmark)

    Nalla, Amarnadh; Draz, Hossam M.; Dole, Anita;


    The aim of this study is to develop a novel method for detection of glycoproteins on polyacrylamide gel. In this method, radio-iodinated-tyrosine (125I-tyrosine) was conjugated to glycoprotein by schiff's base mechanism on the sodium dodecyl sulfate- polyacrylamide gel. Ovalbumin and Concanavalin...

  7. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier. (United States)

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun


    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (PZ-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain.

  8. Requirements for ER-Arrest and Sequential Exit to the Golgi of Tomato Spotted Wilt Virus Glycoproteins

    NARCIS (Netherlands)

    Ribeiro, D.M.O.G.; Goldbach, R.W.; Kormelink, R.J.M.


    The envelope glycoproteins Gn and Gc are major determinants in the assembly of Tomato spotted wilt virus (TSWV) particles at the Golgi complex. In this article, the ER-arrest of singly expressed Gc and the transport of both glycoproteins to the Golgi upon co-expression have been analyzed. While prel

  9. Targeting prostaglandin E2 EP1 receptors prevents seizure-associated P-glycoprotein up-regulation

    NARCIS (Netherlands)

    Pekcec, A.; Unkrüer, B.; Schlichtiger, J.; Soerensen, J.; Hartz, A.M.S.; Bauer, B.; van Vliet, E.A.; Gorter, J.A.; Potschka, H.


    Up-regulation of the blood-brain barrier efflux transporter P-glycoprotein in central nervous system disorders results in restricted brain access and limited efficacy of therapeutic drugs. In epilepsies, seizure activity strongly triggers expression of P-glycoprotein. Here, we identified the prostag

  10. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Vogler, Meike, E-mail: [MRC Toxicology Unit, University of Leicester, LE1 9HN Leicester (United Kingdom); Dickens, David, E-mail: [Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, L69 3GL Liverpool (United Kingdom); Dyer, Martin J.S., E-mail: [MRC Toxicology Unit, University of Leicester, LE1 9HN Leicester (United Kingdom); Owen, Andrew, E-mail: [Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, L69 3GL Liverpool (United Kingdom); Pirmohamed, Munir, E-mail: [Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, L69 3GL Liverpool (United Kingdom); Cohen, Gerald M., E-mail: [MRC Toxicology Unit, University of Leicester, LE1 9HN Leicester (United Kingdom)


    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  11. P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments

    DEFF Research Database (Denmark)

    Ejsing, Thomas B.; Pedersen, Anne D.; Linnet, Kristian


    P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice......P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice...

  12. The DNA sequence of the equine herpesvirus 4 gene encoding glycoprotein gp17/18, the homologue of herpes simplex virus glycoprotein gD. (United States)

    Cullinane, A A; Neilan, J; Wilson, L; Davison, A J; Allen, G


    The nucleotide sequence of the gene to the left of the gI gene of equine herpesvirus 4 (EHV-4) was determined. The gene encodes a peptide of 402 amino acids with an unprocessed M(r) of 45,323. The predicted polypeptide has several features of a glycoprotein including a hydrophobic signal sequence, a membrane spanning domain and four potential N-linked glycosylation sites within the proposed external domain. The predicted amino acid sequence of EHV-4 gD shows 83% identity with that of equine herpesvirus 1 gD. Conservation of the tertiary structure is suggested by the alignment of six cysteine residues with those of the gD of six other alphaherpesviruses. Screening a lambda gt11/EHV-4 expression library with monoclonal antibodies against several of the most abundant EHV-4 glycoproteins unequivocally identified the protein encoded by the EHV-4 gD gene as gp17/18.

  13. Chemical synthesis of glycoproteins with the specific installation of gradient enriched 15N-labeled amino acids for getting insight into glycoprotein behavior. (United States)

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


    We propose a novel partially 15N-labelling method for the amide backbone of a synthetic glycoprotein. By use of a chemical approach utilizing SPPS and NCL, we inserted thirteen 15N-labeled amino acids at specific positions of the protein backbone, while intentionally varying the enrichment of 15N atoms. This idea enables us to discriminate even the same type of amino acid based on the intensities of 1H-15N HSQC signals, thus allowing us to understand 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, T1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied CD spectra and T1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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; 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.

  15. Further Precise Determinations of $\\alpha_s$ from Lattice QCD

    CERN Document Server

    Davies, C T H; Lepage, G P; McCallum, P; Shigemitsu, J; Sloan, J


    We present a new determination of the strong coupling constant from lattice QCD simulations. We use four different short-distance quantities to obtain the coupling, three different (infrared) meson splittings to tune the simulation parameters, and a wide range of lattice spacings, quark masses, and lattice volumes to test for systematic errors. Our final result consists of ten different determinations of $\\alpha^{(3)}_{P}(8.2 GeV)$, which agree well with each other and with our previous results. The most accurate of these, when evolved perturbatively to the $Z^0$ mass, gives obtained from other recent lattice simulations.

  16. Double-real corrections at O(alpha alpha_s) to single gauge boson production

    CERN Document Server

    Bonciani, Roberto; Mondini, Roberto; Vicini, Alessandro


    We consider the O(alpha alpha_s) corrections to single on-shell gauge boson production at hadron colliders. We concentrate on the contribution of all the subprocesses where the gauge boson is accompanied by the emission of two additional real partons and we evaluate the corresponding total cross sections. The latter are divergent quantities, because of soft and collinear emissions, and are expressed as Laurent series in the dimensional regularization parameter. The total cross sections are evaluated by means of reverse unitarity, i.e. expressing the phase-space integrals in terms of two-loop forward box integrals with cuts on the final state particles. The results are reduced to a combination of Master Integrals, which eventually are evaluated in terms of Generalized Polylogarithms. The presence of internal massive lines in the Feynman diagrams, due to the exchange of electroweak gauge bosons, causes the appearance of 14 Master Integrals which were not previously known in the literature and have been evaluate...

  17. Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc (United States)

    Stettner, Eva; Jeffers, Scott Allen; Pérez-Vargas, Jimena; Pehau-Arnaudet, Gerard; Tortorici, M. Alejandra; Jestin, Jean-Luc; England, Patrick; Tischler, Nicole D.; Rey, Félix A.


    Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens. PMID:27783711

  18. Increased Expression of P-Glycoprotein Is Associated With Chlorpyrifos Resistance in the German Cockroach (Blattodea: Blattellidae). (United States)

    Hou, Weiyuan; Jiang, Chu; Zhou, Xiaojie; Qian, Kun; Wang, Lei; Shen, Yanhui; Zhao, Yan


    A principal method for control of the German cockroach, Blattella germanica (L.), is the broad-spectrum organophosphorus insecticide, chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate); however, extensive and repeated application has resulted in the development of resistance to chlorpyrifos in this insect. Evidence suggests that ATP-binding cassette protein transporters, including P-glycoprotein, are involved in insecticide resistance. However, little is known of the role of P-glycoprotein in insecticide resistance in the German cockroach. Here, we developed a chlorpyrifos-resistant strain of German cockroach and investigated the relationship between P-glycoprotein and chlorpyrifos resistance using toxicity assays; inhibition studies with two P-glycoprotein inhibitors, verapamil and quinine; P-glycoprotein-ATPase activity assays; and western blotting analysis. After 23 generations of selection from susceptible strain cockroaches, we obtained animals with high resistance to chlorpyrifos. When P-glycoprotein-ATPase activity was inhibited by verapamil and quinine, we observed enhanced susceptibility to chlorpyrifos in both control and chlorpyrifos-resistant cockroaches. No significant alterations of P-glycoprotein expression or ATPase activity were observed in cockroaches acutely exposed to LD50 doses of chlorpyrifos for 24 h, while P-glycoprotein expression and ATPase activity were clearly elevated in the chlorpyrifos-resistant cockroach strain. Thus, we conclude that P-glycoprotein is associated with chlorpyrifos resistance in the German cockroach and that elevated levels of P-glycoprotein expression and ATPase activity may be an important mechanism of chlorpyrifos resistance in the German cockroach.

  19. Folding model study of the elastic $\\alpha + \\alpha$ scattering at low energies

    CERN Document Server

    Tan, Ngo Hai; Khoa, Dao T


    The folding model analysis of the elastic $\\alpha + \\alpha$ scattering at the incident energies below the reaction threshold of 34.7 MeV (in the lab system) has been done using the well-tested density dependent versions of the M3Y interaction and realistic choices for the $^4$He density. Because the absorption is negligible at the energies below the reaction threshold, we were able to probe the $\\alpha + \\alpha$ optical potential at low energies quite unambiguously and found that the $\\alpha + \\alpha$ overlap density used to construct the density dependence of the M3Y interaction is strongly distorted by the Pauli blocking. This result gives possible explanation of a long-standing inconsistency of the double-folding model in its study of the elastic $\\alpha + \\alpha$ and $\\alpha$-nucleus scattering at low energies using the same realistic density dependent M3Y interaction.

  20. Scale setting for alpha_s beyond leading order

    CERN Document Server

    Hornbostel, K; Morningstar, C J


    We present a general procedure for incorporating higher-order information into the scale-setting prescription of Brodsky, Lepage and Mackenzie. In particular, we show how to apply this prescription when the leading coefficient or coefficients in a series in the strong coupling alpha_s are anomalously small and the original prescription can give an unphysical scale. We give a general method for computing an optimum scale numerically, within dimensional regularization, and in cases when the coefficients of a series are known. We apply it to the heavy quark mass and energy renormalization in lattice NRQCD, and to a variety of known series. Among the latter, we find significant corrections to the scales for the ratio of e+e- to hadrons over muons, the ratio of the quark pole to MSbar mass, the semi-leptonic B-meson decay width, and the top decay width. Scales for the latter two decay widths, expressed in terms of MSbar masses, increase by factors of five and thirteen, respectively, substantially reducing the size...

  1. Recoil-alpha-fission and recoil-alpha-alpha-fission events observed in the reaction Ca-48 + Am-243

    CERN Document Server

    Forsberg, U; Andersson, L -L; Di Nitto, A; Düllmann, Ch E; Gates, J M; Golubev, P; Gregorich, K E; Gross, C J; Herzberg, R -D; Hessberger, F P; Khuyagbaatar, J; Kratz, J V; Rykaczewski, K; Sarmiento, L G; Schädel, M; Yakushev, A; Åberg, S; Ackermann, D; Block, M; Brand, H; Carlsson, B G; Cox, D; Derkx, X; Dobaczewski, J; Eberhardt, K; Even, J; Fahlander, C; Gerl, J; Jäger, E; Kindler, B; Krier, J; Kojouharov, I; Kurz, N; Lommel, B; Mistry, A; Mokry, C; Nazarewicz, W; Nitsche, H; Omtvedt, J P; Papadakis, P; Ragnarsson, I; Runke, J; Schaffner, H; Schausten, B; Shi, Y; Thörle-Pospiech, P; Torres, T; Traut, T; Trautmann, N; Türler, A; Ward, A; Ward, D E; Wiehl, N


    Products of the fusion-evaporation reaction Ca-48 + Am-243 were studied with the TASISpec set-up at the gas-filled separator TASCA at the GSI Helmholtzzentrum f\\"ur Schwerionenforschung. Amongst the detected thirty correlated alpha-decay chains associated with the production of element Z=115, two recoil-alpha-fission and five recoil-alpha-alpha-fission events were observed. The latter are similar to four such events reported from experiments performed at the Dubna gas-filled separator. Contrary to their interpretation, we propose an alternative view, namely to assign eight of these eleven decay chains of recoil-alpha(-alpha)-fission type to start from the 3n-evaporation channel 115-288. The other three decay chains remain viable candidates for the 2n-evaporation channel 115-289.

