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Sample records for human glycoprotein complex

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

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    Francesco Renzi

    2011-06-01

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

  2. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

    DEFF Research Database (Denmark)

    Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

    2012-01-01

    Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable......, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human m......Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81...

  3. Random mutagenesis and screening of complex glycoproteins : expression of human gonadotropins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Linskens, Maarten H.K.; Grootenhuis, Peter D.J.; Blaauw, Mieke; Huisman-de Winkel, Bianca; Ravestein, Arno van; Haastert, Peter J.M. van; Heikoop, Judith C.

    1999-01-01

    The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism, Furthermore, both follicle stimulation hormone and choriogona

  4. Chemical synthesis of a glycoprotein having an intact human complex-type sialyloligosaccharide under the Boc and Fmoc synthetic strategies.

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    Yamamoto, Naoki; Tanabe, Yasutaka; Okamoto, Ryo; Dawson, Philip E; Kajihara, Yasuhiro

    2008-01-16

    The chemical synthesis of complex glycoproteins is an ongoing challenge in protein chemistry. We have examined the synthesis of a single glycoform of monocyte chemotactic protein-3 (MCP-3), a CC-chemokine that consists of 76 amino acids and one N-glycosylation site. A three-segment native chemical ligation strategy was employed using unprotected peptides and glycopeptide. Importantly, the synthesis required the development of methods for the generation of sialylglycopeptide-alphathioesters. For the sialylglycopeptide-alphathioester segment, we examined and successfully implemented approaches using Fmoc-SPPS and Boc-SPPS. To avoid use of hydrogen fluoride, the Boc approach utilized minimal side chain protection and direct thiolysis of the resin bound peptide. Using these strategies, we successfully synthesized a glycoprotein having an intact and homogeneous complex-type sialyloligosaccharide.

  5. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets.

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    Ezumi, Y; Shindoh, K; Tsuji, M; Takayama, H

    1998-07-20

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRgamma, Syk, and PLCgamma2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcRgamma complex.

  6. Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets

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    Ezumi, Yasuharu; Shindoh, Keisuke; Tsuji, Masaaki; Takayama, Hiroshi

    1998-01-01

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex. PMID:9670039

  7. 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.

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    Millot, M C; Hervé, F; Sébille, B

    1995-02-03

    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.

  8. Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone.

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    Aussedat, Baptiste; Fasching, Bernhard; Johnston, Eric; Sane, Neeraj; Nagorny, Pavel; Danishefsky, Samuel J

    2012-02-22

    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.

  9. Role of zona pellucida glycoproteins during fertilization in humans.

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    Gupta, Satish Kumar

    2015-04-01

    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. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

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    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  11. Optimization of human immunodeficiency virus type 1 envelope glycoproteins with V1/V2 deleted, using virus evolution

    NARCIS (Netherlands)

    Bontjer, I.; Land, A.; Eggink, D.; Verkade, E.; Tuin, K.; Baldwin, C.; Pollakis, G.; Paxton, W.A.; Braakman, L.J.; Berkhout, B.; Sanders, R.W.

    2009-01-01

    The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have prove

  12. β2-Glycoprotein I/HLA class II complexes are novel autoantigens in antiphospholipid syndrome.

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    Tanimura, Kenji; Jin, Hui; Suenaga, Tadahiro; Morikami, Satoko; Arase, Noriko; Kishida, Kazuki; Hirayasu, Kouyuki; Kohyama, Masako; Ebina, Yasuhiko; Yasuda, Shinsuke; Horita, Tetsuya; Takasugi, Kiyoshi; Ohmura, Koichiro; Yamamoto, Ken; Katayama, Ichiro; Sasazuki, Takehiko; Lanier, Lewis L; Atsumi, Tatsuya; Yamada, Hideto; Arase, Hisashi

    2015-04-30

    Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy complications. β2-glycoprotein I (β2GPI) complexed with phospholipid is recognized as a major target for autoantibodies in APS; however, less than half the patients with clinical manifestations of APS possess autoantibodies against the complexes. Therefore, the range of autoantigens involved in APS remains unclear. Recently, we found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins to the cell surface via association with their peptide-binding grooves. Furthermore, immunoglobulin G heavy chain/HLA class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we demonstrate that intact β2GPI, not peptide, forms a complex with HLA class II molecules. Strikingly, 100 (83.3%) of the 120 APS patients analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize β2GPI/HLA class II complexes in the absence of phospholipids. In situ association between β2GPI and HLA class II was observed in placental tissues of APS patients but not in healthy controls. Furthermore, autoantibodies against β2GPI/HLA class II complexes mediated complement-dependent cytotoxicity against cells expressing the complexes. These data suggest that β2GPI/HLA class II complexes are a target in APS that might be involved in the pathogenesis.

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

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    Lefièvre, L; Conner, S J; Salpekar, A; Olufowobi, O; Ashton, P; Pavlovic, B; Lenton, W; Afnan, M; Brewis, I A; Monk, M; Hughes, D C; Barratt, C L R

    2004-07-01

    The zona pellucida (ZP) is an extracellular glycoprotein matrix which surrounds all mammalian oocytes. Recent data have shown the presence of four human zona genes (ZP1, ZP2, ZP3 and ZPB). The aim of the study was to determine if all four ZP proteins are expressed and present in the human. cDNA derived from human oocytes were used to amplify by PCR the four ZP genes. In addition, isolated native human ZP were heat-solubilized, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). All four genes were expressed and the respective proteins present in the human ZP. Moreover, a bioinformatics approach showed that the mouse ZPB gene, although present, is likely to encode a non-functional protein. Four ZP genes are expressed in human oocytes (ZP1, ZP2, ZP3 and ZPB) and preliminary data show that the four corresponding ZP proteins are present in the human ZP. Therefore, this is a fundamental difference with the mouse model

  14. Crystal Structure of the Human Cytomegalovirus Glycoprotein B.

    Directory of Open Access Journals (Sweden)

    Heidi G Burke

    2015-10-01

    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.

  15. Uncoupling GP1 and GP2 expression in the Lassa virus glycoprotein complex: implications for GP1 ectodomain shedding

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    Illick Kerry A

    2008-12-01

    Full Text Available Abstract Background Sera from convalescent Lassa fever patients often contains antibodies to Lassa virus (LASV glycoprotein 1 (GP1, and glycoprotein 2 (GP2; Immunization of non-human primates with viral vectors expressing the arenaviral glycoprotein complex (GPC confers full protective immunity against a lethal challenge with LASV. Thus, the development of native or quasi native recombinant LASV GP1 and GP2 as soluble, uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever. To this end, mammalian expression systems were engineered for production and purification of secreted forms of soluble LASV GP1 and GP2 proteins. Results Determinants for mammalian cell expression of secreted uncoupled Lassa virus (LASV glycoprotein 1 (GP1 and glycoprotein 2 (GP2 were established. Soluble GP1 was generated using either the native glycoprotein precursor (GPC signal peptide (SP or human IgG signal sequences (s.s.. GP2 was secreted from cells only when (1 the transmembrane (TM domain was deleted, the intracellular domain (IC was fused to the ectodomain, and the gene was co-expressed with a complete GP1 gene in cis; (2 the TM and IC domains were deleted and GP1 was co-expressed in cis; (3 expression of GP1 was driven by the native GPC SP. These data implicate GP1 as a chaperone for processing and shuttling GP2 to the cell surface. The soluble forms of GP1 and GP2 generated through these studies were secreted as homogeneously glycosylated proteins that contained high mannose glycans. Furthermore, observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC represents a novel aspect of arenaviral glycoprotein expression. Conclusion These results implicate GP1 as a chaperone for the correct processing and shuttling of GP2 to the cell surface, and suggest that native GPC SP plays a role in this process. In the absence of GP1 and GPC SP the GP2 protein may be processed by an alternate pathway that produces

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

    Science.gov (United States)

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

    2010-12-28

    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.

  17. 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: fufengliu@tju.edu.cn, E-mail: ysun@tju.edu.cn; Dong, Xiao-Yan; Sun, Yan, E-mail: fufengliu@tju.edu.cn, E-mail: ysun@tju.edu.cn [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)

    2013-12-14

    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.

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

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

    2013-12-01

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

  19. Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry

    Science.gov (United States)

    2011-07-01

    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. Characterization of phosphine complexes of technetium(III) as transport substrates of the multidrug resistance P-glycoprotein and functional markers of P-glycoprotein at the blood-brain barrier.

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    Luker, G D; Rao, V V; Crankshaw, C L; Dahlheimer, J; Piwnica-Worms, D

    1997-11-18

    The multidrug resistance (MDR1) P-glycoprotein functions as a broad specificity efflux transporter of structurally diverse natural product and xenobiotic compounds. P-glycoprotein also is an important component of the functional blood-brain barrier. To enable further studies of function and modulation of MDR1 P-glycoprotein in vitro and in vivo, two novel phosphine technetium(III) complexes were designed and characterized: trans-[2,2'-(1, 2-ethanediyldiimino)bis(1, 5-methoxy-5-methyl-4-oxo-hexenyl)]bis[methylbis(3-methoxy-1- propyl)ph osphine]Tc(III) (Tc-Q58) and trans-[5,5'-(1,2-ethanediyl diimino)bis(2-ethoxy-2-methyl-3-oxo-4-pentenyl)]bis[dimethyl(3- methox y-1-propyl)phosphine)]Tc(III) (Tc-Q63). In human drug-sensitive KB 3-1 cells and multidrug-resistant KB 8-5 and 8-5-11 derivative cell lines, expressing nonimmunodetectable, low, and high levels of MDR1 P-glycoprotein, respectively, accumulation of Tc-Q58 and Tc-Q63 was inverse to expression of the transporter. Differences between drug-sensitive and multidrug-resistant cells, while detectable at picomolar concentrations of each radiopharmaceutical, were independent of tracer concentration. Ratios of tracer accumulation in KB 3-1 and 8-5 cells were 62.3 and 48.1 for Tc-Q58 and Tc-Q63, respectively. Cell contents of Tc-Q58 and Tc-Q63 were enhanced up to 60-fold in MDR cells by known modulators of MDR1 P-glycoprotein, while drugs not in the multidrug-resistant phenotype had no effect on their accumulation. In KB 8-5 cells, potency of modulators was GF120918 > cyclosporin A > verapamil. Accumulation of Tc-Q58 and Tc-Q63 in Sf9 insect cells infected with a recombinant baculovirus containing human MDR1 P-glycoprotein was reduced in a GF120918-reversible manner (EC50 phosphine-containing metal complexes. As shown with Tc-Q58, these Q complexes can be used to detect transport activity and modulation of MDR1 P-glycoprotein in vitro and to directly monitor the functional status of P-glycoprotein at the blood

  1. Demethoxycurcumin modulates human P-glycoprotein function via uncompetitive inhibition of ATPase hydrolysis activity.

    Science.gov (United States)

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

    2015-01-28

    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.