  2. Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gp13) with homology to herpes simplex virus glycoprotein C. (United States)

    Allen, G P; Coogle, L D


    The molecular structure of the equine herpesvirus type 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was analyzed. The gene is contained within a 1.8-kilobase AccI-EcoRI restriction fragment mapping at map coordinates 0.136 to 0.148 in the UL region of the EHV-1 genome and is transcribed from right to left. Determination of the nucleotide sequence of the DNA fragment revealed a complete transcriptional unit composed of typical regulatory promoter elements upstream to a long open reading frame (1,404 base pairs) that encoded a 468-amino-acid primary translation product of 51 kilodaltons. The predicted protein has the characteristic features of a membrane-spanning protein: an N-terminal signal sequence, a hydrophobic membrane anchor region, a charged C-terminal cytoplasmic tail, and an exterior domain with nine potential N-glycosylation sites. The EHV-1 DNA sequences expressed in lambda gt11 as gp13 epitopes were present in the open reading frame. Amino acid sequences composing a major antigenic site, recognized by 35% of a panel of 42 anti-gp13 monoclonal antibodies, were identified in the N-terminal surface domain of the deduced gp13 molecule. Comparison of the EHV-1 gp13 DNA sequence with that encoding glycoproteins of other alphaherpesviruses revealed no detectable homology. However, a search for homology at the amino acid level showed regions of significant sequence similarity between the amino acids of the carboxy half of EHV-1 gp13 and those of the same region of gC-like glycoproteins of herpes simplex virus (gC-1 and gC-2), pseudorabies herpesvirus (gIII), and varicella-zoster virus (gp66). The sequences of the N-terminal portion of gp13, by contrast, were much less conserved. The results of these studies indicate that EHV-1 gp13 is the structural homolog of herpes simplex virus glycoprotein C and further suggest that the epitope-containing N-terminal amino acid sequences of the herpesvirus gC-like glycoproteins have undergone more extensive evolutionary

  3. Targeted alpha therapy for cancer

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Barry J [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Raja, Chand [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Rizvi, Syed [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Li Yong [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Tsui, Wendy [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Zhang, David [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Song, Emma [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Qu, C F [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Kearsley, John [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Graham, Peter [Centre for Experimental Radiation Oncology, St George Cancer Care Centre, Gray St, Kogarah 2217, NSW (Australia); Thompson, John [Sydney Melanoma Unit, Royal Prince Alfred Hospital, Camperdown 2050 NSW (Australia)


    Targeted alpha therapy (TAT) offers the potential to inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The practicality and efficacy of TAT is tested by in vitro and in vivo studies in melanoma, leukaemia, colorectal, breast and prostate cancers, and by a phase 1 trial of intralesional TAT for melanoma. The alpha-emitting radioisotope used is Bi-213, which is eluted from the Ac-225 generator and chelated to a cancer specific monoclonal antibody (mab) or protein (e.g. plasminogen activator inhibitor-2 PAI2) to form the alpha-conjugate (AC). Stable alpha-ACs have been produced which have been tested for specificity and cytotoxicity in vitro against melanoma (9.2.27 mab), leukaemia (WM60), colorectal (C30.6), breast (PAI2, herceptin), ovarian (PAI2, herceptin, C595), prostate (PAI2, J591) and pancreatic (PAI2, C595) cancers. Subcutaneous inoculation of 1-1.5 million human cancer cells into the flanks of nude mice causes tumours to grow in all mice. Tumour growth is compared for untreated controls, nonspecific AC and specific AC, for local (subcutaneous) and systemic (tail vein or intraperitoneal) injection models. The {sup 213}Bi-9.2.27 AC is injected into secondary skin melanomas in stage 4 patients in a dose escalation study to determine the effective tolerance dose, and to measure kinematics to obtain the equivalent dose to organs. In vitro studies show that TAT is one to two orders of magnitude more cytotoxic to targeted cells than non-specific ACs, specific beta emitting conjugates or free isotopes. In vivo local TAT at 2 days post-inoculation completely prevents tumour formation for all cancers tested so far. Intra-lesional TAT can completely regress advanced sc melanoma but is less successful for breast and prostate cancers. Systemic TAT inhibits the growth of sc melanoma xenografts and gives almost complete control of breast and prostate cancer tumour growth. Intralesional doses up to 450 {mu

  4. Linearized Bekenstein Varying Alpha Models

    CERN Document Server

    Pina-Avelino, P; Oliveira, J C


    We study the simplest class of Bekenstein-type, varying $\\alpha$ models, in which the two available free functions (potential and gauge kinetic function) are Taylor-expanded up to linear order. Any realistic model of this type reduces to a model in this class for a certain time interval around the present day. Nevertheless, we show that no such model is consistent with all existing observational results. We discuss possible implications of these findings, and in particular clarify the ambiguous statement (often found in the literature) that ``the Webb results are inconsistent with Oklo''.

  5. The Alpha Magnetic Spectrometer (AMS)

    Energy Technology Data Exchange (ETDEWEB)

    Alcaraz, J.; Alpat, B.; Ambrosi, G.; Anderhub, H.; Ao, L.; Arefiev, A.; Azzarello, P.; Babucci, E.; Baldini, L.; Basile, M.; Barancourt, D.; Barao, F.; Barbier, G.; Barreira, G.; Battiston, R.; Becker, R.; Becker, U.; Bellagamba, L.; Bene, P.; Berdugo, J.; Berges, P.; Bertucci, B.; Biland, A.; Bizzaglia, S.; Blasko, S.; Boella, G.; Boschini, M.; Bourquin, M.; Brocco, L.; Bruni, G.; Buenerd, M.; Burger, J.D.; Burger, W.J.; Cai, X.D.; Camps, C.; Cannarsa, P.; Capell, M.; Casadei, D.; Casaus, J.; Castellini, G.; Cecchi, C.; Chang, Y.H.; Chen, H.F.; Chen, H.S.; Chen, Z.G.; Chernoplekov, N.A.; Chiueh, T.H.; Chuang, Y.L.; Cindolo, F.; Commichau, V.; Contin, A. E-mail:; Crespo, P.; Cristinziani, M.; Cunha, J.P. da; Dai, T.S.; Deus, J.D.; Dinu, N.; Djambazov, L.; DAntone, I.; Dong, Z.R.; Emonet, P.; Engelberg, J.; Eppling, F.J.; Eronen, T.; Esposito, G.; Extermann, P.; Favier, J.; Fiandrini, E.; Fisher, P.H.; Fluegge, G.; Fouque, N.; Galaktionov, Yu.; Gervasi, M.; Giusti, P.; Grandi, D.; Grimm, O.; Gu, W.Q.; Hangarter, K.; Hasan, A.; Hermel, V.; Hofer, H.; Huang, M.A.; Hungerford, W.; Ionica, M.; Ionica, R.; Jongmanns, M.; Karlamaa, K.; Karpinski, W.; Kenney, G.; Kenny, J.; Kim, W.; Klimentov, A.; Kossakowski, R.; Koutsenko, V.; Kraeber, M.; Laborie, G.; Laitinen, T.; Lamanna, G.; Laurenti, G.; Lebedev, A.; Lee, S.C.; Levi, G.; Levtchenko, P.; Liu, C.L.; Liu, H.T.; Lopes, I.; Lu, G.; Lu, Y.S.; Luebelsmeyer, K.; Luckey, D.; Lustermann, W.; Mana, C.; Margotti, A.; Mayet, F.; McNeil, R.R.; Meillon, B.; Menichelli, M.; Mihul, A.; Mourao, A.; Mujunen, A.; Palmonari, F.; Papi, A.; Park, I.H.; Pauluzzi, M.; Pauss, F.; Perrin, E.; Pesci, A.; Pevsner, A.; Pimenta, M.; Plyaskin, V.; Pojidaev, V.; Postolache, V.; Produit, N.; Rancoita, P.G.; Rapin, D.; Raupach, F.; Ren, D.; Ren, Z.; Ribordy, M.; Richeux, J.P.; Riihonen, E.; Ritakari, J.; Roeser, U.; Roissin, C.; Sagdeev, R.; Sartorelli, G.; Schultz von Dratzig, A.; Schwering, G.; Scolieri, G.; Seo, E.S.; Shoutko, V.


    The Alpha Magnetic Spectrometer (AMS) is a large acceptance (0.65 sr m{sup 2}) detector designed to operate in the International Space Station (ISS) for three years. The purposes of the experiment are to search for cosmic antimatter and dark matter and to study the composition and energy spectrum of the primary cosmic rays. A 'scaled-down' version has been flown on the Space Shuttle Discovery for 10 days in June 1998. The complete AMS is programmed for installation on the ISS in October 2003 for an operational period of 3 yr. This contribution reports on the experimental configuration that will be installed on the ISS.

  6. The Alpha Magnetic Spectrometer (AMS)

    CERN Document Server

    Alcaraz, J; Ambrosi, G; Anderhub, H; Ao, L; Arefev, A; Azzarello, P; Babucci, E; Baldini, L; Basile, M; Barancourt, D; Barão, F; Barbier, G; Barreira, G; Battiston, R; Becker, R; Becker, U; Bellagamba, L; Bene, P; Berdugo, J; Berges, P; Bertucci, B; Biland, A; Bizzaglia, S; Blasko, S; Bölla, G; Boschini, M; Bourquin, Maurice; Brocco, L; Bruni, G; Buénerd, M; Burger, J D; Burger, W J; Cai, X D; Camps, C; Cannarsa, P; Capell, M; Casadei, D; Casaus, J; Castellini, G; Cecchi, C; Chang, Y H; Chen, H F; Chen, H S; Chen, Z G; Chernoplekov, N A; Tzi Hong Chiueh; Chuang, Y L; Cindolo, F; Commichau, V; Contin, A; Crespo, P; Cristinziani, M; Cunha, J P D; Dai, T S; Deus, J D; Dinu, N; Djambazov, L; Dantone, I; Dong, Z R; Emonet, P; Engelberg, J; Eppling, F J; Eronen, T; Esposito, G; Extermann, P; Favier, Jean; Fiandrini, E; Fisher, P H; Flügge, G; Fouque, N; Galaktionov, Yu; Gervasi, M; Giusti, P; Grandi, D; Grimm, O; Gu, W Q; Hangarter, K; Hasan, A; Hermel, V; Hofer, H; Huang, M A; Hungerford, W; Ionica, M; Ionica, R; Jongmanns, M; Karlamaa, K; Karpinski, W; Kenney, G; Kenny, J; Kim, W; Klimentov, A; Kossakowski, R; Koutsenko, V F; Kraeber, M; Laborie, G; Laitinen, T; Lamanna, G; Laurenti, G; Lebedev, A; Lee, S C; Levi, G; Levchenko, P M; Liu, C L; Liu, H T; Lopes, I; Lu, G; Lü, Y S; Lübelsmeyer, K; Luckey, D; Lustermann, W; Maña, C; Margotti, A; Mayet, F; McNeil, R R; Meillon, B; Menichelli, M; Mihul, A; Mourao, A; Mujunen, A; Palmonari, F; Papi, A; Park, I H; Pauluzzi, M; Pauss, Felicitas; Perrin, E; Pesci, A; Pevsner, A; Pimenta, M; Plyaskin, V; Pozhidaev, V; Postolache, V; Produit, N; Rancoita, P G; Rapin, D; Raupach, F; Ren, D; Ren, Z; Ribordy, M; Richeux, J P; Riihonen, E; Ritakari, J; Röser, U; Roissin, C; Sagdeev, R; Sartorelli, G; Schwering, G; Scolieri, G; Seo, E S; Shoutko, V; Shoumilov, E; Siedling, R; Son, D; Song, T; Steuer, M; Sun, G S; Suter, H; Tang, X W; Ting, Samuel C C; Ting, S M; Tornikoski, M; Torsti, J; Ulbricht, J; Urpo, S; Usoskin, I; Valtonen, E; Vandenhirtz, J; Velcea, F; Velikhov, E P; Verlaat, B; Vetlitskii, I; Vezzu, F; Vialle, J P; Viertel, Gert M; Vitè, Davide F; Gunten, H V; Wallraff, W; Wang, B C; Wang, J Z; Wang, Y H; Wiik, K; Williams, C; Wu, S X; Xia, P C; Yan, J L; Yan, L G; Yang, C G; Yang, M; Ye, S W; Yeh, P; Xu, Z Z; Zhang, H Y; Zhang, Z P; Zhao, D X; Zhu, G Y; Zhu, W Z; Zhuang, H L; Zichichi, A; Zimmermann, B


    The Alpha Magnetic Spectrometer (AMS) is a large acceptance (0.65 sr m sup 2) detector designed to operate in the International Space Station (ISS) for three years. The purposes of the experiment are to search for cosmic antimatter and dark matter and to study the composition and energy spectrum of the primary cosmic rays. A 'scaled-down' version has been flown on the Space Shuttle Discovery for 10 days in June 1998. The complete AMS is programmed for installation on the ISS in October 2003 for an operational period of 3 yr. This contribution reports on the experimental configuration that will be installed on the ISS.

  7. Orthopositronium lifetime at O({alpha}) and O({alpha}{sup 3} ln {alpha}) in closed form

    Energy Technology Data Exchange (ETDEWEB)

    Kniehl, B.A.; Kotikov, A.V.; Veretin, O.L. [Hamburg Univ. (Germany). 2. Inst. fuer Theoretische Physik


    Recently, the O({alpha}) and O({alpha}{sup 3} ln{alpha}) radiative corrections to the orthopositronium lifetime have been presented in closed analytical form, in terms of basic irrational numbers that can be evaluated numerically to arbitrary precision [Phys. Rev. Lett. 101, 193401 (2008)]. Here, we present the details of this calculation and reveal the nature of these new constants. We also list explicit transformation formulas for generalized polylogarithms of weight four, which may be useful for other applications. (orig.)

  8. Virulence determinants within the E2 glycoprotein of Classical Swine Fever Virus

    DEFF Research Database (Denmark)

    Johnston, Camille Melissa; Fahnøe, Ulrik; Lohse, Louise

    Classical Swine Fever is a highly contagious disease of pigs caused by Classical Swine Fever Virus (CSFV), a member of the pestivirus genus within the family Flaviviridae. The E2 glycoprotein of CSFV has been shown to be an important factor for the virulence of the virus. In a recent study, we ha...

  9. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators. (United States)

    Loo, T W; Clarke, D M


    There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

  10. Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model

    DEFF Research Database (Denmark)

    Andersson, O.; Badisco, L.; Hansen, A. H.;


    In earlier studies insects were proposed as suitable models for vertebrate blood–brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain b...

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

    Directory of Open Access Journals (Sweden)

    Kamel El Omari


    Full Text Available 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.

  12. Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers

    NARCIS (Netherlands)

    Zhang, Xinzheng; Sheng, Ju; Austin, S. Kyle; Hoornweg, Tabitha E.; Smit, Jolanda M.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.


    Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However,

  13. Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

    NARCIS (Netherlands)

    Konings, WN; Poelarends, GJ


    Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural a

  14. Monoclonal Antibodies for Dengue Virus prM Glycoprotein Protect Mice against Lethal Dengue Infection (United States)


    Nile virus and a prelysozomal endosome prM glycoprotein of dengue virus can also be required for viral replication . PrM Mabs 2H2 protective bodies can prevent lethal alphavirus encepha- niques to preserve immunogenicity, to deter- litis. Nature 297: 70-72. UI:82173237 mine whether

  15. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B. (United States)

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


    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 forms 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.

  16. Structure of three acidic O-linked carbohydrate chains of porcine zona pellucida glycoproteins

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Hokke, C.H.; Damm, J.B.L.; Kamerling, J.P.


    Structural analysis by ID and 2D 1H NMR spectroscopy of three acidic O-linked oligosaccharide alditols, released from porcine zona pellucida glycoproteins by alkaline borohydride treatment, afforded the following structures: Gal beta l-4(6SO4-)GlcNAc beta l-3Gal beta l-4GlcNAc beta 1-3Gal beta 1-3Ga

  17. Identification of key structural characteristics of Schisandra chinensis lignans involved in P-glycoprotein inhibition. (United States)

    Slanina, Jiří; Páchniková, Gabriela; Carnecká, Martina; Porubová Koubíková, Ludmila; Adámková, Lenka; Humpa, Otakar; Smejkal, Karel; Slaninová, Iva


    The aim of the present study was to determine the structural requirements for dibenzocyclooctadiene lignans essential for P-glycoprotein inhibition. Altogether 15 structurally related lignans isolated from Schisandra chinensis or prepared by modification of their backbone were investigated, including three pairs of enantiomers. P-Glycoprotein inhibition was quantified using a doxorubicin accumulation assay in human promyelotic leukemia HL60/MDR cells overexpressing P-glycoprotein. A preliminary quantitative structure-activity relationship analysis revealed three main structural features involved in P-glycoprotein inhibition: a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group. The most effective inhibitors, (-)-gomisin N (1) and (+)-deoxyschizandrin [(+)-2], were selected for further evaluation of their effects. Both these lignans restored the cytotoxic effect of doxorubicin in HL60/MDR cells and when combined with a subtoxic concentration of this compound increased the proportion of G2/M cells significantly, which is a usual response to treatment with this anticancer drug.

  18. Irradiation of rat brain reduces P-glycoprotein expression and function

    NARCIS (Netherlands)

    Bart, J.; Nagengast, W.B.; Coppes, R.P.; Wegman, T.D.; Graaf, W.T.A. van der; Groen, H.J.; Vaalburg, W.; Vries, E.G.F. de; Hendrikse, N.H.


    The blood-brain barrier (BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P-glycoprotein (P-gp), expressed on brain capillary endothelial cells, is part of the BBB. P-gp expression on capillary endothelium decreases 5 days after brain irradiation,

  19. Irradiation of rat brain reduces P-glycoprotein expression and function

    NARCIS (Netherlands)

    Bart, J.; Nagengast, W. B.; Coppes, R. P.; Wegman, T. D.; van der Graaf, W. T. A.; Groen, H. J. M.; Vaalburg, W.; de Vries, E. G. E.; Hendrikse, N. H.


    The blood - brain barrier ( BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P- glycoprotein ( P- gp), expressed on brain capillary endothelial cells, is part of the BBB. P- gp expression on capillary endothelium decreases 5 days after brain irrad

  20. Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models

    NARCIS (Netherlands)

    Kramer, Gertjan; Wegdam, Wouter; Donker-Koopman, Wilma; Ottenhoff, Roelof; Gaspar, Paulo; Verhoek, Marri; Nelson, Jessica; Gabriel, Tanit; Kallemeijn, Wouter; Boot, Rolf G.; Laman, Jon D.; Vissers, Johannes P. C.; Cox, Timothy; Pavlova, Elena; Moran, Mary Teresa; Aerts, Johannes M.; van Eijk, Marco


    Gaucher disease is caused by inherited deficiency of lysosomal glucocere-brosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL

  1. Elevation of glycoprotein nonmetastatic melanoma protein B in type 1 Gaucher disease patients and mouse models

    NARCIS (Netherlands)

    Kramer, G.; Wegdam, W.; Donker-Koopman, W.; Ottenhoff, R.; Gaspar, P.; Verhoek, M.; Nelson, J.; Gabriel, T.; Kallemeijn, W.W.; Boot, R.G.; Laman, J.D.; Vissers, J.P.; Cox, T.; Pavlova, E.; Moran, M.T.; Aerts, J.M.F.G.; Eijk, van M.C.


    Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL1

  2. Interactions between P-glycoprotein substrates and other cationic drugs at the hepatic excretory level

    NARCIS (Netherlands)

    Smit, JW; Duin, E; Steen, H; Roggeveld, J; Meijer, DKF


    1 In the present study it was tested whether known P-glycoprotein (P-gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds tr

  3. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits (United States)

    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.


    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. Effect of luteolin on glycoproteins metabolism in 1, 2-dimethylhydrazine induced experimental colon carcinogenesis

    Directory of Open Access Journals (Sweden)

    Manju Vaiyapuri


    carcinogenesis. Thus, the present study indicates that luteolin has protected the cell surface and maintained the structural integrity of the cell membranes during DMH induced colon carcinogenesis. Keywords: Colon cancer, 1, 2-dimethylhydrazine, luteolin, glycoproteins Received: 23 January 2009 / Received in revised form: 17 February 2009, Accepted: 28 February 2009, Published online: 3 March 2009

  5. Entactin: ultrastructural localization of an ubiquitous basement membrane glycoprotein in mouse skin

    DEFF Research Database (Denmark)

    Horiguchi, Y; Fine, J D; Ljubimov, A V;


    Entactin is a recently described sulfated glycoprotein component of mouse endodermal cell-derived extracellular matrix and is present in a number of basement membranes. It has been ultrastructurally localized to both lamina densa and adjacent epithelial cell membranes in rodent kidney. In the pre...

  6. Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1 (United States)

    Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these i...

  7. A pro-inflammatory glycoprotein biomarker is associated with lower bilirubin in metabolic syndrome

    NARCIS (Netherlands)

    Dullaart, Robin P. F.; Gruppen, Eke G.; Connelly, Margery A.; Lefrandt, Joop D.


    Objectives: Bilirubin exerts anti-oxidative and anti-inflammatory properties which may beneficially influence the development of cardio-metabolic disorders. A nuclear magnetic resonance (NMR) spectroscopy-based glycoprotein biomarker, designated GlycA, whose signal originates from several glycosylat

  8. A vesicular stomatitis pseudovirus expressing the surface glycoproteins of influenza A virus. (United States)

    Cheresiz, S V; Kononova, A A; Razumova, Yu V; Dubich, T S; Chepurnov, A A; Kushch, A A; Davey, R; Pokrovsky, A G


    Pseudotyped viruses bearing the glycoprotein(s) of a donor virus over the nucleocapsid core of a surrogate virus are widely used as safe substitutes for infectious virus in virology studies. Retroviral particles pseudotyped with influenza A virus glycoproteins have been used recently for the study of influenza hemagglutinin and neuraminidase-dependent processes. Here, we report the development of vesicular-stomatitis-virus-based pseudotypes bearing the glycoproteins of influenza A virus. We show that pseudotypes bearing the hemagglutinin and neuraminidase of H5N1 influenza A virus mimic the wild-type virus in neutralization assays and sensitivity to entry inhibitors. We demonstrate the requirement of NA for the infectivity of pseudotypes and show that viruses obtained with different NA proteins are significantly different in their transduction activities. Inhibition studies with oseltamivir carboxylate show that neuraminidase activity is required for pseudovirus production, but not for the infection of target cells with H5N1-VSV pseudovirus. The HA-NA-VSV pseudoviruses have high transduction titers and better stability than the previously reported retroviral pseudotypes and can replace live influenza virus in the development of neutralization assays, screening of potential antivirals, and the study of different HA/NA reassortants.

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

    Energy Technology Data Exchange (ETDEWEB)

    Backovic, Marija [Northwestern Univ., Evanston, IL (United States); Longnecker, Richard [Northwestern Univ., Chicago, IL (United States); Jardetzky, Theodore S [Northwestern Univ., Evanston, IL (United States)


    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 forms 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.