  2. Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays.

    Science.gov (United States)

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

    2016-03-15

    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.

  3. 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.

    2006-01-01

    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.

  4. The Dystrophin-Glycoprotein Complex in the Prevention of Muscle Damage

    OpenAIRE

    2011-01-01

    Muscular dystrophies are genetically diverse but share common phenotypic features of muscle weakness, degeneration, and progressive decline in muscle function. Previous work has focused on understanding how disruptions in the dystrophin-glycoprotein complex result in muscular dystrophy, supporting a hypothesis that the muscle sarcolemma is fragile and susceptible to contraction-induced injury in multiple forms of dystrophy. Although benign in healthy muscle, contractions in dystrophic muscle ...

  5. Expression of dystrophin-glycoprotein complex at the skeletal muscle sarcolemma in Duchenne muscular dystrophy

    OpenAIRE

    Zhao, Lei; Chao-ping HU; Wang, Yi; Shui-zhen ZHOU; Shi, Yi-Yun; Xi-hua LI

    2015-01-01

    Background  Eccentric exercise or high tension exercise could cause damage to skeletal muscle structure, resulting in deficiency of dystrophin and secondary loss of dystrophin-glycoprotein complex (DGC) from the sarcolemma, which indicated that down-regulation of dystrophin was one of the key points of skeletal muscle injury from eccentric exercise. Duchenne muscular dystrophy (DMD) is caused by mutations of DMD gene, resulting in the absence of dystrophin, which means that skeletal muscles o...

  6. Electrophoretic demonstration of glycoproteins, lipoproteins, and phosphoproteins in human and bovine enamel

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D; Bøg-Hansen, T C;

    1990-01-01

    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....

  7. The human P-glycoprotein transporter enhances the type I interferon response to Listeria monocytogenes infection.

    Science.gov (United States)

    Sigal, Nadejda; Kaplan Zeevi, Millie; Weinstein, Shiri; Peer, Dan; Herskovits, Anat A

    2015-06-01

    Human multidrug efflux transporters are known for their ability to extrude antibiotics and toxic compounds out of cells, yet accumulating data indicate they have additional functions in diverse physiological processes not related to drug efflux. Here, we show that the human multidrug transporter P-glycoprotein (P-gp) (also named MDR1 and ABCB1) is transcriptionally induced in the monocytic cell line THP-1 upon infection with the human intracellular bacterial pathogen Listeria monocytogenes. Notably, we found that P-gp is important for full activation of the type I interferon response elicited against L. monocytogenes bacteria. Both inhibition of P-gp function by verapamil and inhibition of its transcription using mRNA silencing led to a reduction in the magnitude of the type I response in infected cells. This function of P-gp was specific to type I interferon cytokines elicited against cytosolic replicating bacteria and was not observed in response to cyclic di-AMP (c-di-AMP), a molecule that was shown to be secreted by L. monocytogenes during infection and to trigger type I interferons. Moreover, P-gp was not involved in activation of other proinflammatory cytokines, such as those triggered by vacuolar-restricted L. monocytogenes or lipopolysaccharide (LPS). Taken together, these findings demonstrate a role for P-gp in proper development of an innate immune response against intracellular pathogens, highlighting the complexity in employing therapeutic strategies that involve inhibition of multidrug resistance (MDR) efflux pumps.

  8. A transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins through N-glycosylation.

    Science.gov (United States)

    Hu, Jia-Biao; Zhang, Peng; Wang, Mei-Xian; Zhou, Fang; Niu, Yan-Shan; Miao, Yun-Gen

    2012-08-01

    Glycoproteins have been implicated in a wide variety of important biochemical and biological functions, including protein stability, immune function, enzymatic function, cellular adhesion and others. Unfortunately, there is no therapeutic protein produced in insect system to date, due to the expressed glycoproteins are paucimannosidic N-glycans, rather than the complex, terminally sialylated N-glycans in mammalian cells. In this paper, we cloned the necessary genes in glycosylation of mammalian cells, such as N-acetylglucosaminyltransferase II (Gn-TII), galactosyltransferases (Gal-Ts), 2,6-Sial-T (ST6 GalII)and 2,3-Sial-T (ST3GalIII), and transformed them to silkworm genome of BmN cell line through transgenesis to establish a transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins. The study supplied a new insect cell line which is practically to produce "bisected" complex N-glycans like in mammalian cells.

  9. Modification-specific proteomic analysis of glycoproteins in human body fluids by mass spectrometry

    DEFF Research Database (Denmark)

    Bunkenborg, Jakob; Hägglund, Per; Jensen, Ole Nørregaard

    2007-01-01

    -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...

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

    NARCIS (Netherlands)

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

    1998-01-01

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

  11. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D. (NIH)

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  12. Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers.

    OpenAIRE

    Rey, M A; Laurent, A G; McClure, J; Krust, B; Montagnier, L; Hovanessian, A G

    1990-01-01

    An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of...

  13. Glycoproteins of the carcinoembryonic antigen (CEA) family are expressed in sweat and sebaceous glands of human fetal and adult skin.

    Science.gov (United States)

    Metze, D; Bhardwaj, R; Amann, U; Eades-Perner, A M; Neumaier, M; Wagener, C; Jantscheff, P; Grunert, F; Luger, T A

    1996-01-01

    The carcinoembryonic antigen (CEA) family comprises a group of glycoproteins including the classical CEA, nonspecific cross-reacting antigens (NCA), and biliary glycoprotein (BGP). CEA glycoproteins have been identified in many glandular and mucosal tissues. In view of their putative role in cell adhesion, protein sorting, and signal transduction, CEA glycoproteins are thought to be involved in embryogenesis, architectual integrity, and secretory mechanisms of glandular epithelia. Since there are few data available on the expression of CEA-like proteins in human skin, the aim of this study was to immunohistochemically specify and localize the CEA glycoproteins in cutaneous adult and fetal glands using a panel of well-characterized antibodies. The secretory parts of eccrine sweat glands expressed CEA, NCA-90, and BGP, whereas apocrine glands remained unreactive for CEA glycoproteins. The ductal epithelia of both eccrine and apocrine glands contained CEA and NCA-90. Sebaceous glands were stained for BGP only. Electron microscopy of sweat glands showed CEA glycoprotein expression in cytoplasmic organelles and on microvilli lining the ductal surface. In sebaceous glands, BGP were demonstrated in small vesicles and along the cell membranes of differentiating sebocytes. Fetal development of cutaneous glands was associated with early expression of CEA glycoproteins. Additionally, mice transgenic for human CEA were shown to express CEA in sweat glands. The overall distribution of CEA glycoproteins in cutaneous glands was consistent with that in epithelia of other glandular tissues.

  14. The Dystrophin-Glycoprotein Complex in the Prevention of Muscle Damage

    Directory of Open Access Journals (Sweden)

    Jessica D. Gumerson

    2011-01-01

    Full Text Available Muscular dystrophies are genetically diverse but share common phenotypic features of muscle weakness, degeneration, and progressive decline in muscle function. Previous work has focused on understanding how disruptions in the dystrophin-glycoprotein complex result in muscular dystrophy, supporting a hypothesis that the muscle sarcolemma is fragile and susceptible to contraction-induced injury in multiple forms of dystrophy. Although benign in healthy muscle, contractions in dystrophic muscle may contribute to a higher degree of muscle damage which eventually overwhelms muscle regeneration capacity. While increased susceptibility of muscle to mechanical injury is thought to be an important contributor to disease pathology, it is becoming clear that not all DGC-associated diseases share this supposed hallmark feature. This paper outlines experimental support for a function of the DGC in preventing muscle damage and examines the evidence that supports novel functions for this complex in muscle that when impaired, may contribute to the pathogenesis of muscular dystrophy.

  15. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    Full Text Available The henipaviruses, represented by Hendra (HeV and Nipah (NiV viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4 was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  16. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Science.gov (United States)

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L; Fusco, Deborah L; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S; Bishop-Lilly, Kimberly A; Broder, Christopher C; Nikolov, Dimitar B

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  17. Multiple Drug Transport Pathways through Human P-Glycoprotein.

    Science.gov (United States)

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

    2015-07-21

    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.

  18. Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein.

    Science.gov (United States)

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

    2015-01-01

    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.

  19. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

    Science.gov (United States)

    Loo, T W; Clarke, D M

    1997-01-10

    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.

  20. Structure of a human rhinovirus complexed with its receptor molecule.

    OpenAIRE

    1993-01-01

    Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. ...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    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 such ...... such that involve cell cultures, binding proteins present in sera might interfere in the experiments....

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

    Science.gov (United States)

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

    2017-01-01

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

  3. 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

    2014-01-01

    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...

  4. Lefty Glycoproteins in Human Embryonic Stem Cells: Extracellular Delivery Route and Posttranslational Modification in Differentiation.

    Science.gov (United States)

    Khalkhali-Ellis, Zhila; Galat, Vasiliy; Galat, Yekaterina; Gilgur, Alina; Seftor, Elisabeth A; Hendrix, Mary J C

    2016-09-19

    Lefty is a member of transforming growth factor-beta (TGF-β) superfamily and a potent antagonist of the TGF-β/Nodal/Activin signaling pathway. Lefty is critical in sustaining self-renewal/pluripotency status, and implicated in the differentiation of embryonic stem cells (ESCs). However, emerging studies depict Lefty as a multifaceted protein involved in myriad cellular events. Lefty proteins (human Lefty A and B) are secreted glycoproteins, but their mode of secretion and the significance of their "glycan" moiety remain mostly unexplored. By employing an in vitro system of human ESCs (hESCs), we observed that Lefty protein(s) are encased in exosomes for extracellular release. The exosomal- and cell-associated Lefty diverge in their proteolytic processing, and possess N-glycan structures of high mannose and complex nature. Differentiation of hESCs to mesenchymal cells (MSCs) or neuronal progenitor cells (NPCs) entails distinct changes in the Lefty A/Lefty B gene(s), and protein expression. Specifically, the proteolytic cleavage and N-glycan composition of the cell-associated and exosomal Lefty differ in the differentiated progenies. These modifications affected Lefty's inhibitory effect on Nodal signaling in aggressive melanoma cells. The microheterogeneity in the processing and glycosylation of Lefty protein(s) between hESCs, MSCs, and NPCs could present efficient means of diversifying the endogenous functions of Lefty. Whether Lefty's diverse functions in embryonic patterning, as well as its diffusion range in the extracellular environment, are similarly affected remains to be determined. Our studies underscore the potential relevance of Lefty-packaged exosomes for combating debilitating diseases such as cancer.

  5. Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells.