  10. Human intestinal P-glycoprotein activity estimated by the model substrate digoxin

    DEFF Research Database (Denmark)

    Larsen, U L; Hyldahl Olesen, L; Nyvold, Charlotte Guldborg;


    P-glycoprotein (Pgp) plays a part in the intestinal uptake of xenobiotics and has been associated with susceptibility to ulcerative colitis. The aim of this study was to examine Pgp activity in relation to age, gender, medical treatment (rifampicin or ketoconazole) and the multidrug resistance (M...

  11. Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins. (United States)

    Cova, Marta; Oliveira-Silva, Rui; Ferreira, José Alexandre; Ferreira, Rita; Amado, Francisco; Daniel-da-Silva, Ana Luísa; Vitorino, Rui


    Protein post-translational modifications (PTMs) have increasingly become a research field of incredible importance to fully understand the regulation of biological processes in health and disease. Among PTMs, glycosylation is one of the most studied for which contributed the development and improvement of enrichment techniques. Nowadays, glycoprotein enrichment methods are based on lectin affinity, covalent interactions, and hydrophilic interaction liquid chromatography (HILIC). Nonetheless, the nanotechnology era has fetched new methods to enrich glycoproteins from complex samples as human biological fluids. For instance, magnetic nanoparticles (MNPs) are being used as an interesting enrichment approach allowing a better characterization of glycoproteins and glycopeptides.In this chapter, we describe an enrichment method based on MNPs functionalized with lectins (Concavalin A, wheat germ agglutinin, and Maackia amurensis lectin) to enrich specific sets of glycoproteins from biological fluids. Moreover, it is proposed a bioinformatic strategy to deal with data retrieved from mass spectrometry analysis of enriched samples aiming the identification of relevant biological processes modulated by a given stimuli and, ultimately, of new biomarkers for disease screening/management.

  12. Stabilization of HIV-1 envelope glycoprotein trimers to induce neutralizing antibodies

    NARCIS (Netherlands)

    de Taeye, S.W.


    HIV-1 has evolved various tricks to prevent the development of a potent humoral immune response. The only target for neutralizing antibodies (NAbs) is the HIV-1 envelope glycoprotein (Env), which is the sole viral protein embedded in the viral membrane. It consists of three gp41 subunits and three g

  13. Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer

    NARCIS (Netherlands)

    Walls, Alexandra C; Tortorici, M Alejandra; Bosch, Berend-Jan; Frenz, Brandon; Rottier, Peter J M; DiMaio, Frank; Rey, Félix A; Veesler, David


    The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion f

  14. St. John's Wort constituents modulate P-glycoprotein transport activity at the blood-brain barrier.

    NARCIS (Netherlands)

    Ott, M.; Huls, M.; Cornelius, M.G.; Fricker, G.


    PURPOSE: The purpose of this study was to investigate the short-term signaling effects of St. John's Wort (SJW) extract and selected SJW constituents on the blood-brain barrier transporter P-glycoprotein and to describe the role of PKC in the signaling. METHODS: Cultured porcine brain capillary endo

  15. Modification-specific proteomic analysis of glycoproteins in human body fluids by mass spectrometry

    DEFF Research Database (Denmark)

    Bunkenborg, Jakob; Hägglund, Per; Jensen, Ole Nørregaard


    -glycosylated proteins in body fluids and other complex samples. An approach for identification of N-glycosylated proteins and mapping of their glycosylation sites is described. In this approach, glycoproteins are initially selectively purified by lectin chromatography. Following tryptic digestion, glycopeptides...

  16. Virulence determinants within the E2 glycoprotein of Classical Swine Fever Virus

    DEFF Research Database (Denmark)

    Johnston, Camille Melissa; Fahnøe, Ulrik; Lohse, Louise

    Classical Swine Fever is a highly contagious disease of pigs caused by Classical Swine Fever Virus (CSFV), a member of the pestivirus genus within the family Flaviviridae. The E2 glycoprotein of CSFV has been shown to be an important factor for the virulence of the virus. In a recent study, we have...

  17. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion

    Directory of Open Access Journals (Sweden)

    Natalie K. Garcia


    Full Text Available The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

  18. A simple method to discriminate between beta(2)-glycoprotein I- and prothrombin-dependent lupus anticoagulants

    NARCIS (Netherlands)

    Simmelink, MJA; Derksen, RHWM; Arnout, J; De Groot, PG


    Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin. In the present study, we describe a method to

  19. Selective inhibition of herpes simplex virus glycoprotein synthesis by a benz-amidinohydrazone derivative

    Energy Technology Data Exchange (ETDEWEB)

    Campadelli-Fiume, G.; Sinibaldi-Vallebona, P.; Mannini-Palenzona, A. (Bologna Univ. (Italy). Ist. di Microbiologia e Virologia); Cavrini, V. (Bologna Univ. (Italy). Ist. di Chimica Farmaceutica e Tossicologica)


    1 H-benz(f)indene-1.3(2H)dione-bis-amidinohydrazone (benzhydrazone) inhibited incorporation of /sup 14/C-glucosamine, /sup 14/C-fucose and /sup 14/C-mannose into glycoproteins of HEp-2 cells infected with various strains of herpes simplex virus 1 (HSV-1) and impaired RNA and protein synthesis to a low extent. These biochemical effects are very similar to those induced by glycosylation inhibitors such as tunicamycin, D-glucosamine and 2-deoxy-D-glucose. In contrast to these inhibitors, benzhydrazone reduced HSV glycoprotein synthesis selectively since it did not significantly modify i) the saccharide uptake into glycoproteins of uninfected and of Sindbis virus-infected cells, ii) viral growth and cell fusion in paramyxovirus-infected cells, two activities which depend on viral glycoprotein synthesis. Benzhydrazone had only minor effects on the overall metabolism of uninfected cells, since it did not alter cell growth rate, and amino acid, uridine, and hexose incorporations were about 80 per cent those of untreated cells.

  20. Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

    NARCIS (Netherlands)

    West, Christopher M.; Van Der Wel, Hanke; Chinoy, Zoiesha; Boons, Geert Jan; Gauthier, Ted J.; Taylor, Carol M.; Xu, Yuechi


    Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1, 4(

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

    NARCIS (Netherlands)

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


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

  2. Blood-brain barrier P-glycoprotein function is not impaired in early Parkinson's disease

    NARCIS (Netherlands)

    Bartels, A. L.; van Berckel, B. N. M.; Lubberink, M.; Luurtsema, G.; Lammertsma, A. A.; Leenders, K. L.


    The cause of Parkinson's disease (PD) is unknown. Genetic susceptibility and exposure to environmental toxins contribute to specific neuronal loss in PD. Decreased blood-brain barrier (BBB) P-glycoprotein (P-gp) efflux function has been proposed as a possible causative link between toxin exposure an

  3. The role of p-glycoprotein in psychiatric disorders : a reliable guard of the brain?

    NARCIS (Netherlands)

    De Klerk, Onno L; Bosker, Fokko J; Luurtsema, Gert; Nolte, Ilja M; Dierckx, Rudi; Den Boer, Johan A; Potschka, Heidrun


    A major component in the protection of the brain against blood-borne toxic influences is the multispecific efflux pump P-glycoprotein. This pump, a 170 kD protein, located at the luminal side of the capillary endothelial cells, has a large capacity and is capable of extruding a wide array of structu

  4. Blood-Brain Barrier P-Glycoprotein Function in Neurodegenerative Disease

    NARCIS (Netherlands)

    Bartels, A. L.


    Protection of the brain is strengthened by active transport and ABC transporters. P-glycoprotein (P-gp) at the blood-brain barrier (BBB) functions as an active efflux pump by extruding a substrate from the brain, which is important for maintaining loco-regional homeostasis in the brain and protectio

  5. Quantitative assessment of p-glycoprotein expression and function using confocal image analysis. (United States)

    Hamrang, Zahra; Arthanari, Yamini; Clarke, David; Pluen, Alain


    P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

  6. Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

    NARCIS (Netherlands)

    Tomlinson, M. G.; Calaminus, S. D.; Berlanga, O.; Bori-Sanz, T.; Meyaard, L.; Watson, S. P.; Auger, J.M.


    Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR-gamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective: To determine why GPVI-FcR gamma signals poorly, or not at all, in response to

  7. P-glycoprotein regulating biphasic insulin secretion in rat pancreatic beta cells

    Institute of Scientific and Technical Information of China (English)

    TANG Yun-zhao; LI Dai-qing; SUN Fu-jun; LI Li; YU De-min


    Background A 65-kD mdr1(multi-drug resistance protein 1,P-glycoprotein)-like protein has been suggested to be the regulatory protein to the chloride channel protein 3(CIC-3)mediating insulin granules acidification and release in mouse pancreatic beta cells.But the protein has not been deeply investigated.In this study,we identified existence of the 65-kda protein in rat islets and preliminarily explored its biological functions.Methods Total RNAs of rat kidneys served as positive controls,and pancreas,islets and INS-1 cells were extracted for reverse-transcript PCR(RT-PCR),respectively.The cDNAs were run with specific primers selected from the mRNA of abcblb encoding P-glycoprotein.All PCR products were visualized in agarose gel electrophoresis and sequenced.Homogenates of rat islets and INS-1 cells were applied to SDS-PAGE.P-glycoprotein was detected by a specific monoclonal antibody,C219.Biphasic insulin release was measured in static incubations of rat islets with radioimmunology assay.Results Compared with positive control,expression of the P-glycoprotein mRNA segments were detected in the islets,INS-1 cells and pancreas.Sequence analysis confirmed that the PCR products were matched with mRNA of P-glycoprotein.A 65-kda protein was recognized by the antibody in the islets homogenate but not in that of INS-1 cells in Western-blotting.Instead,the homogenate of INS-1 cells contained a 160-kda protein recognized by the antibody.Insulin secretion of rat islets were stimulated by high glucose(16.7mmol/L),and showed biphasic curve during 60-minute incubation.After co-incubation with cyclosporine A(CsA),specific inhibitor to P-glycoprotein,the second phase of insulin secretion was reduced significantly while the first phase was not influenced.Conclusions The 65-kda protein expressed in rat islets is most likely a mini-P-glycoprotein.It may play a key role regulating biphasic insulin release.

  8. Blood-brain barrier P-glycoprotein function in Alzheimer's disease. (United States)

    van Assema, Daniëlle M E; Lubberink, Mark; Bauer, Martin; van der Flier, Wiesje M; Schuit, Robert C; Windhorst, Albert D; Comans, Emile F I; Hoetjes, Nikie J; Tolboom, Nelleke; Langer, Oliver; Müller, Markus; Scheltens, Philip; Lammertsma, Adriaan A; van Berckel, Bart N M


    A major pathological hallmark of Alzheimer's disease is accumulation of amyloid-β in senile plaques in the brain. Evidence is accumulating that decreased clearance of amyloid-β from the brain may lead to these elevated amyloid-β levels. One of the clearance pathways of amyloid-β is transport across the blood-brain barrier via efflux transporters. P-glycoprotein, an efflux pump highly expressed at the endothelial cells of the blood-brain barrier, has been shown to transport amyloid-β. P-glycoprotein function can be assessed in vivo using (R)-[(11)C]verapamil and positron emission tomography. The aim of this study was to assess blood-brain barrier P-glycoprotein function in patients with Alzheimer's disease compared with age-matched healthy controls using (R)-[(11)C]verapamil and positron emission tomography. In 13 patients with Alzheimer's disease (age 65 ± 7 years, Mini-Mental State Examination 23 ± 3), global (R)-[(11)C]verapamil binding potential values were increased significantly (P = 0.001) compared with 14 healthy controls (aged 62 ± 4 years, Mini-Mental State Examination 30 ± 1). Global (R)-[(11)C]verapamil binding potential values were 2.18 ± 0.25 for patients with Alzheimer's disease and 1.77 ± 0.41 for healthy controls. In patients with Alzheimer's disease, higher (R)-[(11)C]verapamil binding potential values were found for frontal, parietal, temporal and occipital cortices, and posterior and anterior cingulate. No significant differences between groups were found for medial temporal lobe and cerebellum. These data show altered kinetics of (R)-[(11)C]verapamil in Alzheimer's disease, similar to alterations seen in studies where P-glycoprotein is blocked by a pharmacological agent. As such, these data indicate that P-glycoprotein function is decreased in patients with Alzheimer's disease. This is the first direct evidence that the P-glycoprotein transporter at the blood-brain barrier is compromised in sporadic

  9. Selective sorting of alpha-granule proteins. (United States)

    Italiano, J E; Battinelli, E M


    One of the main functions of blood platelets is to secrete a variety of substances that can modify a developing thrombus, regulate the growth of the vasculature, promote wound repair, and contribute to cell-adhesive events. A majority of this vast array of secreted proteins are stored in alpha-granules. Until recently, it was assumed that platelets contained one homogeneous population of alpha-granules that undergo complete de-granulation during platelet activation. This review focuses on the mechanisms of alpha-granule biogenesis and secretion, with a particular emphasis on recent findings that clearly demonstrate that platelets contain distinct subpopulations of alpha-granules that undergo differential release during activation. We consider the implications of this new paradigm of platelet secretion, discuss mechanisms of alpha-granule biogenesis, and review the molecular basis of transport and delivery of alpha-granules to assembling platelets.