    Science.gov (United States)

    Yang, Xiaojing; Tao, Shujuan; Orlando, Ron; Brockhausen, Inka; Kan, Frederick W K

    2012-09-01

    Oviduct-specific glycoprotein (OVGP1) is a major mucin-like glycoprotein synthesized and secreted exclusively by non-ciliated secretory cells of mammalian oviduct. In vitro functional studies showed that OVGP1 plays important roles during fertilization and early embryo development. We have recently produced recombinant human oviduct-specific glycoprotein (rhOVGP1) in human embryonic kidney 293 (HEK293) cells. The present study was undertaken to characterize the structures and determine the biosynthetic pathways of the N- and O-glycans of rhOVGP1. Treatment of the stable rhOVGP1-expressing HEK293 cells with either GalNAcα-Bn to block O-glycan extension, tunicamycin to block N-glycosylation, or neuraminidase increased the electrophoretic mobility of rhOVGP1. A detailed analysis of O- and N-linked glycans of rhOVGP1 by mass spectrometry showed a broad range of many simple and complex glycan structures. In order to identify the enzymes involved in the glycosylation of rhOVGP1, we assayed glycosyltransferase activities involved in the assembly of O- and N-glycans in HEK293 cells, and compared these to those from the immortalized human oviductal cells (OE-E6/E7). Our results demonstrate that HEK293 and OE-E6/E7 cells exhibit a similar spectrum of glycosyltransferase activities that can synthesize elongated and sialylated O-glycans with core 1 and 2 structures, as well as complex multiantennary N-glycans. It is anticipated that the knowledge gained from the present study will facilitate future studies of the role of the glycans of human OVGP1 in fertilization and early embryo development.

  6. 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

    2004-01-01

    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.

  7. Chemoenzymatic synthesis of hydrophobic glycoprotein: synthesis of saposin C carrying complex-type carbohydrate.

    Science.gov (United States)

    Hojo, Hironobu; Tanaka, Hiromasa; Hagiwara, Masashi; Asahina, Yuya; Ueki, Akiharu; Katayama, Hidekazu; Nakahara, Yuko; Yoneshige, Azusa; Matsuda, Junko; Ito, Yukishige; Nakahara, Yoshiaki

    2012-11-02

    The complex-type N-linked octasaccharide oxazoline having LacNAc as the nonreducing end sugar was efficiently synthesized using the benzyl-protected LacNAc, mannose, and β-mannosyl GlcNAc units as key building blocks. To achieve a highly β-selective glycosylation with the LacNAc unit, the N-trichloroacetyl group was used for the protection of the amino group in the LacNAc unit. After complete assembly of these units and deprotection, the obtained free sugar was successfully derivatized into the corresponding sugar oxazoline. On the other hand, the N-acetylglucosaminylated saposin C, a hydrophobic lipid-binding protein, was chemically synthesized by the native chemical ligation reaction. On the basis of the previous results related to the synthesis of the nonglycosylated saposin C, the O-acyl isopeptide structure was introduced to the N-terminal peptide thioester carrying GlcNAc to improve its solubility toward aqueous organic solvents. The ligation reaction efficiently proceeded with the simultaneous O- to N-acyl shift at the O-acyl isopeptide moiety. After the removal of the cysteine-protecting group and folding, saposin C carrying GlcNAc was successfully obtained. The synthetic sugar oxazoline was then transferred to this glycoprotein using the mutant of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) (glycosynthase), and the saposin C carrying the complex-type nonasaccharide was successfully obtained.

  8. Humoral immune response to the entire human immunodeficiency virus envelope glycoprotein made in insect cells

    Energy Technology Data Exchange (ETDEWEB)

    Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.; Langlois, A.J.; Lyerly, H.K.; Carson, H.; Krohn, K.; Ranki, A.; Gallo, R.C.; Bolognesi, D.P.; Putney, S.D.

    1987-10-01

    The human immunodeficiency virus envelope gene was expressed in insect cells by using a Baculovirus expression vector. The protein has an apparent molecular mass of 160 kDa, appears on the surface of infected insect cells, and does not appear to be cleaved to glycoproteins gp120 and gp41. Goats immunized with the 160-kDa protein have high titers of antibody that neutralizes virus infection as measured by viral gene expression or cell cytolysis. In addition, immune sera can block fusion of human immunodeficiency virus-infected cells in culture. Both neutralization and fusion-blocking activities are bound to and eluted from immobilized gp120.

  9. Dissection of the Antibody Response against Herpes Simplex Virus Glycoproteins in Naturally Infected Humans

    Science.gov (United States)

    Huang, Zhen-Yu; Whitbeck, J. Charles; Ponce de Leon, Manuel; Lou, Huan; Wald, Anna; Krummenacher, Claude; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally

  10. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Directory of Open Access Journals (Sweden)

    Annasara Lenman

    2015-02-01

    Full Text Available Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR protein. Human adenovirus type 52 (HAdV-52 is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK binds to CAR, and the knob domain of the short fiber (52SFK binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

  11. A defined peptide that inhibits the formation of the glycoprotein IIb and IIIa complex.

    Science.gov (United States)

    Chiang, Thomas M; Zhu, Jiaqian

    2005-01-01

    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. Zinc-alpha2-glycoprotein expression as a marker of differentiation in human oral tumors.

    Science.gov (United States)

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

    1999-03-22

    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.

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

    OpenAIRE

    2009-01-01

    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...

  14. P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin

    OpenAIRE

    1995-01-01

    Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P- selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P- selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein- dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes...

  15. Glycoprotein Ib-IX-V Complex Transmits Cytoskeletal Forces That Enhance Platelet Adhesion.

    Science.gov (United States)

    Feghhi, Shirin; Munday, Adam D; Tooley, Wes W; Rajsekar, Shreya; Fura, Adriane M; Kulman, John D; López, Jose A; Sniadecki, Nathan J

    2016-08-09

    Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

  16. Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

    Science.gov (United States)

    Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

    2017-04-01

    The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National

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

    Science.gov (United States)

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  18. Multiscale molecular dynamics simulations of lipid interactions with P-glycoprotein in a complex membrane.

    Science.gov (United States)

    Domicevica, Laura; Koldsø, Heidi; Biggin, Philip C

    2017-09-02

    P-glycoprotein (P-gp) can transport a wide range of very different hydrophobic organic molecules across the membrane. Its ability to extrude molecules from the cell creates delivery problems for drugs that target proteins in the central nervous system (CNS) and also causes drug-resistance in many forms of cancer. Whether a drug will be susceptible to export by P-gp is difficult to predict and currently this is usually assessed with empirical and/or animal models. Thus, there is a need to better understand how P-gp works at the molecular level in order to fulfil the 3Rs: Refinement, reduction and replacement of animals in research. As structural information increasingly becomes available, our understanding at the molecular level improves. Proteins like P-gp are however very dynamic entities and thus one of the most appropriate ways to study them is with molecular dynamics simulations, especially as this can capture the influence of the surrounding environment. Recent parameterization developments have meant that it is now possible to simulate lipid bilayers that more closely resemble in vivo membranes in terms of their composition. In this report we construct a complex lipid bilayer that mimics the composition of brain epithelial cells and examine the interactions of it with P-gp. We find that the negatively charged phosphatidylserine lipids in the inner leaflet of the membrane tend to form an annulus around P-gp. We also observed the interaction of cholesterol with three distinct areas of the P-gp. Potential of mean force (PMF) calculations suggest that a crevice between transmembrane helices 10 and 12 has particularly favourable interaction energy for cholesterol. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Expression of dystrophin-glycoprotein complex at the skeletal muscle sarcolemma in Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Lei ZHAO

    2015-07-01

    Full Text Available Background  Eccentric exercise or high tension exercise could cause damage to skeletal muscle structure, resulting in deficiency of dystrophin and secondary loss of dystrophin-glycoprotein complex (DGC from the sarcolemma, which indicated that down-regulation of dystrophin was one of the key points of skeletal muscle injury from eccentric exercise. Duchenne muscular dystrophy (DMD is caused by mutations of DMD gene, resulting in the absence of dystrophin, which means that skeletal muscles of DMD patients after birth are in the natural state of actual path of force transmission which carried high tension from eccentric exercise. This paper investigated systematically whether expression of DGC is associated with progressive muscle weakness in natural history of DMD, and analyzed the expression of DGC at the sarcolemma of 197 confirmed DMD cases (9 days-12 years old.  Methods  The expression of α- and β-dystroglycan (DG, α-, β-, γ- and δ-sarcoglycan (SG and syntrophin at the sarcolemma of DMD patients was analyzed by immunofluorescent staining.  Results  The study showed that there was no relationship between lack of proteins and progressive muscle weakness with increasing age, although expression of α- and β-DG, α-, β-, γ- and δ-SG and syntrophin at the sarcolemma at different stages of 197 DMD patients (9 days-12 years old had different degrees of deficiency.  Conclusions  Deficiency of DGC may occur before birth and DMD patients were recommended to avoid further damage to skeletal muscles from eccentric exercise and high-resistance movement in activities of daily life and rehabilitation training. DOI: 10.3969/j.issn.1672-6731.2015.06.006

  20. Purification and characterization of a heteromultimeric glycoprotein from Artocarpus heterophyllus latex with an inhibitory effect on human blood coagulation.

    Science.gov (United States)

    Siritapetawee, Jaruwan; Thammasirirak, Sompong

    2011-01-01

    Plant latex has many health benefits and has been used in folk medicine. In this study, the biological effect of Artocarpus heterophyllus (jackfruit) latex on human blood coagulation was investigated. By a combination of heat precipitation and ion-exchange chromatography, a heat stable heteromultimeric glycoprotein (HSGPL1) was purified from jackfruit milky latex. The apparent molecular masses of the monomeric proteins on SDS/PAGE were 33, 31 and 29 kDa. The isoelectric points (pIs) of the monomers were 6.63, 6.63 and 6.93, respectively. Glycosylation and deglycosylation tests confirmed that each subunit of HSGPL1 formed the native multimer by sugar-based interaction. Moreover, the multimer of HSGPL1 also resisted 2-mercaptoethanol action. Peptide mass fingerprint analysis indicated that HSGPL1 was a complex protein related to Hsps/chaperones. HSGPL1 has an effect on intrinsic pathways of the human blood coagulation system by significantly prolonging the activated partial thrombin time (APTT). In contrast, it has no effect on the human extrinsic blood coagulation system using the prothrombin time (PT) test. The prolonged APTT resulted from the serine protease inhibitor property of HSGPL1, since it reduced activity of human blood coagulation factors XI(a) and α-XII(a).