  10. The role of alpha-glucosidase in germinating barley grains. (United States)

    Stanley, Duncan; Rejzek, Martin; Naested, Henrik; Smedley, Mark; Otero, Sofía; Fahy, Brendan; Thorpe, Frazer; Nash, Robert J; Harwood, Wendy; Svensson, Birte; Denyer, Kay; Field, Robert A; Smith, Alison M


    The importance of α-glucosidase in the endosperm starch metabolism of barley (Hordeum vulgare) seedlings is poorly understood. The enzyme converts maltose to glucose (Glc), but in vitro studies indicate that it can also attack starch granules. To discover its role in vivo, we took complementary chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an α-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the endosperm 10 d after imbibition, implying inhibition of maltase activity. Three of the four inhibitors also reduced starch degradation and seedling growth, but the fourth did not affect these parameters. Inhibition of starch degradation was apparently not due to inhibition of amylases. Inhibition of seedling growth was primarily a direct effect of the inhibitors on roots and coleoptiles rather than an indirect effect of the inhibition of endosperm metabolism. It may reflect inhibition of glycoprotein-processing glucosidases in these organs. In transgenic seedlings carrying an RNA interference silencing cassette for HvAgl97, α-glucosidase activity was reduced by up to 50%. There was a large decrease in the Glc-to-maltose ratio in these lines but no effect on starch degradation or seedling growth. Our results suggest that the α-glucosidase HvAGL97 is the major endosperm enzyme catalyzing the conversion of maltose to Glc but is not required for starch degradation. However, the effects of three glucosidase inhibitors on starch degradation in the endosperm indicate the existence of unidentified glucosidase(s) required for this process.

  11. The alpha,alpha-(1-->1) linkage of trehalose is key to anhydrobiotic preservation. (United States)

    Albertorio, Fernando; Chapa, Vanessa A; Chen, Xin; Diaz, Arnaldo J; Cremer, Paul S


    This study compares the efficacy of six disaccharides and glucose for the preservation of solid supported lipid bilayers (SLBs) upon exposure to air. Disaccharide molecules containing an alpha,alpha-(1-->1) linkage, such as alpha,alpha-trehalose and alpha,alpha-galacto-trehalose, were found to be effective at retaining bilayer structure in the absence of water. These sugars are known to crystallize in a clam shell conformation. Other saccharides, which are found to crystallize in more open structures, did not preserve the SLB structure during the drying process. These included the nonreducing sugar, sucrose, as well as maltose, lactose, and the monosaccharide, glucose. In fact, even close analogs to alpha,alpha-trehalose, such as alpha,beta-trehalose, which connects its glucopyranose rings via a (1-->1) linkage in an axial, equatorial fashion, permitted nearly complete delamination and destruction of supported bilayers upon exposure to air. Lipids with covalently attached sugar molecules such as ganglioside GM1, lactosyl phosphatidylethanolamine, and glucosylcerebroside were also ineffective at preserving bilayer structure. The liquid crystalline-to-gel phase transition temperature of supported phospholipid bilayers was tested in the presence of sugars in a final set of experiments. Only alpha,alpha-trehalose and alpha,alpha-galacto-trehalose depressed the phase transition temperature, whereas the introduction of other sugar molecules into the bulk solution caused the phase transition temperature of the bilayer to increase. These results point to the importance of the axial-axial linkage of disaccharides for preserving SLB structure.

  12. Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeffrey E.; Fusco, Marnie L.; Abelson, Dafna M.; Hessell, Ann J.; Burton, Dennis R. [Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Saphire, Erica Ollmann, E-mail: [Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)


    Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing of the trimeric ebolavirus glycoprotein are described. The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008 ▶), Nature (London), 454, 177–182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the

  13. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture (United States)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  14. Hamster zona pellucida is formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4. (United States)

    Izquierdo-Rico, M J; Jimenez-Movilla, M; Llop, E; Perez-Oliva, A B; Ballesta, J; Gutierrez-Gallego, R; Jimenez-Cervantes, C; Aviles, M


    The zona pellucida (ZP) is an extracellular glycoprotein matrix that surrounds all mammalian oocytes. Recent data have shown the presence of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) in the ZP of human and rat rather than the three glycoproteins proposed in the mouse model. In the hamster (Mesocricetus auratus), it was previously described that ZP was composed of three different glycoproteins, called ZP1, ZP2, and ZP3, even though only ZP2 and ZP3 have been cloned thus far. The aim of the study was to determine whether hamster might also express four, rather than three, ZP proteins. The full-length cDNAs encoding hamster ZP glycoproteins 1 and 4 were isolated using rapid amplification cDNA ends (RACE). The cDNA of ZP1 contains an open reading frame of 1851 nucleotides encoding a polypeptide of 616 amino acid residues. The amino acid sequence of ZP1 revealed a high homology with other mammalian species like human (66%), rat (80%), and mouse (80%). The cDNA of ZP4 contains an open reading frame of 1632 nucleotides encoding a polypeptide of 543 amino acid residues. The deduced amino acid sequence of ZP4 revealed high overall homology with rat (82%) and human (78%). Subsequent mass spectrometric analysis of the hamster ZP allowed identification of peptides from all four glycoproteins. The data presented in this study provide evidence, for the first time, that the hamster ZP matrix is composed of four glycoproteins.

  15. Role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in acute lung injury in rats

    DEFF Research Database (Denmark)

    Shanley, T P; Schmal, H; Friedl, H P


    The role of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the pathogenesis of acute lung injury in rats after intrapulmonary deposition of IgG immune complexes or intratracheal administration of LPS has been assessed. Critical to these studies was the cloning and functional expression...... of rat MIP-1 alpha. The resulting product shared 92% and 90% homology with the known murine sequence at the cDNA level and protein level, respectively. Recombinant rat MIP-1 alpha exhibited dose-dependent chemotactic activity for both rat and human monocytes and neutrophils, which could be blocked...... by anti-murine MIP-1 alpha Ab. Rat MIP-1 alpha mRNA and protein expression were determined as a function of time in both injury models. A time-dependent increase in MIP-1 alpha mRNA in lung extracts was observed in both models. In the LPS model, MIP-1 alpha protein could also be detected...

  16. $\\alpha_{s}$ from the (revised) ALEPH data for $\\tau$ decay

    CERN Document Server

    Boito, Diogo; Maltman, Kim; Osborne, James; Peris, Santiago


    We present a new analysis of $\\alpha_s$ from hadronic $\\tau$ decays based on the recently revised ALEPH data. The analysis is based on a strategy which we previously applied to the OPAL data. We critically compare our strategy to the one traditionally used and comment on the main differences. Our analysis yields the values $\\alpha_s(m_\\tau^2)=0.296\\pm 0.010$ using fixed-order perturbation theory, and $\\alpha_s(m_\\tau^2)=0.310\\pm 0.014$ using contour-improved perturbation theory. Averaging these values with our previously obtained values from the OPAL data, we find $\\alpha_s(m_\\tau^2)=0.303\\pm 0.009$, respectively, $\\alpha_s(m_\\tau^2)=0.319\\pm 0.012$, as the most reliable results for $\\alpha_s$ from $\\tau$ decays currently available.

  17. EEG, alpha waves and coherence (United States)

    Ascolani, Gianluca

    This thesis addresses some theoretical issues generated by the results of recent analysis of EEG time series proving the brain dynamics are driven by abrupt changes making them depart from the ordinary Poisson condition. These changes are renewal, unpredictable and non-ergodic. We refer to them as crucial events. How is it possible that this form of randomness be compatible with the generation of waves, for instance alpha waves, whose observation seems to suggest the opposite view the brain is characterized by surprisingly extended coherence? To shed light into this apparently irretrievable contradiction we propose a model based on a generalized form of Langevin equation under the influence of a periodic stimulus. We assume that there exist two different forms of time, a subjective form compatible with Poisson statistical physical and an objective form that is accessible to experimental observation. The transition from the former to the latter form is determined by the brain dynamics interpreted as emerging from the cooperative interaction among many units that, in the absence of cooperation would generate Poisson fluctuations. We call natural time the brain internal time and we make the assumption that in the natural time representation the time evolution of the EEG variable y(t) is determined by a Langevin equation perturbed by a periodic process that in this time representation is hardly distinguishable from an erratic process. We show that the representation of this random process in the experimental time scale is characterized by a surprisingly extended coherence. We show that this model generates a sequence of damped oscillations with a time behavior that is remarkably similar to that derived from the analysis of real EEG's. The main result of this research work is that the existence of crucial events is not incompatible with the alpha wave coherence. In addition to this important result, we find another result that may help our group, or any other research

  18. Carbohydrate composition of the alpha-subunit of human choriogonadotropin (hCG alpha) and the free alpha molecules produced in pregnancy: most free alpha and some combined hCG alpha molecules are fucosylated. (United States)

    Blithe, D L


    The carbohydrate compositions of pregnancy-derived hCG alpha (dissociated from intact hCG) and free alpha-subunit were analyzed using a combination of chemical analysis, lectin affinity chromatography, and glycosidase sensitivity. For direct compositional analysis, parallel samples were hydrolyzed in trifluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both hCG alpha and free alpha. Free alpha contained 2.5-fold higher amounts of sialic acid and galactose as well as a higher amount of N-acetylglucosamine than did hCG alpha. Free alpha also contained a 6-fold higher amount of fucose than did hCG alpha (1.2 vs. 0.2 residues of fucose/molecule). Serial fractionation of intact hCG alpha and free alpha molecules by lectin affinity chromatography indicated that virtually all of the hCG alpha-subunits contained at least one Concanavalin-A (Con-A)-binding site, whereas as many as 32% of the free alpha molecules could not bind to Con-A. Chromatography on Lens culinaris (Lch) resulted in 12% binding of hCG alpha and approximately 72% binding of free alpha (80-85% of the Con-A-bound free alpha and 47-48% of the Con-A-nonbound free alpha bound to Lch). Endoglycosidase-H (endo-H) treatment of hCG alpha released a portion of the oligosaccharides. The endo-H-released material appeared to be a monoantennary hybrid based on DEAE-binding properties and carbohydrate composition. In contrast to hCG alpha, free alpha was completely resistant to endo-H treatment. Incubation of endo-H-resistant hCG alpha with glycopeptidase-A resulted in the release of two components, which could be separated into monoantennary and biantennary

  19. TNF-alpha and annexin A2: inflammation in thrombotic primary antiphospholipid syndrome. (United States)

    Bećarević, Mirjana


    Antiphospholipid syndrome (APS) is characterized by thromboses and/or pregnancy losses. Laboratory criterion for the diagnosis of APS is the presence of antiphospholipid antibodies (anticardiolipin, anti-beta2-glycoprotein I (aβ2gpI) and lupus anticoagulant). On the one hand, the latest classification criteria for the diagnosis of APS emphasized that thrombotic manifestations of the syndrome should be without any signs of an inflammatory process, while on the other hand, some recent reports have suggested that APS is a "pro-inflammatory state." This article is focused on the importance of TNF-alpha and annexin A2 (anxA2) for patients with vascular (thrombotic) manifestations of the primary APS. The classic antithrombotic and antiplatelet therapy does not protect APS patients from the development of recurrent thrombosis. Therefore, an urgent need for the introduction of new therapeutic approaches in the treatment of APS patients is obvious. This review provides a rationale for the necessity for the use of immunomodulatory medications that could interfere with β2gpI binding to its receptor(s), such as anxA2, and/or inhibit TNF-alpha activity.

  20. Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone. (United States)

    Aussedat, Baptiste; Fasching, Bernhard; Johnston, Eric; Sane, Neeraj; Nagorny, Pavel; Danishefsky, Samuel J


    Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.

  1. Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation. (United States)

    Zeinyeh, Waël; Mahiout, Zahia; Radix, Sylvie; Lomberget, Thierry; Dumoulin, Axel; Barret, Roland; Grenot, Catherine; Rocheblave, Luc; Matera, Eva-Laure; Dumontet, Charles; Walchshofer, Nadia


    Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

  2. Unexpected tolerance of glycosylation by UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferase revealed by electron capture dissociation mass spectrometry: carbohydrate as potential protective groups. (United States)

    Yoshimura, Yayoi; Matsushita, Takahiko; Fujitani, Naoki; Takegawa, Yasuhiro; Fujihira, Haruhiko; Naruchi, Kentarou; Gao, Xiao-Dong; Manri, Naomi; Sakamoto, Takeshi; Kato, Kentaro; Hinou, Hiroshi; Nishimura, Shin-Ichiro


    UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs, EC, a family of key enzymes that initiate posttranslational modification with O-glycans in mucin synthesis by introduction of alpha-GalNAc residues, are structurally composed of a catalytic domain and a lectin domain. It has been known that multiple Ser/Thr residues are assigned in common mucin glycoproteins as potential O-glycosylation sites and more than 20 distinct isoforms of this enzyme family contribute to produce densely O-glycosylated mucin glycoproteins. However, it seems that the functional role of the lectin domain of ppGalNAcTs remains unclear. We considered that electron capture dissociation mass spectrometry (ECD-MS), a promising method for highly selective fragmentation at peptide linkages of glycopeptides to generate unique c and z series of ions, should allow for precise structural characterization to uncover the mechanism in O-glycosylation of mucin peptides by ppGalNAcTs. In the present study, it was demonstrated that a system composed of an electrospray source, a linear RFQ ion trap that isolates precursor ions, the ECD device, and a TOF mass spectrometer is a nice tool to identify the preferential O-glycosylation sites without any decomposition of the carbohydrate moiety. It should be noted that electrons used for ECD are accelerated within a range from 1.75 to 9.75 eV depending on the structures of glycopeptides of interest. We revealed for the first time that additional installation of a alpha-GalNAc residue at potential glycosylation sites by ppGalNAcT2 proceeds smoothly in various unnatural glycopeptides having alpha-Man, alpha-Fuc, and beta-Gal residues as well as alpha-GalNAc residues. The results may suggest that ppGalNAcT2 did not differentiate totally presubstituted sugar residues in terms of configuration of functional groups, d-, l-configuration, and even alpha-, beta-stereochemistry at an anomeric carbon atom when relatively short synthetic


    NARCIS (Netherlands)



    The enzymatic hydrolyses of a variety of alpha-substituted mandelic and lactic esters using pig liver esterase (PLE) have been investigated. High to moderate enantioselectivity was found for various alpha-substituted mandelic esters, whereas PLE showed low to no enantioselectivity for alpha-substitu

  4. Matching coefficients for alpha_s and m_b to O(alpha_s^2) in the MSSM

    CERN Document Server

    Bauer, A; Salomon, J


    We compute the exact two-loop matching coefficients for the strong coupling constant alpha_s and the bottom-quark mass m_b within the Minimal Supersymmetric Standard Model (MSSM), taking into account O(alpha_s^2) contributions from Supersymmetric Quantum Chromodynamics (SQCD). We find that the explicit mass pattern of the supersymmetric particles has a significant impact on the predictions of alpha_s and m_b at high energies. Further on, the three-loop corrections exceed the uncertainty due to the current experimental accuracy. In case of the the running bottom-quark mass, they can reach in the large tan(beta) regime up to 30% from the tree-level value.

  5. Local Varying-Alpha Theories

    CERN Document Server

    Barrow, John D


    In a recent paper we demonstrated how the simplest model for varying alpha may be interpreted as the effect of a dielectric material, generalized to be consistent with Lorentz invariance. Unlike normal dielectrics, such a medium cannot change the speed of light, and its dynamics obey a Klein-Gordon equation. This work immediately suggests an extension of the standard theory, even if we require compliance with Lorentz invariance. Instead of a wave equation, the dynamics may satisfy a local algebraic relation involving the permittivity and the properties of the electromagnetic field, in analogy with more conventional dielectric (but still preserving Lorentz invariance). We develop the formalism for such theories and investigate some phenomenological implications. The problem of the divergence of the classical self-energy can be solved, or at least softened, in this framework. Some interesting new cosmological solutions for the very early universe are found, including the possibility of a bounce, inflation and e...

  6. Alpha Shape Topology of the Cosmic Web

    NARCIS (Netherlands)

    Weygaert, Rien van de; Platen, Erwin; Vegter, Gert; Eldering, Bob; Kruithof, Nico


    We study the topology of the Megaparsec Cosmic Web on the basis of the Alpha Shapes of the galaxy distribution. The simplicial complexes of the alpha shapes are used to determine the set of Betti numbers (βk, k = 1, . . . , D), which represent a complete characterization of the topology of a manifol

  7. The ALPHA detector : Module Production and Assembly

    CERN Document Server

    Andresen, G; Bowe, P D; Cesar, C L; Chapman, S; Charlton, M; Fajans, J; Fujiwara, M C; Gill, D R; Hangst, J S; Hydomako, R; Jenkins, M J; Kurchaninov, L; Madsen, N; Nolan, P; Olchanski, K; Olin, A; Povilus, A; Robicheaux, F; Sarid, E; Silveira, D M; Storey, J W; Thompson, R I; van der Werf, D P; Wurtele, J S; Yamazaki, Y; Ashkezari, M D; Baquero-Ruiz, M; Butler, E; Deller, A; Eriksson, S; Friesen, T; Gutierrez, A; Hardy, W N; Hayden, M E; Humphries, A J; Jonsell, S; McKenna, J T K; Menary, S; Pusa, P; Sampson, J; Seddon, D; Seif el Nasr, S; So, C; Thornhill, J; Wells, D; Jorgensen, L V


    ALPHA is one of the experiments situated at CERN's Antiproton Decelerator (AD). A Silicon Vertex Detector (SVD) is placed to surround the ALPHA atom trap. The main purpose of the SVD is to detect and locate antiproton annihilation events by means of the emitted charged pions. The SVD system is presented with special focus given to the design, fabrication and performance of the modules.

  8. Bayesian Meta-Analysis of Coefficient Alpha (United States)

    Brannick, Michael T.; Zhang, Nanhua


    The current paper describes and illustrates a Bayesian approach to the meta-analysis of coefficient alpha. Alpha is the most commonly used estimate of the reliability or consistency (freedom from measurement error) for educational and psychological measures. The conventional approach to meta-analysis uses inverse variance weights to combine…

  9. Remote Associates Test and Alpha Brain Waves (United States)

    Haarmann, Henk J.; George, Timothy; Smaliy, Alexei; Dien, Joseph


    Previous studies found that performance on the remote associates test (RAT) improves after a period of incubation and that increased alpha brain waves over the right posterior brain predict the emergence of RAT insight solutions. We report an experiment that tested whether increased alpha brain waves during incubation improve RAT performance.…

  10. ALPHA experiment facility and Prof. Jeffrey Hangst.

    CERN Multimedia

    Maximilien Brice


    Picture 01-07: General views of the ALPHA experiment Picture 5: Andrea Gutierrez, a PhD student from UBC, transfers liquid helium from a storage dewar into the cryostat containing the superconducting magnetic trap used by the ALPHA experiment.Picture 08-11: Jeffery Hangst, spokesperson for ALPHA Picture 12: The ALPHA silicon detector, which surrounds the trapping resion and is used for imaging antiproton annihilations (Credit University of Liverpool) Picture 13: Untrapped antihydrogen atoms annihilating on the inner surface of the ALPHA trap. These are measured by the ALPHA annihilation detector. The events are concentrated at the electrode radius of about 22.3 mm. The coordinates are defined in the Nature article, Figure 1b. Picture 14: The electrodes (gold) for the ALPHA Penning trap being inserted into the vacuum chamber and cryostat assembly. This is the trap used to combine or "mix" positrons and antiprotons to make antihydrogen. (Credit: Niels Madsen ALPHA/Swansea.) Picture 15: Top, a diagram of the...

  11. Measurement of the angle alpha at BABAR

    CERN Document Server

    Pérez, A


    We present recent measurements of the CKM angle alpha using data collected by the BABAR detector at the PEP-II asymmetric-energy e^+e^- collider at the SLAC National Accelerator Laboratory, operating at the Upsilon(4S) resonance. We present constraints on alpha from B->pipi, B->rhorho and B->rhopi decays.

  12. Atypical Alpha Asymmetry in Adults with ADHD (United States)

    Hale, T. Sigi; Smalley, Susan L.; Hanada, Grant; Macion, James; McCracken, James T.; McGough, James J.; Loo, Sandra K.


    Introduction: A growing body of literature suggests atypical cerebral asymmetry and interhemispheric interaction in ADHD. A common means of assessing lateralized brain function in clinical populations has been to examine the relative proportion of EEG alpha activity (8-12 Hz) in each hemisphere (i.e., alpha asymmetry). Increased rightward alpha…

  13. Psychiatric Symptoms in Alpha-Mannosidosis (United States)

    Malm, D.; Pantel, J.; Linaker, O. M.


    Alpha-mannosidosis is characterized by mild to moderate intellectual disability (ID), moderate to severe neurosensory hearing loss, frequent infections, psychomotor disturbances and skeletal dysmorphism. For the first time, a panel of nine alpha-mannosidosis patients with psychiatric symptoms is presented. The clinical picture has several…

  14. Confidence Intervals for Cronbach's Coefficient Alpha Values

    NARCIS (Netherlands)

    A.J. Koning (Alex); Ph.H.B.F. Franses (Philip Hans)


    textabstractCoefficient Alpha, which is widely used in empirical research, estimates the reliability of a test consisting of parallel items. In practice it is difficult to compare values of alpha across studies as it depends on the number of items used. In this paper we provide a simple solution, wh

  15. Teaching Calculus with Wolfram|Alpha (United States)

    Dimiceli, Vincent E.; Lang, Andrew S. I. D.; Locke, LeighAnne


    This article describes the benefits and drawbacks of using Wolfram|Alpha as the platform for teaching calculus concepts in the lab setting. It is a result of our experiences designing and creating an entirely new set of labs using Wolfram|Alpha. We present the reasoning behind our transition from using a standard computer algebra system (CAS) to…

  16. Anomalous atomic volume of alpha-Pu

    DEFF Research Database (Denmark)

    Kollar, J.; Vitos, Levente; Skriver, Hans Lomholt


    .3%. The comparison between the LDA and GGA results show that the anomalously large atomic volume of alpha-Pu relative to alpha-Np can be ascribed to exchange-correlation effects connected with the presence of low coordinated sites in the structure where the f electrons are close to the onset of localization...