  1. Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects

    Directory of Open Access Journals (Sweden)

    Momoh Mambu

    2010-11-01

    Full Text Available Abstract Background Lassa hemorrhagic fever (LHF is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. With a high rate of infection that may lead to morbidity and mortality, understanding how the virus interacts with the host's immune system is of great importance for generating vaccines and therapeutics. Previous work by our group identified a soluble isoform of the Lassa virus (LASV GP1 (sGP1 in vitro resulting from the expression of the glycoprotein complex (GPC gene 12. Though no work has directly been done to demonstrate the function of this soluble isoform in arenaviral infections, evidence points to immunomodulatory effects against the host's immune system mediated by a secreted glycoprotein component in filoviruses, another class of hemorrhagic fever-causing viruses. A significant fraction of shed glycoprotein isoforms during viral infection and biogenesis may attenuate the host's inflammatory response, thereby enhancing viral replication and tissue damage. Such shed glycoprotein mediated effects were previously reported for Ebola virus (EBOV, a filovirus that also causes hemorrhagic fever with nearly 90% fatality rates 345. The identification of an analogous phenomenon in vivo could establish a new correlate of LHF infection leading to the development of sensitive diagnostics targeting the earliest molecular events of the disease. Additionally, the reversal of potentially untoward immunomodulatory functions mediated by sGP1 could potentiate the development of novel therapeutic intervention. To this end, we investigated the presence of sGP1 in the serum of suspected LASV patients admitted to the Kenema Government Hospital (KGH Lassa Fever Ward (LFW, in Kenema, Sierra Leone that tested positive for viral antigen or displayed classical signs of Lassa fever. Results It is reasonable to expect that a narrow window exists for

  2. Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene.

    Science.gov (United States)

    Merritt, C M; Board, P G

    1988-06-15

    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.

  3. Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity

    Energy Technology Data Exchange (ETDEWEB)

    Halaban, R.; Moellmann, G. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1990-06-01

    Melanogenesis is regulated in large part by tyrosinase, and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmentated (B{sup lt}/B{sup lt}) coat versus the wild-type black (B/B). The authors show that the b locus codes for a glycoprotein with the activity of a catalase (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, they conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. The studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the B{sup lt} mutation renders the protein susceptible to rapid proteolytic degradation.

  4. Interaction of a human blood group Sd(a-) Tamm-Horsfall glycoprotein with applied lectins.

    Science.gov (United States)

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

    1996-04-22

    Unlike the human blood group Sd(a+) Tamm-Horsfall glycoprotein (THGP), the Sd(a-) one lacks terminal GalNAcbeta1--> residues at the nonreducing ends. The binding properties of this glycoprotein and its asialo product with lectins were characterized by quantitative precipitin (QPA) and precipitin inhibition assays. Among 20 lectins tested by QPA, both native and asialo Sd(a-) THGP reacted best with Abrus precatorius and Ricinus communis and completely precipitated the lectin added. They also precipitated well Wistaria floribunda (WFA), Glycine max (SBA), Bauhinia purpurea alba, abrin-a and ricin, all of which recognize the Galbeta1--> 4GlcNAcbeta1--> sequence, although at different strength. The lectin-glycan interactions were inhibited by Galbeta1--> 4GlcNAc and Galbeta1--> 4Glc. When the precipitability of Sd(a-) THGP was compared with that of the Sd(a+) phenotype, the native Sd(a-) THGP exhibited a 40% lesser affinity for WFA, SBA, WGA and mistletoe lectin-I (ML-I). Mapping the precipitation and inhibition profiles of the present study and the results of THGP Sd(a+), it is concluded that Sd(a-) THGP showed a strongly diminished affinity for GalNAcbeta1--> active lectins (SBA and WFA) than the Sd(a+) phenotype.

  5. Recognition by human sera of a variable region of the surface glycoprotein of HTLV-I.

    Science.gov (United States)

    Edouard, E; Londos-Gagliardi, D; Busetta, B; Geoffre, S; Dalbon, P; Moreau, J P; Guillemain, B

    1994-04-01

    The region comprised between the amino acids 175 and 199 of the HTLV-I envelope surface glycoprotein is one of the immunodominant domains of this molecule. In this region, which is well recognized by sera from HTLV-I infected patients, a substitution of the proline at position 192 by a serine has been described in some isolates. Because this mutation could modify the secondary structure of the glycoprotein molecule, we studied the inference of the presence of proline or serine on the recognition of the region 175-199 by human sera. For this, three peptides have been synthetized (a 25-mer 175-199 corresponding to the sequence of the ATK prototype, and two internal 10-mer 190-Pro-199 and 190-Ser-199 having a proline or a serine at position 192) and tested by immunosorbent assay. While most sera reacted with 190-Pro-199 and with 190-Ser-199 synthetic peptides, a differential recognition was observed according to the pathology associated to HTLV-I infection. Moreover sera corresponding to patients infected with a virus harboring a serine at position 192 were found to recognize only the 10-mer with a serine. These data indicates that HTLV-I is subject to antigenic variability.

  6. Bortezomib (PS-341 treatment decreases inflammation and partially rescues the expression of the dystrophin-glycoprotein complex in GRMD dogs.

    Directory of Open Access Journals (Sweden)

    Karla P C Araujo

    Full Text Available Golden retriever muscular dystrophy (GRMD is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs with the proteasome inhibitor bortezomib, and three were control dogs (CD. Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-β1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and β-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some

  7. Determining postmortem interval using glycoproteinous adhesion deposits by Balanus improvisus on human skeletal and dental remains.

    Science.gov (United States)

    Bytheway, Joan A; Pustilnik, Stephen M

    2013-01-01

    An anthropological analysis was conducted on skeletal and dental remains brought to the Galveston County Medical Examiner's office. The skeletal remains were dry, fragmented, and absent of typical fluvial characteristics. During microscopic examination, semitransparent, circular objects were discovered on the dentition, the mandible, tibial plateau, and distal femur. The objects were glycoproteinous adhesions deposited by the acorn barnacle, Balanus improvisus. B. improvisus is an intertidal barnacle found in estuaries in Galveston Bay. Basal diameter of the adhesions on the dentition were significantly smaller than those found on the postcranial bones (p = 0.010), indicating two consecutive cohorts adhered to the bone and dentition. As settlement typically occurs once a year, this would indicate that the remains were in the fluvial environment for at least 375-410 days. It is important in geographic areas that have prevalent fluvial environments that human remains, particularly dentition, are microscopically examined for marine life evidence. © 2012 American Academy of Forensic Sciences.

  8. Evolution of human alpha 1-acid glycoprotein genes and surrounding Alu repeats.

    Science.gov (United States)

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

    1990-04-01

    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.

  9. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

  10. The human glycoprotein salivary agglutinin inhibits the interaction of dc-sign and langerin with oral micro-organisms

    NARCIS (Netherlands)

    Boks, M.A.; Gunput, S.T.G.; Kosten, I.; Gibbs, S.; van Vliet, S.J.; Ligtenberg, A.J.M.; van Kooyk, Y.

    2016-01-01

    Salivary agglutinin (SAG), also known as gp340 or SALSA, is a glycoprotein encoded by the Deleted in Malignant Brain Tumours 1 gene and is abundantly present in human saliva. SAG aggregates bacteria and viruses, thereby promoting their clearance from the oral cavity. The mucosa lining the oral cavit

  11. Differential expression of sphingolipids in P-glycoprotein or multidrug resistance-related protein 1 expressing human neuroblastoma cell lines

    NARCIS (Netherlands)

    Dijkhuis, AJ; Douwes, J; Kamps, W; Sietsma, H; Kok, JW

    2003-01-01

    The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established. SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in S

  12. Differential expression of sphingolipids in P-glycoprotein or multidrug resistance-related protein 1 expressing human neuroblastoma cell lines

    NARCIS (Netherlands)

    Dijkhuis, AJ; Douwes, J; Kamps, W; Sietsma, H; Kok, JW

    2003-01-01

    The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established. SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in

  13. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    Science.gov (United States)

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  14. Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

    Directory of Open Access Journals (Sweden)

    Yannan Qin

    2014-11-01

    Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

  15. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Russell, Rodney S.; Goossens, Vera

    2008-01-01

    The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human...... monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...... neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a...

  16. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Tiwari, Vaibhav [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Darmani, Nissar A.; Thrush, Gerald R. [Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2009-12-18

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  17. Oxidized Low-Density Lipoprotein-β2-Glycoprotein I Complex But Not Free Oxidized LDL Is Associated With the Presence and Severity of Coronary Artery Disease.

    Science.gov (United States)

    Bliden, Kevin P; Chaudhary, Rahul; Lopez, Luis R; Damrongwatanasuk, Rongras; Guyer, Kirk; Gesheff, Martin G; Franzese, Christopher J; Kaza, Himabindu; Tantry, Udaya S; Gurbel, Paul A

    2016-09-01

    Oxidized low-density lipoprotein (oxLDL) and β2-glycoprotein I (β2GPI) have been identified in human atherosclerotic lesions and when complexed have been implicated as a pro-atherothrombotic antigen. We examined the association of free oxLDL and oxLDL-β2GPI complex in patients with coronary artery disease who underwent elective cardiac catheterization. Serum was collected from patients with suspected coronary artery disease immediately before elective cardiac catheterization who were either treated (n = 385) or not treated (n = 150) with statins and from healthy volunteers (n = 134). OxLDL and oxLDL-β2GPI complex levels were determined by enzyme-linked immunosorbent assay. Disease severity was defined angiographically as none-minimal (75%) luminal diameter obstruction of any major coronary vessel. Both oxLDL and oxLDL-β2GPI complex were lower in patients on statins (p LDL4 and triglycerides increased with oxLDL-β2GPI complex quartiles (p = 0.001). OxLDL-β2GPI complex (>0.32 U/ml) was predictive of severe atherosclerosis by receiver-operating characteristic curve analysis in statin-naive patients (area under the curve 0.66, p = 0.002). In conclusion, oxLDL-β2GPI appears more predictive of coronary artery disease severity than oxLDL alone in statin-naive patients.

  18. Analysis of COPII Vesicles Indicates a Role for the Emp47-Ssp120 Complex in Transport of Cell Surface Glycoproteins.

    Science.gov (United States)

    Margulis, Neil G; Wilson, Joshua D; Bentivoglio, Christine M; Dhungel, Nripesh; Gitler, Aaron D; Barlowe, Charles

    2016-03-01

    Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium-binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47-Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.

  19. In vitro transport assays of rufinamide, pregabalin, and zonisamide by human P-glycoprotein.

    Science.gov (United States)

    Chan, Pui Shan; Zhang, Chunbo; Zuo, Zhong; Kwan, Patrick; Baum, Larry

    2014-03-01

    Epilepsy is resistant to treatment with antiepileptic drugs (AEDs) in about one third of epilepsy patients. AED export by P-glycoprotein (Pgp) overexpressed in the blood-brain barrier may contribute to AED resistance. The Pgp transport status of many of the recently approved AEDs remains unknown. We investigated whether several new AEDs - zonisamide (ZNS), pregabalin (PGB), and rufinamide (RFM) - are human Pgp substrates. MDCKII and LLC-PK1 cells transfected with the human MDR1 gene, which encodes the Pgp protein, were used in concentration equilibrium transport assays (CETA) to determine the substrate status of ZNS, PGB, and RFM. For each drug, an equal concentration was added to apical and basal chambers, and the concentration in both chambers was measured up to 4h. RFM, ZNS, and PGB were not transported by MDR1-transfected cells from basolateral to apical sides in CETA. ZNS, RFM, and PGB are not substrates of human Pgp. These data suggest that resistance to these drugs may not be attributed to increased Pgp activity in resistant patients. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Inhibition of P-glycoprotein activity in human leukemic cells by mifepristone.