  17. Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Congjun; Evans, Chheng-Orn [Department of Neurosurgery and Laboratory of Molecular Neurosurgery and Biotechnology, Emory University, School of Medicine, Atlanta, Georgia (United States); Stevens, Victoria L. [Epidemiology and Surveillance Research, American Cancer Society, Atlanta, Georgia (United States); Owens, Timothy R. [Emory University, School of Medicine, Atlanta, Georgia (United States); Oyesiku, Nelson M., E-mail: [Department of Neurosurgery and Laboratory of Molecular Neurosurgery and Biotechnology, Emory University, School of Medicine, Atlanta, Georgia (United States)


    We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

  18. Effect of ammonium chloride and tunicamycin on the glycoprotein content and infectivity of herpes simplex virus type 1

    Energy Technology Data Exchange (ETDEWEB)

    Kousoulas, K.G.; Bzik, D.J.; DeLuca, N.; Person, S.


    Infectious virions of MP, a syncytial strain of herpes simplex virus type 1, are formed in the presence of 50 mM NH/sub 4/Cl. Underglycosylated virion glycoproteins are synthesized in infected cells and are incorporated into virions in the presence of the same concentration of NH/sub 4/Cl. We conclude that fully glycosylated glycoproteins are not required for viral infectivity. Virus particles, deficient in glycosylated glycoproteins, are assembled in the presence of tunicamycin but they are not infectious. The decrease in infectivity could be due to the decreased amount of the gB or possibly other peptides and/or to the lack of the high-mannose saccharides of precursor glycoproteins. 32 references, 4 figures.

  19. Brain Barriers and a Subpopulation of Astroglial Progenitors of Developing Human Forebrain Are Immunostained for the Glycoprotein YKL-40

    DEFF Research Database (Denmark)

    Bjørnbak, Camilla; Brøchner, Christian B; Larsen, Lars A


    YKL-40, a glycoprotein involved in cell differentiation, has been associated with neurodevelopmental disorders, angiogenesis, neuroinflammation and glioblastomas. We evaluated YKL-40 protein distribution in the early human forebrain using double-labeling immunofluorescence and immunohistochemistry...

  20. Use of P-glycoprotein gene probes to investigate anthelmintic resistance in Haemonchus contortus and comparison with Onchocerca volvulus

    NARCIS (Netherlands)

    Kwa, M.S.G.; Okoli, M.N.; Schulz-Key, H.; Okongkwo, P.O.; Roos, M.H.


    A P-glycoprotein gene probe from the sheep parasitic nematode Haemonchus contortus was developed and used to analyse restriction fragment length polymorphisms between susceptible isolates and isolates resistant to either benzimidazole; levamisole and benzimidazole; or benzimidazole, ivermectin and c

  1. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J


    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  2. Alpha macroglobulins and the low-density-lipoprotein-related protein alpha-2-macroglobulin receptor in experimental renal fibrosis

    NARCIS (Netherlands)

    van Goor, H; Diamond, [No Value; Ding, GH; Kaysen, GA


    In this study, we evaluated the location of non-specific proteinase inhibitors and their receptor in experimental glomerular and interstitial fibrosis. The alpha macroglobulins alpha-2-macroglobulin (alpha 2M) and alpha-1-inhibitor 3 (alpha 1I3) are proteinase inhibitors, including metalloproteinase

  3. Radiative corrections to the muonium hyperfine structure; 1, the $\\alpha^{2}$ (Z$\\alpha$) correction

    CERN Document Server

    Kinoshita, T


    This is the first of a series of papers on a systematic application of the NRQED bound state theory of Caswell and Lepage to higher-order radiative corrections to the hyperfine structure of the muonium ground state. This paper describes the calculation of the \\alpha^2 (Z\\alpha) radiative correction. Our result for the complete \\alpha^2 (Z\\alpha) correction is 0.424(4) kHz, which reduces the theoretical uncertainty significantly. The remaining uncertainty is dominated by that of the numerical evaluation of the nonlogarithmic part of the \\alpha (Z\\alpha )^2 term and logarithmic terms of order \\alpha^4. These terms will be treated in the subsequent papers.

  4. The fratricide of alpha-Omega dynamos by their alpha-squared siblings

    CERN Document Server

    Hubbard, Alexander; Brandenburg, Axel


    Context. Helically forced magneto-hydrodynamic shearing-sheet turbulence can support different large-scale dynamo modes, although the {\\alpha} - {\\Omega} mode is generally expected to dominate because it is the fastest growing. In an {\\alpha} - {\\Omega} dynamo, most of the field amplification is produced by the shear. As differential rotation is an ubiquitous source of shear in astrophysics, such dynamos are believed to be the source of most astrophysical large-scale magnetic fields. Aims. We study the stability of oscillatory migratory {\\alpha} - {\\Omega} type dynamos in turbulence simulations. Methods. We use shearing-sheet simulations of hydromagnetic turbulence that is helically forced at a wavenumber that is about three times larger than the lowest wavenumber in the domain so that both {\\alpha} - {\\Omega} and {\\alpha}2 dynamo action is possible. Results. After initial dominance and saturation, the {\\alpha} - {\\Omega} mode is found to be destroyed by an orthogonal {\\alpha}2 mode sustained by the helical t...

  5. Mobilities of the inner three core residues and the Man(alpha 1--6) branch of the glycan at Asn78 of the alpha-subunit of human chorionic gonadotropin are restricted by the protein. (United States)

    van Zuylen, C W; de Beer, T; Leeflang, B R; Boelens, R; Kaptein, R; Kamerling, J P; Vliegenthart, J F


    Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone involved in the maintenance of the corpus luteum in early pregnancy. The free alpha-subunit of hCG has a biological activity of its own, namely, stimulation of prolactin secretion from term pregnancy decidual cells [Blithe, D. L., et al. (1991) Endocrinology 129, 2257-2259]. Glycosylation at Asn78 of the alpha-subunit is required for the stability of the protein, but the exact nature of the stabilizing effect is not known. In our previous study, it was indicated that GlcNAc-1 at Asn78 has a reduced mobility, whereas the glycan at Asn52 is highly mobile [De Beer, T., et al. (1996) Eur. J. Biochem. 241, 229-242]. In the present investigation, it is shown that the PNGase F susceptibility of the Asn52-linked glycan in the free alpha-subunit is absent in the heterodimer. Thus, the high mobility of the glycan at Asn52 may be characteristic for the free alpha-subunit. For accurate modeling of alpha hCG, knowledge of the behavior of each of the glycans is essential. In this context, the mobility of the glycans and their interactions with the protein are explored by NMR spectroscopy using desialylated, partially deglycosylated free alpha-subunit (as-pd alpha) carrying glycans at Asn78 only. NOEs between GlcNAc-2 and several amino acid residues indicate that GlcNAc-2 is involved in stabilizing alpha hCG. From the values of 13C relaxation parameters T2 and T1 rho of the constituting monosaccharide residues, it was concluded that the inner three residues have a severely restricted mobility. The Man-4 and Man-4' residues of the diantennary oligosaccharide exhibit a similar relaxation behavior, suggesting that the Man-4' branch occurs in a single conformation of the C5-C6 linkage of Man-3 instead of in rapidly interconverting conformations that are known to exist for this linkage for the free oligosaccharide.

  6. The Laminin 511/521 Binding Site on the Lutheran Blood Group Glycoprotein is Located at theFlexible Junction of Ig Domains 2 and 3

    Energy Technology Data Exchange (ETDEWEB)

    Mankelow, Tosti J.; Burton, Nicholas; Stedansdottir, Fanney O.; Spring, Frances A.; Parsons, Stephen F.; Pesersen, Jan S.; Oliveira, Cristiano L.P.; Lammie, Donna; Wess, Timothy; Mohandas, Narla; Chasis, Joel A.; Brady, R. Leo; Anstee, David J.


    The Lutheran blood group glycoprotein, first discovered on erythrocytes, is widely expressed in human tissues. It is a ligand for the {alpha}5 subunit of Laminin 511/521, an extracellular matrix protein. This interaction may contribute to vasocclusive events that are an important cause of morbidity in sickle cell disease. Using X-ray crystallography, small angle X-ray scattering and site directed mutagenesis we show that the extracellular region of Lutheran forms an extended structure with a distinctive bend between the second and third immunoglobulin-like domains. The linker between domains 2 and 3 appears to be flexible and is a critical determinant in maintaining an overall conformation for Lutheran that is capable of binding to Laminin. Mutagenesis studies indicate that Asp312 of Lutheran and the surrounding cluster of negatively charged residues in this linker region form the Laminin binding site. Unusually, receptor binding is therefore not a function of the domains expected to be furthermost from the plasma membrane. These studies imply that structural flexibility of Lutheran may be essential for its interaction with Laminin and present a novel opportunity for the development of therapeutics for sickle cell disease.

  7. Localization of the glycoprotein Cb42 in larvae of the screwworm fly Chrysomya bezziana (Diptera: Calliphoridae

    Directory of Open Access Journals (Sweden)

    Creig Eisemanni


    Full Text Available The glycoprotein Cb-peritrophin-42 was localized in third instar larvae of Chrysomya bezziana using primary antibodies raised against a non-glycosylated bacterial recombinant form of this protein. Both immunofluorescent antibody techniques on unfixed whole mounts of gut tissues and immunogold electron microscopy techniques on ultra-thin sections of fixed and embedded tissues were employed. The protein was shown to be exposed over the whole of both surfaces of the peritrophic membrane and to occur throughout its thickness. Immunogold labelling indicated that Cb-peritrophin-42 was expressed in the peritrophic membrane-secreting cells of the cardia, a specialized peritrophic membrane-forming organ situated at the junction of the foregut and midgut. The accessibility of Cb-peritrophin-42 present in intact peritrophic membrane to the primary antibodies used in the immunofluorescent antibody localization indicates that this glycoprotein is a potential molecular target for vaccination of host animals against larvae of Chrysomya bezziana.

  8. Partial Characterization of a Vicilin-Like Glycoprotein from Seeds of Flowering Tobacco (Nicotiana sylvestris

    Directory of Open Access Journals (Sweden)

    Jared Q. Gerlach


    Full Text Available A vicilin-like glycoprotein from the seeds of Nicotiana sylvestris, flowering tobacco, has been identified using nanoLC/ESI-MS/MS. Sequences from a fragment of protein demonstrated homology with vicilins from other members of the Solanaceae family, notably potato (Solanum demissum. Reducing and nonreducing SDS-PAGE analyses of the identified protein indicated that fragments resulting from in situ proteolytic processing are joined by intrachain disulphide bonds. Staining with Con A lectin was specifically inhibited by mannose suggested the presence of -linked glycosylation which was confirmed by carbohydrate compositional analysis of PVDF-bound protein subunits. HPAEC-PAD analysis of the monosaccharides released from the glycoprotein by acid hydrolysis revealed glucosamine and mannose. -acetylglucosamine termination of attached oligosaccharides was further verified by inhibitable WGA lectin staining. Immunostaining of PVDF-bound N. sylvestris proteins with antibodies against G. max total protein demonstrated cross-staining at masses corresponding to fragments from the proteolytically processed protein subunits.