    Science.gov (United States)

    Fardel, O; Courtois, A; Drenou, B; Lamy, T; Lecureur, V; le Prisé, P Y; Fauchet, R

    1996-08-01

    The antiprogestatin drug mifepristone has previously been shown to potentiate anti-cancer drug activity in rodent multidrug-resistant cell lines through inhibition of P-glycoprotein (P-gp) function. In order to characterize P-gp-mifepristone interactions in human tumoral cells, we have studied the effect of the antiprogestatin agent on P-gp activity in human CD34+ leukemic cells known to display high levels of P-gp-related drug efflux. P-gp-mediated transport of the fluorescent dye rhodamine 123 occurring in the CD34+ KG1a myeloid leukemia cell line was found to be strongly inhibited by mifepristone in a dose-dependent manner. Similarly to verapamil, a well-known chemosensitizer agent, the antiprogestatin drug increased doxorubicin cytotoxicity in KG1a cells. Mifepristone, when used at a 10 microM concentration thought to be achievable in vivo without major toxicity, was also able to markedly decrease cellular rhodamine 123 efflux occurring in CD34+ blast cells isolated from six patients suffering from myeloid acute leukemias. These results thus indicate that mifepristone can strongly inhibit P-gp activity in human cells, including tumoral cells freshly isolated from patients, therefore suggesting that the clinical use of this compound may contribute to down-modulate P-gp-mediated drug resistance.

  1. Pneumocystis carinii major surface glycoprotein induces interleukin-8 and monocyte chemoattractant protein-1 release from a human alveolar epithelial cell line

    DEFF Research Database (Denmark)

    Benfield, T L; Lundgren, Bettina; Shelhamer, J H

    1999-01-01

    BACKGROUND: The major surface glycoprotein (MSG) is an abundant, immunogenic glycoprotein located on the surface of Pneumocystis carinii. Little is known about the proinflammatory effects of MSG. DESIGN: We have investigated the effect of human MSG on the secretion of the chemokines interleukin 8...

  2. A Con A- purified hydatid glycoprotein fraction effectively diagnoses human hydatidosis.

    Science.gov (United States)

    Kamel, Manal M; Maher, Kesmat M; Rabia, Ibrahim; Helmy, Ahmed H; El-Adawi, Azza I; Mousa, Mousa A; Mahgoub, Abeer M

    2006-12-01

    Diagnosis and quantification of Echinococcus granulosus infection in man and animal hosts are centralized to feasible control. This study included 93 serum samples, 25 sure positive hydatid cases confirmed surgically, 7 suspected cases diagnosed by indirect haemagglutination IHA and 41 cases other parasitic infections (15 S. mansoni, 8 Fasciola, 7 Ascaris, 5 H. nana & 6 Ancylostoma) diagnosed by microscopic examination and were negative by ELISA and/or IHA for anti-hydatid antibody. Twenty negative serum samples served as healthy controls. Six types of hydatid fluid antigens (crude, host-free & Con-A purified) of human and camel origin were subjected to electrophoretic separation (SDS-PAGE) and immunoblotting (EITB). The anti-hydatid IgG was detected in sera of the different groups for evaluation of sensitivity, specificity and diagnostic efficacy of each type of antigens. Detection of circulating hydatid antigen (CAg) was performed using anti rabbit hyperimmune sera raised against Con-A purified either human or camel hydatid antigen. SDS-PAGE revealed several bands ranging from 55-185 kDa with 10 kDa band shared by all antigens. The specific bands revealed by EITB for Con-A purified camel and human antigens were at 80, 110 & 55, 110 kDa respectively. ELISA highest sensitivity (96.9%) was by using host-free Con-A purified glycoprotein fraction of human hydatid antigen. Highest specificity (98.4%) was recorded upon use of either Con-A purified camel or human antigen with 94.5% & 97.7% diagnostic efficacy respectively. Detection of circulating antigen by polyclonal antibodies against Con-A purified human hydatid antigen revealed 91.8% specificity.

  3. DUSP1 induces paclitaxel resistance through the regulation of p-glycoprotein expression in human ovarian cancer cells.

    Science.gov (United States)

    Kang, Yu-Seon; Seok, Hyun-Jeong; Jeong, Eun-Jeong; Kim, Yuna; Yun, Seok-Joong; Min, Jeong-Ki; Kim, Sun Jin; Kim, Jang-Seong

    2016-09-01

    The heterogeneity and genetic instability of ovarian cancer cells often lead to the development of drug resistance, closely related with the increased cancer-related mortality. In this study, we investigated the role of dual-specificity phosphatase 1 (DUSP1) in the development of the resistance in human ovarian cancer cells against paclitaxel. Overexpression of DUSP1 in HeyA8 human ovarian cancer cells (HeyA8-DUSP1) up-regulated the expression of the drug efflux pump, p-glycoprotein. Consequently, HeyA8-DUSP1 cells are highly resistant to paclitaxel, with the resistance comparable to that of a multi-drug resistance cell line (HeyA8-MDR). Moreover, over expression of DUSP1 significantly increased the activation of p38 MAPK, leaving the activation of ERK1/2 and JNK1/2 unaffected. Pharmacological suppression of p38 MAPK activity prevents the up-regulation of p-glycoprotein expression and the consequent resistance against paclitaxel in HeyA8-DUSP1 cells. By contrast, HeyA8-MDR cells expressed a significantly higher level of DUSP1, but treatment with small interference RNA against DUSP1 significantly suppressed the expression of p-glycoprotein and the resistance against paclitaxel in HeyA8-MDR cells. Ectopic expression of MKK3, an upstream activator of p38 MAPK, significantly up-regulated the expression of p-glycoprotein and increased the consequent resistance against paclitaxel in HeyA8 cells. Collectively, these data indicated that DUSP1 may induce the resistance against paclitaxel through the p38 MAPK-mediated overexpression of p-glycoprotein in human ovarian cancer cells.

  4. Disorder in Complex Human System

    Science.gov (United States)

    Akdeniz, K. Gediz

    2011-11-01

    Since the world of human and whose life becomes more and more complex every day because of the digital technology and under the storm of knowledge (media, internet, governmental and non-governmental organizations, etc...) the simulation is rapidly growing in the social systems and in human behaviors. The formation of the body and mutual interactions are left to digital technological, communication mechanisms and coding the techno genetics of the body. Deconstruction begins everywhere. The linear simulation mechanism with modern realities are replaced by the disorder simulation of human behaviors with awareness realities. In this paper I would like to introduce simulation theory of "Disorder Sensitive Human Behaviors". I recently proposed this theory to critique the role of disorder human behaviors in social systems. In this theory the principle of realty is the chaotic awareness of the complexity of human systems inside of principle of modern thinking in Baudrillard's simulation theory. Proper examples will be also considered to investigate the theory.

  5. In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon.

    Science.gov (United States)

    Casali, P; Sissons, J G; Buchmeier, M J; Oldstone, M B

    1981-09-01

    Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive, both erythrocyte-rosetting and non-erythrocyte-rosetting, as assessed by both positive and negative selection experiments. The induction of nonspecific CMC by viral glycoproteins either in the soluble state or inserted into artificial membranes could be segregated from the CMC associated with whole virions. First, on kinetics studies, purified viral glycoproteins induced CMC more rapidly than did whole virus. Second, viral glycoprotein-produced response occurred in the absence of detectable release of interferon into the culture medium, whereas CMC activity due to whole virions was associated with interferon release. The fact that purified measles virus glycoproteins integrated into artificial membrane bilayers were as efficient as their soluble counterparts in inducing CMC suggests that the hydrophobic portion of the glycoproteins was not involved in the induction and expression of the lytic activity. Purified glycoproteins from lymphocytic choriomeningitis virus behave similarly, although this virus is unrelated to measles virus. It is inferred that interferon-independent CMC induced by viral glycoproteins might account for some of the biological reactions occurring early in the control of a viral infection.

  6. Discovery and Characterization of Phage Display-Derived Human Monoclonal Antibodies against RSV F Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zhifeng Chen

    Full Text Available Respiratory syncytial virus (RSV is a leading cause of lower respiratory tract infection in infants, the elderly and in immunosuppressed populations. The vast majority of neutralizing antibodies isolated from human subjects target the RSV fusion (F glycoprotein, making it an attractive target for the development of vaccines and therapeutic antibodies. Currently, Synagis® (palivizumab is the only FDA approved antibody drug for the prevention of RSV infection, and there is a great need for more effective vaccines and therapeutics. Phage display is a powerful tool in antibody discovery with the advantage that it does not require samples from immunized subjects. In this study, Morphosys HuCAL GOLD® phage libraries were used for panning against RSV prefusion and postfusion F proteins. Panels of human monoclonal antibodies (mAbs against RSV F protein were discovered following phage library panning and characterized. Antibodies binding specifically to prefusion or postfusion F proteins and those binding both conformations were identified. 3B1 is a prototypic postfusion F specific antibody while 2E1 is a prototypic prefusion F specific antibody. 2E1 is a potent broadly neutralizing antibody against both RSV A and B strains. Epitope mapping experiments identified a conformational epitope spanning across three discontinuous sections of the RSV F protein, as well as critical residues for antibody interaction.

  7. Machupo virus glycoprotein determinants for human transferrin receptor 1 binding and cell entry.

    Directory of Open Access Journals (Sweden)

    Sheli R Radoshitzky

    Full Text Available Machupo virus (MACV is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1. TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors.

  8. P-glycoprotein expression and amyloid accumulation in human aging and Alzheimer's disease: preliminary observations.

    Science.gov (United States)

    Chiu, Catherine; Miller, Miles C; Monahan, Renée; Osgood, Doreen P; Stopa, Edward G; Silverberg, Gerald D

    2015-09-01

    P-glycoprotein (P-gp), part of the blood-brain barrier, limits drug access to the brain and is the target for therapies designed to improve drug penetration. P-gp also extrudes brain amyloid-beta (Aβ). Accumulation of Aβ is a hallmark of Alzheimer's disease (AD). Aβ accumulates in normal aging and in AD primarily due to decreased Aβ clearance. This is a preliminary report on the relative protein and messenger RNA expression of P-gp in human brains, ages 20-100 years, including AD subjects. In these preliminary studies, cortical endothelial P-gp expression decreased in AD compared with controls (p P-gp expression in human aging are similar to aging rats. Microvessel P-gp messenger RNA remained unchanged with aging and AD. Aβ plaques were found in 42.8% of normal subjects (54.5% of those older than 50 years). A qualitative analysis showed that P-gp expression is lower than the group mean in subjects older than 75 years but increased if younger. Decreased P-gp expression may be related to Aβ plaques in aging and AD. Downregulating P-gp to allow pharmaceuticals into the central nervous system may increase Aβ accumulation.