  9. Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix

    DEFF Research Database (Denmark)

    Hogan, B L; Taylor, A; Kurkinen, M;


    Two sulphated glycoproteins (sgps) of apparent molecular weight (Mr) 180,000 and 150,000, are synthesized by murine PYS and PF HR9 parietal endoderm and Swiss 3T3 cells. The Mr 150,000 sgp has a similar chemical structure to the sulphated glycoprotein, C, synthesized and laid down in Reichert......'s membrane by mouse embryo parietal endoderm cells (Hogan, B. L.M., A. Taylor, and A.R. Cooper, 1982, Dev. Biol., 90:210-214). Both the Mr 180,000 and 150,000 sgps are deposited in the detergent-insoluble matrix of cultured cells, but they do not apparently undergo any disulphide-dependent intermolecular...

  10. Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

    Directory of Open Access Journals (Sweden)

    José L. Affranchino


    Full Text Available 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.

  11. Recent advances in the study of active endogenous retrovirus envelope glycoproteins in the mammalian placenta

    Institute of Scientific and Technical Information of China (English)

    Yufei; Zhang; Jing; Shi; Shuying; Liu


    Endogenous retroviruses(ERVs) are a component of the vertebrate genome and originate from exogenous infections of retroviruses in the germline of the host. ERVs have coevolved with their hosts over millions of years. Envelope glycoproteins of endogenous retroviruses are often expressed in the mammalian placenta, and their potential function has aroused considerable research interest, including the manipulation of maternal physiology to benefit the fetus. In most mammalian species, trophoblast fusion in the placenta is an important event, involving the formation of a multinucleated syncytiotrophoblast layer to fulfill essential fetomaternal exchange functions. The key function in this process derives from the envelope genes of endogenous retroviruses, namely syncytins, which show fusogenic properties and placenta-specific expression. This review discusses the important role of the recognized endogenous retrovirus envelope glycoproteins in the mammalian placenta.

  12. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology. (United States)

    Chua, Chong Long; Sam, I-Ching; Chan, Yoke Fun


    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection.

  13. [Contents and its change during storage of alpha-solanine and alpha-chaconine in potatoes]. (United States)

    Shindo, Tetsuya; Ushiyama, Hirofumi; Kan, Kimiko; Yasuda, Kazuo; Saito, Kazuo


    Contents of alpha-solanine and alpha-chaconine in native species of potato (May Queen, Danshaku and Waseshiro), and in species (Jagakids Red '90 (Red) and Jagakids Purple '90 (Purple)) on the market, and their change during storage at room temparature were investigated. alpha-Solanine and alpha-chaconine were extracted from potatoes with methanol, cleaned up by using a Sep-Pak Plus C18 cartridge, and then subjected to HPLC. The recoveries of alpha-solanine and alpha-chaconine from potatoes were both more than 96%, and the quantitation limits were both 2 microg/g. alpha-Solanine and alpha-chaconine were detected in periderm in all samples at the levels of 260-320 microg/g in May Queen,190-240 microg/g in Danshaku, 43-63 microg/g in Waseshiro, 140-200 microg/g in Red and 84-130 microg/g in Purple, respectively. alpha-Solanine and alpha-chaconine were detected in the cortex in all samples of May Queen and Danshaku at the levels of 2.7-12 microg/g and 5.8-31 microg/g, respectively. Contents of alpha-solanine and alpha-chaconine in the cortex of May Queen and Danshaku were less than 10% of those in the periderm. When potatoes were stored for 90 days at room temparature in a dark place, no marked change in the contents of alpha-solanine and alpha-chaconine was observed in any of the potato samples.

  14. Involvement of central alpha1-adrenoceptors on renal responses to central moxonidine and alpha-methylnoradrenaline. (United States)

    de Andrade, Carina A F; de Andrade, Glaucia M F; De Paula, Patricia M; De Luca, Laurival A; Menani, José V


    Moxonidine (alpha2-adrenoceptor/imidazoline receptor agonist) injected into the lateral ventricle induces diuresis, natriuresis and renal vasodilation. Moxonidine-induced diuresis and natriuresis depend on central imidazoline receptors, while central alpha1-adrenoceptors are involved in renal vasodilation. However, the involvement of central alpha1-adrenoceptors on diuresis and natriuresis to central moxonidine was not investigated yet. In the present study, the effects of moxonidine, alpha-methylnoradrenaline (alpha2-adrenoceptor agonist) or phenylephrine (alpha1-adrenoceptor agonist) alone or combined with previous injections of prazosin (alpha1-adrenoceptor antagonist), yohimbine or RX 821002 (alpha2-adrenoceptor antagonists) intracerebroventricularly (i.c.v.) on urinary sodium, potassium and volume were investigated. Male Holtzman rats (n = 5-18/group) with stainless steel cannula implanted into the lateral ventricle and submitted to gastric water load (10% of body weight) were used. Injections of moxonidine (20 nmol) or alpha-methylnoradrenaline (80 nmol) i.c.v. induced natriuresis (196 +/- 25 and 171 +/- 30, respectively, vs. vehicle: 101 +/- 9 microEq/2 h) and diuresis (9.0 +/- 0.4 and 12.3 +/- 1.6, respectively, vs. vehicle: 5.2 +/- 0.5 ml/2 h). Pre-treatment with prazosin (320 nmol) i.c.v. abolished the natriuresis (23 +/- 4 and 76 +/- 11 microEq/2 h, respectively) and diuresis (5 +/- 1 and 7.6 +/- 0.8 ml/2 h, respectively) produced by i.c.v. moxonidine or alpha-methylnoradrenaline. RX 821002 (320 nmol) i.c.v. abolished the natriuretic effect of alpha-methylnoradrenaline, however, yohimbine (320 nmol) did not change renal responses to moxonidine. Phenylephrine (80 nmol) i.c.v. induced natriuresis and kaliuresis that were blocked by prazosin. Therefore, the present data suggest that moxonidine and alpha-methylnoradrenaline acting on central imidazoline receptors and alpha2-adrenoceptors, respectively, activate central alpha1-adrenergic mechanisms to

  15. Mapping High-Velocity H-alpha and Lyman-alpha Emission from Supernova 1987A (United States)

    France, Kevin; McCray, Richard; Fransson, Claes; Larsson, Josefin; Frank, Kari A.; Burrows, David N.; Challis, Peter; Kirshner, Robert P.; Chevalier, Roger A.; Garnavich, Peter; Heng, Kevin; Lawrence, Stephen S.; Lundqvist, Peter; Smith, Nathan; Sonneborn, George


    We present new Hubble Space Telescope images of high-velocity H-alpha and Lyman-alpha emission in the outer debris of SN 1987A. The H-alpha images are dominated by emission from hydrogen atoms crossing the reverse shock. For the first time we observe emission from the reverse shock surface well above and below the equatorial ring, suggesting a bipolar or conical structure perpendicular to the ring plane. Using the H-alpha imaging, we measure the mass flux of hydrogen atoms crossing the reverse shock front, in the velocity intervals (-7,500 < V(sub obs) < -2,800 km/s) and (1,000 < V(sub obs) < 7,500 km/s), ?M(sub H) = 1.2 × 10(exp -3) M/ y. We also present the first Lyman-alpha imaging of the whole remnant and new Chandra X-ray observations. Comparing the spatial distribution of the Lyman-alpha and X-ray emission, we observe that the majority of the high-velocity Lyman-alpha emission originates interior to the equatorial ring. The observed Lyman-alpha/H-alpha photon ratio, R(L-alpha/H-alpha) approx. = 17, is significantly higher than the theoretically predicted ratio of approx. = 5 for neutral atoms crossing the reverse shock front. We attribute this excess to Lyman-alpha emission produced by X-ray heating of the outer debris. The spatial orientation of the Lyman-alpha and X-ray emission suggests that X-ray heating of the outer debris is the dominant Lyman-alpha production mechanism in SN 1987A at this phase in its evolution.

  16. Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets. (United States)

    Artemenko, Elena O; Yakimenko, Alena O; Pichugin, Alexey V; Ataullakhanov, Fazly I; Panteleev, Mikhail A


    In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbβ3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.

  17. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity.


    Stanley, P.


    The production of glycoproteins with carbohydrates of defined structure and minimal heterogeneity is important for functional studies of mammalian carbohydrates. To facilitate such studies, several Chinese hamster ovary mutants that carry between two and four glycosylation mutations were developed. All of the lines grew readily in culture despite the drastic simplification of their surface carbohydrates. Therefore, both endogenous glycoproteins and those introduced by transfection can be obta...

  18. P-glycoprotein alters blood–brain barrier penetration of antiepileptic drugs in rats with medically intractable epilepsy

    Directory of Open Access Journals (Sweden)

    Ma A


    Full Text Available Aimei Ma,1,* Cuicui Wang,2,3,* Yinghui Chen,2,3 Weien Yuan4 1Department of Neurology, The People's Hospital of Shanxi Province, Taiyuan, 2Department of Neurology, Jinshan Hospital, Fudan University, 3Department of Neurology, Shanghai Medical College, Shanghai, 4School of Pharmacy, Shanghai JiaoTong University, Shanghai, People's Republic of China *These authors contributed equally to this work Abstract: P-glycoprotein is one of the earliest known multidrug transporters and plays an important role in resistance to chemotherapeutic drugs. In this study, we detected levels of P-glycoprotein and its mRNA expression in a rat brain model of medically intractable epilepsy established by amygdala kindling and drug selection. We investigated whether inhibition of P-glycoprotein affects the concentration of antiepileptic drugs in cortical extracellular fluid. We found that levels of P-glycoprotein and its mRNA expression were upregulated in epileptic cerebral tissue compared with cerebral tissue from normal rats. The concentrations of two antiepileptic drugs, carbamazepine and phenytoin, were very low in the cortical extracellular fluid of rats with medically intractable epilepsy, and were restored after blockade of P-glycoprotein by verapamil. These results show that increased P-glycoprotein levels alter the ability of carbamazepine and phenytoin to penetrate the blood–brain barrier and reduce the concentrations of these agents in extracellular cortical fluid. High P-glycoprotein levels may be involved in resistance to antiepileptic drugs in medically intractable epilepsy. Keywords: P-glycoprotein, medically intractable epilepsy, antiepileptic drugs, amygdala kindling, verapamil

  19. [Influence of low-intensity laser radiation on the formation of liquid crystalline structures in a solution of glycoproteins]. (United States)

    Skopinov, S A; Iakovleva, S V; Denisova, E A; Vazina, A A; Zheleznaia, L A


    Liquid-crystalline structure formation in glycoprotein solutions irradiated by helium-neon laser in the presence of hydrogen peroxide was observed by both polarizing microscopy and spectrophotometry. High molecular weight (2.10(6) Da) and heavily glycosylated (about 80%) glycoprotein was isolated from the mucus layer of pig small intestine. Remarkable changes of both optic parameters of the solutions and the morphology of liquid-crystalline structures were detected in irradiated samples compared to the non-irradiated ones.

  20. Immunization with Cytomegalovirus Envelope Glycoprotein M and Glycoprotein N DNA Vaccines can Provide Mice with Complete Protection against a Lethal Murine Cytomegalovirus Challenge

    Institute of Scientific and Technical Information of China (English)

    Huadong Wang; Yanfeng Yao; Chaoyang Huang; Quanjiao Chen; Jianjun Chen; Ze Chen


    Human cytomegalovirus virions contain three major glycoprotein complexes (gC Ⅰ,Ⅱ,Ⅲ),all of which are required for CMV infectivity.These complexes also represent major antigenic targets for anti-viral immune responses.The gC Ⅱ complex consists of two glycoproteins,gM and gN.In the current study,DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model.Humoral and cellular immune responses,spleen viral titers,and mice survival and body-weight changes were examined.The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection,whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response,and provided mice with complete protection against a lethal MCMV challenge.This study provides the first in vivo evidence that the gC Ⅱ (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.