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

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

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

  10. Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization.

    Science.gov (United States)

    Arroteia, Kélen Fabíola; Barbieri, Mainara Ferreira; Souza, Gustavo Henrique Martins Ferreira; Tanaka, Hiromitsu; Eberlin, Marcos Nogueira; Hyslop, Stephen; Alvares, Lúcia Elvira; Pereira, Luís Antonio Violin Dias

    2014-01-01

    The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

  11. Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

    Science.gov (United States)

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

    1979-04-18

    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.

  12. Structure of the Ebola Virus Glycoprotein Bound to An Antibody From a Human Survivor

    Energy Technology Data Exchange (ETDEWEB)

    Lee, J.E.; Fusco, M.L.; Hessell, A.J.; Oswald, W.B.; Burton, D.R.; Saphire, E.O.

    2009-05-20

    Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and fusion of viral and host membranes. Here we report the crystal structure of EBOV GP in its trimeric, pre-fusion conformation (GP1+GP2) bound to a neutralizing antibody, KZ52, derived from a human survivor of the 1995 Kikwit outbreak. Three GP1 viral attachment subunits assemble to form a chalice, cradled by the GP2 fusion subunits, while a novel glycan cap and projected mucin-like domain restrict access to the conserved receptor-binding site sequestered in the chalice bowl. The glycocalyx surrounding GP is likely central to immune evasion and may explain why survivors have insignificant neutralizing antibody titres. KZ52 recognizes a protein epitope at the chalice base where it clamps several regions of the pre-fusion GP2 to the amino terminus of GP1. This structure provides a template for unraveling the mechanism of EBOV GP-mediated fusion and for future immunotherapeutic development.

  13. Evaluation of P-glycoprotein (Pgp) expression in human osteosarcoma by high-throughput tissue microarray.

    Science.gov (United States)

    Gao, Yan; Liao, Yunfei; Shen, Jacson K; Feng, Yong; Choy, Edwin; Cote, Gregory; Harmon, David; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

    2016-09-01

    Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well-established cause of MDR involves overexpression of P-glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high-throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre-operative chemotherapy in osteosarcoma patients. Eighteen of the fifty-seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606-1612, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  14. Levistolide A overcomes P-glycoprotein-mediated drug resistance in human breast carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Fei CHEN; Tao WANG; Jia WANG; Zi-qiang WANG; Ming QIAN

    2008-01-01

    Aim:The aim of the present study was to investigate the reversing effect of levistolide A (LA) on P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in human breast carcinoma Bcap37/MDR1 cells. Methods:After chemotherapeu-tic drugs (adriamycin or vincristine) used alone or in combination with LA, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-lium bromide assay and cell cycle distribution by flow cytometry. RT-PCR was used to detect MDR1 gene transcription and the Western blot assay was used to assess P-gp expression and the cleavages of poly(ADP-ribose) polymerase and caspase-3. Apoptosis was detected by terminal transferase-mediated dUTP nick end-labeling assay. Moreover, the P-gp function was evaluated by the intracellu-lar accumulation of the P-gp substrate detected by flow cytometry. Results:We found the subcytotoxic doses of LA significantly enhanced adriamycin- or vinc-ristine-induced G2/M arrest and apoptosis. These effects were consistent with the ability of LA to inhibit P-gp function. Moreover, LA dramatically enhanced the verapamil (VER) ability to reverse drug resistance. Conclusion:LA has the poten-tial to be developed as a novel P-gp modulator. Furthermore, the combination of LA and VER might represent a more sufficient but less toxic anti-MDR regimen.

  15. Genetic diversity and molecular evolution of the major human metapneumovirus surface glycoproteins over a decade.

    Science.gov (United States)

    Papenburg, Jesse; Carbonneau, Julie; Isabel, Sandra; Bergeron, Michel G; Williams, John V; De Serres, Gaston; Hamelin, Marie-Ève; Boivin, Guy

    2013-11-01

    Human metapneumovirus (HMPV) is a recently discovered paramyxovirus that is a major cause of respiratory infections worldwide. We aim to describe the molecular evolution of the HMPV F (fusion) and G (attachment) surface glycoproteins because they are targets for vaccines, monoclonal antibodies and antivirals currently in development. Nasopharyngeal aspirates were collected in children genetic lineages (A1, A2a, A2b, B1 and B2). Multiple lineages circulated each year in Quebec City. With the exception of B1, each of the 5 subgroups was the predominant lineage during ≥1 season. The A1 lineage was not detected since 2002-2003 in our local cohort. There was no evidence of inter- or intragenic recombination. HMPV-F was highly conserved, whereas HMPV-G exhibited greater diversity. HMPV-F demonstrated strong evidence of purifying selection, both overall and in an abundance of negatively selected amino acid sites. In contrast, sites under diversifying selection were detected in all HMPV-G lineages (range, 4-15), all of which were located in the ectodomain. Predominant circulating HMPV lineages vary by year. HMPV-F is highly constrained and undergoes significant purifying selection. Given its high genetic variability, we found a modest number of positively selected sites in HMPV-G. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Truncation of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein defines elements required for fusion, incorporation, and infectivity.

    Science.gov (United States)

    Yue, Ling; Shang, Liang; Hunter, Eric

    2009-11-01

    The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a "snorkeling" model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.

  17. Convergent evolution of pregnancy-specific glycoproteins in human and horse.

    Science.gov (United States)

    Aleksic, Denis; Blaschke, Lisa; Mißbach, Sophie; Hänske, Jana; Weiß, Wiebke; Handler, Johannes; Zimmermann, Wolfgang; Cabrera-Sharp, Victoria; Read, Jordan E; de Mestre, Amanda M; O'Riordan, Ronan; Moore, Tom; Kammerer, Robert

    2016-09-01

    Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be related to PSGs of rodents and primates. We identified seven genes that encode secreted PSG-like CEACAMs Phylogenetic analyses indicate that they evolved independently from an equine CEACAM1-like ancestor rather than from a common PSG-like ancestor with rodents and primates. Significantly, expression of PSG-like genes (CEACAM44, CEACAM48, CEACAM49 and CEACAM55) was found in non-invasive as well as invasive trophoblast cells such as purified chorionic girdle cells and endometrial cup cells. Chorionic girdle cells are highly invasive trophoblast cells that invade the endometrium of the mare where they form endometrial cups and are in close contact with maternal immune cells. Therefore, the microenvironment of invasive equine trophoblast cells has striking similarities to the microenvironment of trophoblast cells in hemochorial placentas, suggesting that equine PSG-like CEACAMs and rodent and primate PSGs have undergone convergent evolution. This is supported by our finding that equine PSG-like CEACAM49 exhibits similar activity to certain rodent and human PSGs in a functional assay of platelet-fibrinogen binding. Our results have implications for understanding the evolution of PSGs and their functions in maternal-fetal interactions. © 2016 Society for Reproduction and Fertility.

  18. Tariquidar Is an Inhibitor and Not a Substrate of Human and Mouse P-glycoprotein.

    Science.gov (United States)

    Weidner, Lora D; Fung, King Leung; Kannan, Pavitra; Moen, Janna K; Kumar, Jeyan S; Mulder, Jan; Innis, Robert B; Gottesman, Michael M; Hall, Matthew D

    2016-02-01

    Since its development, tariquidar (TQR; XR9576; N-[2-[[4-[2-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide) has been widely regarded as one of the more potent inhibitors of P-glycoprotein (P-gp), an efflux transporter of the ATP-binding cassette (ABC) transporter family. A third-generation inhibitor, TQR exhibits high affinity for P-gp, although it is also a substrate of another ABC transporter, breast cancer resistance protein (BCRP). Recently, several studies have questioned the mechanism by which TQR interfaces with P-gp, suggesting that TQR is a substrate for P-gp instead of a noncompetitive inhibitor. We investigated TQR and its interaction with human and mouse P-gp to determine if TQR is a substrate of P-gp in vitro. To address these questions, we used multiple in vitro transporter assays, including cytotoxicity, flow cytometry, accumulation, ATPase, and transwell assays. A newly generated BCRP cell line was used as a positive control that demonstrates TQR-mediated transport. Based on our results, we conclude that TQR is a potent inhibitor of both human and mouse P-gp and shows no signs of being a substrate at the concentrations tested. These in vitro data further support our position that the in vivo uptake of [(11)C]TQR into the brain can be explained by its high-affinity binding to P-gp and by it being a substrate of BCRP, followed by amplification of the brain signal by ionic trapping in acidic lysosomes.

  19. Development of patatin knockdown potato tubers using RNA interference (RNAi technology, for the production of human-therapeutic glycoproteins

    Directory of Open Access Journals (Sweden)

    Ko Jeong-Heon

    2008-04-01

    Full Text Available Abstract Background Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines. Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels. Results Based upon the sequence similarity within the multi-gene family of patatins, a highly conserved target sequence (635 nts of patatin gene pat3-k1 [GenBank accession no. DQ114421] in potato plants (Solanum tuberosum L. was amplified for the construction of a patatin-specific hairpin RNAi (hpRNAi vector. The CaMV 35S promoter-driven patatin hpRNAi vector was transformed into the potato cultivar Desiree by Agrobacterium-mediated transformation. Ten transgenic potato lines bearing patatin hpRNA were generated. The effects of RNA interference were characterized at both the protein and mRNA levels using 1D and 2D SDS/PAGE and quantitative real-time RT-PCR analysis. Dependent upon the patatin hpRNAi line, patatins decreased by approximately 99% at both the protein and mRNA levels. However, the phenotype (e.g. the number and size of potato tuber, average tuber weight, growth pattern, etc. of hpRNAi lines was not distinguishable from wild-type potato plants under both in vitro and ex vitro growth conditions. During glycoprotein purification, patatin-knockdown potato tubers allowed rapid purification of other potato glycoproteins with less contamination of patatins. Conclusion Patatin-specific hpRNAi effectively

  20. Glycoproteins: Occurrence and Significance

    Science.gov (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.

  1. The Human Glycoprotein Salivary Agglutinin Inhibits the Interaction of DC-SIGN and Langerin with Oral Micro-Organisms.

    Science.gov (United States)

    Boks, Martine A; Gunput, Sabrina T G; Kosten, Ilona; Gibbs, Susan; van Vliet, Sandra J; Ligtenberg, Antoon J M; van Kooyk, Yvette

    2016-01-01

    Salivary agglutinin (SAG), also known as gp340 or SALSA, is a glycoprotein encoded by the Deleted in Malignant Brain Tumours 1 gene and is abundantly present in human saliva. SAG aggregates bacteria and viruses, thereby promoting their clearance from the oral cavity. The mucosa lining the oral cavity contains dendritic cells (DC) and Langerhans cells (LC), which express the C-type lectin receptors (CLR) DC-SIGN and Langerin, respectively. Both DC-SIGN and Langerin recognise mannose and fucose carbohydrate structures on pathogens and self-glycoproteins to regulate immunity and homeostasis. The purpose of this study was to investigate whether SAG interacts with these CLR and whether this interferes with the binding to oral pathogens. We show that whole parotid saliva and SAG, when coated to microplates, strongly interact with DC-SIGN and Langerin, probably via mannose and fucose structures. Also, primary human DC and LC bind parotid saliva and SAG via DC-SIGN and Langerin, respectively. Furthermore, SAG binding to DC-SIGN or Langerin prevented binding to the micro-organisms Candida albicans and Escherichia coli which express mannose and fucose-containing glycan structures. Thus, binding of saliva glycoprotein SAG to DC-SIGN and Langerin may inhibit pathogen-DC/LC interactions, and could prove to be a new immunomodulatory mechanism of SAG.

  2. Immunological responses to envelope glycoprotein 120 from subtypes of human immunodeficiency virus type 1.

    Science.gov (United States)

    Gilljam, G; Svensson, A; Ekström, A; Wahren, B

    1999-07-01

    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.

  3. Human P-glycoprotein exhibits reduced affinity for substrates during a catalytic transition state.

    Science.gov (United States)

    Ramachandra, M; Ambudkar, S V; Chen, D; Hrycyna, C A; Dey, S; Gottesman, M M; Pastan, I

    1998-04-01

    Human P-glycoprotein (Pgp), a plasma membrane protein that confers multidrug resistance, functions as an ATP-dependent drug efflux pump. Pgp contains two ATP binding/utilization sites and exhibits ATPase activity that is stimulated in the presence of substrates and modulating agents. The mechanism of coupling of ATP hydrolysis to drug transport is not known. To understand the role of ATP hydrolysis in drug binding, it is necessary to develop methods for purifying and reconstituting Pgp that retains properties including stimulation of ATPase activity by known substrates to an extent similar to that in the native membrane. In this study, (His)6-tagged Pgp was expressed in Trichoplusia ni (High Five) cells using the recombinant baculovirus system and purified by metal affinity chromatography. Upon reconstitution into phospholipid vesicles, purified Pgp exhibited specific binding to analogues of substrates and ATP in affinity labeling experiments and displayed a high level of drug-stimulated ATPase activity (specific activity ranging from 4.5 to 6.5 micromol min-1 mg-1). The ATPase activity was inhibited by ADP in a competitive manner, and by vanadate and N-ethylmaleimide at low concentrations. Vanadate which is known to inhibit ATPase activity by trapping MgADP at the catalytic site inhibited photoaffinity labeling of Pgp with substrate analogues, [125I]iodoarylazidoprazosin and [3H]azidopine, only under ATP hydrolysis conditions. Because vanadate-trapped Pgp is known to resemble the ADP and phosphate-bound catalytic transition state, our findings indicate that ATP hydrolysis results in a conformation with reduced affinity for substrates. A catalytic transition conformation with reduced affinity would essentially result in substrate dissociation and supports a model for drug transport in which an ATP hydrolysis-induced conformational change leads to drug release toward the extracellular medium.

  4. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    Science.gov (United States)

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  5. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The Lysosomal Degradation Pathway

    Science.gov (United States)

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S.; Ambudkar, Suresh V.

    2015-01-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7 ± 1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1± 0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp expressing cancer cells towards chemotherapeutic drugs. PMID:26057472

  6. Antigenic characterization of the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor incorporated into nanodiscs

    Science.gov (United States)

    Witt, Kristen C.; Castillo-Menendez, Luis; Ding, Haitao; Espy, Nicole; Zhang, Shijian; Kappes, John C.; Sodroski, Joseph

    2017-01-01

    The entry of human immunodeficiency virus (HIV-1) into host cells is mediated by the viral envelope glycoproteins (Envs), which are derived by the proteolytic cleavage of a trimeric gp160 Env precursor. The mature Env trimer is a major target for entry inhibitors and vaccine-induced neutralizing antibodies. Env interstrain variability, conformational flexibility and heavy glycosylation contribute to evasion of the host immune response, and create challenges for structural characterization and vaccine development. Here we investigate variables associated with reconstitution of the HIV-1 Env precursor into nanodiscs, nanoscale lipid bilayer discs enclosed by membrane scaffolding proteins. We identified detergents, as well as lipids similar in composition to the viral lipidome, that allowed efficient formation of Env-nanodiscs (Env-NDs). Env-NDs were created with the full-length Env precursor and with an Env precursor with the majority of the cytoplasmic tail intact. The self-association of Env-NDs was decreased by glutaraldehyde crosslinking. The Env-NDs exhibited an antigenic profile expected for the HIV-1 Env precursor. Env-NDs were recognized by broadly neutralizing antibodies. Of note, neutralizing antibody epitopes in the gp41 membrane-proximal external region and in the gp120:gp41 interface were well exposed on Env-NDs compared with Env expressed on cell surfaces. Most Env epitopes recognized by non-neutralizing antibodies were masked on the Env-NDs. This antigenic profile was stable for several days, exhibiting a considerably longer half-life than that of Env solubilized in detergents. Negative selection with weak neutralizing antibodies could be used to improve the antigenic profile of the Env-NDs. Finally, we show that lipid adjuvants can be incorporated into Env-NDs. These results indicate that Env-NDs represent a potentially useful platform for investigating the structural, functional and antigenic properties of the HIV-1 Env trimer in a membrane context

  7. Computational analysis of BACE1-ligand complex crystal structures and linear discriminant analysis for identification of BACE1 inhibitors with anti P-glycoprotein binding property.

    Science.gov (United States)

    Manoharan, Prabu; Chennoju, Kiranmai; Ghoshal, Nanda

    2017-01-12

    More than 100 years of research on Alzheimer's disease didn't yield a potential cure for this dreadful disease. Poor Blood Brain Barrier (BBB) permeability and P-glycoprotein binding of BACE1 inhibitors are the major causes for the failure of these molecules during clinical trials. The design of BACE1 inhibitors with a balance of sufficient affinity to the binding site and little or no interaction with P-glycoproteins is indispensable. Identification and understanding of protein-ligand interactions are essential for ligand optimization process. Structure-based drug design (SBDD) efforts led to a steady accumulation of BACE1-ligand crystal complexes in the PDB. This study focuses on analyses of 153 BACE1-ligand complexes for the direct contacts (hydrogen bonds and weak interactions) observed between protein and ligand and indirect contacts (water-mediated hydrogen bonds), observed in BACE1-ligand complex crystal structures. Intraligand hydrogen bonds were analyzed, with focus on ligand P-glycoprotein efflux. The interactions are dissected specific to subsites in the active site and discussed. The observed protein-ligand and intraligand interactions were used to develop the linear discriminant model for the identification of BACE1 inhibitors with less or no P-glycoprotein binding property. Excellent statistical results and model's ability to correctly predict a new data-set with an accuracy of 92% is achieved. The results are retrospectively analyzed to give input for the design of potential BACE1 inhibitors.

  8. A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Pearl Laurence H

    2006-02-01

    Full Text Available Abstract Background The antiphospholipid syndrome (APS, characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI of human beta 2 glycoprotein I (β2GPI is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL, which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. Methods Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b and expressed in BL21(DE3 Escherichia coli (E. coli. By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. Results Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β2GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. Conclusion By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the

  9. In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon

    OpenAIRE

    1981-01-01

    Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive...

  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;

    2007-01-01

    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. Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

    NARCIS (Netherlands)

    Konings, WN; Poelarends, GJ

    2002-01-01

    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

  12. Antibodies to Glycoproteins Shared by Human Peripheral Nerve and Campylobacter jejuni in Patients with Multifocal Motor Neuropathy

    Directory of Open Access Journals (Sweden)

    Ljubica Suturkova

    2013-01-01

    Full Text Available We have tested serum samples from 24 patients with multifocal motor neuropathy (MMN for reactivity to ganglioside GM1 and to Gal(β1–3GalNAc-bearing glycoproteins isolated from human peripheral nerve and from Campylobacter jejuni (Cj serotype O:19. IgM anti-GM1 antibodies were detected by ELISA in 11 patients (45.8% with MMN and in only one subject (4% from the control group. Western blots showed positive reactivity of sera from 6 patients (25% with MMN to several Gal(β1–3GalNAc-bearing glycoproteins from human peripheral nerve and from Cj O:19 isolates. Sera from three patients (12.5% with MMN showed positively reactive bands with similar electrophoretic mobility in all isolates (60–62 kDa, 48–51 kDa, 42 kDa, and 38 kDa. All six patients showed positive reactivity to 48–52 kDa protein isolated from human peripheral nerve. Increased titer of IgG antibodies to 60–62 kDa protein isolated from Cj O:19 associated with Guillain-Barré syndrome was detected in three patients, and their serum showed also IgG positive reactivity to peripheral nerve antigen with the same electrophoretic mobility. One of these patients had a previous history of Cj infection which suggests the possibility that Cj may be also involved in the pathogenesis of MMN.

  13. Topology and Function of Human p-Glycoprotein in Multidrug Resistant Breast Cancer Cells

    Science.gov (United States)

    1999-08-01

    analysis and both drug transport and regulation of swelling-activated chloride currents were examined. To date our results are incomplete to draw...P-glycoprotein, topology, 15. NUMBER.OF PAGES Breast Cancer 32 swelling-activated chloride currents 16. PRICE CODE 17. SECURITY CLASSIFICATION 18...important proteins such as the cystic fibrosis transmembrane conductance regulator ( CFTR ) (1;2). This superfamily is generally characterized by a

  14. 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

    2004-01-01

    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......-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein...... (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein...

  15. Further characterization of some heterophile agglutinins reacting with alkali-labile carbohydrate chains of human erythrocyte glycoproteins.

    Science.gov (United States)

    Dahr, W; Uhlenbruck, G; Bird, G W

    1975-01-01

    The nature of the receptor sites for several agglutinins is characterized by hemagglutination inhibition assays. The inhibitory activity of human erythrocytes glycoproteins, from which sialic acid, sialic acid and galactose or alkali-labile oligosaccharides have been removed, is compared to the inhibitory effect of compounds with known structure. It is shown that the lectin from Arachis hypogea and anti-T bind to alkali-labile galactosyl-residues. Agglutinins from Bauhinia purpurea and variegata (non- or N-specific), Maclura aurantiaca, Iberis amara, sempervirens, umbellata hybrida and umbellata nana (M- or nonspecific), Moluccella laevis (A- plus N-specific), Helix pomatia, Helix aspersa, Helix lucorum and Caucasotachea atrolabiata interact with alkali-labile N-acetylgalactosamine. The results obtained with the anti-A agglutinins from various snails suggest that human erythrocyte glycoproteins contain, besides the alkali-labile tetrasaccharide, a peptide-linked sialyl-N-acetyl-galactosaminyl-residue. The investigations do not allow a precise definition of the receptor sites for the lectins having M- or N-specificity.

  16. Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

    Science.gov (United States)

    Hunt, J S; McGiven, A R; Groufsky, A; Lynn, K L; Taylor, M C

    1985-05-01

    Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

  17. Preliminary exploration of HLA-A 1101-restricted human cytomegalovirus glycoprotein B-specific CD8⁺ T cells in allogeneic stem-cell transplant recipients.

    Science.gov (United States)

    Liu, Anbing; Hu, Jianhua; Wu, Wei; Huang, Yaping; Liang, Hanying; Wang, Huiqi; Yang, Rong; Fan, Jun

    2014-08-08

    T-cell responses directed against human cytomegalovirus (HCMV) glycoprotein B (gB) contribute to protective immunity against HCMV infection in both animal models and humans. However, the gB-specific human CD8(+) T cell responses remain poorly understood. gB antigen-specific CD8(+) T cells were stained with seven major histocompatibility complex (MHC)-peptide pentamers in 16 human leukocyte antigen (HLA)-A 1101-positive, HCMV-seropositive patients following hematopoietic stem cell transplantation (HSCT). Of these seven pentamers, the most frequent CD8(+) T-cell responses were directed against the gB332-340 peptide. These gB332-340-specific CD8(+) T cells were strongly associated with the presence of plasma HCMV immunoglobulin M in all HSCT recipients and exhibited a probable causal relationship with the level of pp65 antigenemia. Together, these data suggest a role for gB332-340-specific CD8(+) T cells in HCMV reactivation after HSCT. Furthermore, the pentamer assay may be valuable in detecting antigen-specific CD8(+) T cells.

  18. Complex interplay between the P-glycoprotein multidrug efflux pump and the membrane: its role in modulating protein function

    Directory of Open Access Journals (Sweden)

    Frances Jane Sharom

    2014-03-01

    Full Text Available Multidrug resistance in cancer is linked to expression of the P-glycoprotein multidrug transporter (Pgp, ABCB1, which exports many structurally diverse compounds from cells. Substrates first partition into the bilayer and then interact with a large flexible binding pocket within the transporter’s transmembrane regions. Pgp has been described as a hydrophobic vacuum cleaner or an outwardly-directed drug/lipid flippase. Recent X-ray crystal structures have shed some light on the nature of the drug-binding pocket and suggested routes by which substrates can enter it from the membrane. Detergents have profound effects on Pgp function, and several appear to be substrates. Biochemical and biophysical studies in vitro, some using purified reconstituted protein, have explored the effects of the membrane environment. They have demonstrated that Pgp is involved in a complex relationship with its lipid environment, which modulates the behaviour of its substrates, as well as various functions of the protein, including ATP hydrolysis, drug binding and drug transport. Membrane lipid composition and fluidity, phospholipid headgroup and acyl chain length all influence Pgp function. Recent studies focusing on thermodynamics and kinetics have revealed some important principles governing Pgp-lipid and substrate-lipid interactions, and how these affect drug binding and transport. In some cells, Pgp is associated with cholesterol-rich microdomains which may modulate its functions. The relationship between Pgp and cholesterol remains an open question; however it clearly affects several aspects of its function in addition to substrate-membrane partitioning. The action of Pgp modulators appears to depend on their membrane permeability, and membrane fluidizers and surfactants reverse drug resistance, likely via an indirect mechanism. A detailed understanding of how the membrane affects Pgp substrates and Pgp’s catalytic cycle may lead to new strategies to combat

  19. Alloantibodies to human platelet glycoprotein antigens (HPA) and HLA class 1 in a cross section of Nigerian antenatal women.

    Science.gov (United States)

    Jeremiah, Zaccheaus Awortu; Atiegoba, Anne Ifeanyi; Mgbere, Osaro

    2011-01-01

    The prevalence of antibodies to human platelet antigens (HPA) and human leukocyte antigens (HLA) class 1 antigens among Nigerian pregnant women has not been reported in our country. This study was therefore aimed at screening the obstetric population for evidence of alloimmunization due to human platelet and HLA class 1 antigens. One hundred and forty four (144) pregnant women attending the obstetric clinic of Military Hospital, Port Harcourt, participated in the study. Their sera were tested for antibodies to HPA and HLA class 1 antigens using GTI PakPlus solid phase ELISA Kit. The total prevalence rate of antibody production was 60.5% (87 out of 144). Among the positive samples, 60 had platelet glycoprotein specific antibodies (41.7%) and 27 had HLA class 1 antibodies (18.8%). In 39.6% of the pregnant women, both platelet specific antibodies and HLA class 1 antibodies appeared. The prevalence of platelet specific glycoprotein antibodies were obtained as follows: GP 11b/111a 12 (8.3%), GP 1a/11a 35 (20.8%), GP Ib/IX 18 (12.5%) and GP IV 9 (6.3%). The prevalence of each platelet antibody subgroup was obtained as follows: anti-HPA-1a,-3a,-4a (4.2%), anti-HPA-1b,-3b,-4a (4.2%), anti-HPA-30 5a and anti-GP Ib/IX (12.5% each), anti-HPA-5b (8.3%) and anti-GP IV (6.3%). A high prevalence rate of human platelet arid cytotoxic antibodies has been observed in our obstetric population. There is need to establish platelet serology laboratory for the proper antenatal and postnatal management of pregnant mothers in this region.

  20. Preparation and characterization of glycoprotein-resistant starch complex as a coating material for oral bioadhesive microparticles for colon-targeted polypeptide delivery.

    Science.gov (United States)

    Situ, Wenbei; Li, Xiaoxi; Liu, Jia; Chen, Ling

    2015-04-29

    For effective oral delivery of polypeptide or protein and enhancement their oral bioavailability, a new resistant starch-glycoprotein complex bioadhesive carrier and an oral colon-targeted bioadhesive delivery microparticle system were developed. A glycoprotein, concanavalin A (Con A), was successfully conjugated to the molecules of resistant starch acetate (RSA), leading to the formation of resistant starch-glycoprotein complex. This Con A-conjugated RSA film as a coating material showed an excellent controlled-release property. In streptozotocin (STZ)-induced type II diabetic rats, the insulin-loaded microparticles coated with this Con A-conjugated RSA film exhibited good hypoglycemic response for keeping the plasma glucose level within the normal range for totally 44-52 h after oral administration with different insulin dosages. Oral glucose tolerance tests indicated that successive oral administration of these colon-targeted bioadhesive microparticles with insulin at a level of 50 IU/kg could achieve a hypoglycemic effect similar to that by injection of insulin at 35 IU/kg. Therefore, the potential of this new Con A-conjugated RSA film-coated microparticle system has been demonstrated to be capable of improving the oral bioavailability of bioactive proteins and peptides.

  1. Constitutive and functional association of the platelet collagen receptor glycoprotein VI-Fc receptor gamma-chain complex with membrane rafts.

    Science.gov (United States)

    Ezumi, Yasuharu; Kodama, Kumi; Uchiyama, Takashi; Takayama, Hiroshi

    2002-05-01

    The platelet collagen receptor glycoprotein (GP) VI-Fc receptor gamma-chain (FcRgamma) complex transduces signals in an immunoreceptorlike manner. We examined a role for the Triton X-100-insoluble membrane rafts in GPVI-FcRgamma complex signaling. Methyl-beta-cyclodextrin (MbetaCD)-induced disruption of the membrane rafts inhibited not only platelet aggregation and secretion but also tyrosine phosphorylation of signaling molecules on stimulation through the GPVI-FcRgamma complex. The GPVI-FcRgamma complex was constitutively associated with membrane rafts wherein the Src family kinases and LAT were also present. Their association was not affected by the complex engagement but was highly sensitive to MbetaCD treatment. Thus, we provide the first evidence that the GPVI-FcRgamma complex is constitutively and functionally associated with membrane rafts.

  2. 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

    2012-01-01

    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.

  3. Investigation of age-related decline of microfibril-associated glycoprotein-1 in human skin through immunohistochemistry study

    Directory of Open Access Journals (Sweden)

    Zheng Q

    2013-12-01

    Full Text Available Qian Zheng, Siming Chen, Ying Chen, John Lyga, Russell Wyborski, Uma SanthanamGlobal Research and Development, Avon Products Inc., Suffern, New York, USAAbstract: During aging, the reduction of elastic and collagen fibers in dermis can lead to skin atrophy, fragility, and aged appearance, such as increased facial wrinkling and sagging. Microfibril-associated glycoprotein-1 (MAGP-1 is an extracellular matrix protein critical for elastic fiber assembly. It integrates and stabilizes the microfibril and elastin matrix network that helps the skin to endure mechanical stretch and recoil. However, the observation of MAGP-1 during skin aging and its function in the dermis has not been established. To better understand age-related changes in the dermis, we investigated MAGP-1 during skin aging and photoaging, using a combination of in vitro and in vivo studies. Gene expression by microarray was performed using human skin biopsies from young and aged female donors. In addition, immunofluorescence analysis on the MAGP-1 protein was performed in dermal fibroblast cultures and in human skin biopsies. Specific antibodies against MAGP-1 and fibrillin-1 were used to examine protein expression and extracellular matrix structure in the dermis via biopsies from donors of multiple age groups. A reduction of the MAGP-1 gene and protein levels were observed in human skin with increasing age and photoexposure, indicating a loss of the functional MAGP-1 fiber network and a lack of structural support in the dermis. Loss of MAGP-1 around the hair follicle/pore areas was also observed, suggesting a possible correlation between MAGP-1 loss and enlarged pores in aged skin. Our findings demonstrate that a critical “pre-elasticity” component, MAGP-1, declines with aging and photoaging. Such changes may contribute to age-related loss of dermal integrity and perifollicular structural support, which may lead to skin fragility, sagging, and enlarged pores

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

    Science.gov (United States)

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

    2016-11-29

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

  5. Typing of core and backbone domains of mucin-type oligosaccharides from human ovarian-cyst glycoproteins by 500-MHz 1H-NMR spectroscopy

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Mutsaers, J.H.G.M.; Halbeek, H. van; Wu, A.M.; Kabat, E.A.

    1986-01-01

    Human blood-group A active glycoproteins from ovarian-cyst fluid were subjected to Smith degradation and subsequent beta-elimination. The resulting oligosaccharide-alditols represent the core and backbone domains of the O-linked carbohydrate chains. Nine of these, ranging in size from disaccharides

  6. Protein and glycoprotein abnormalities in platelets from human Chediak-Higashi syndrome: polyacrylamide gel electrophoretic study of platelets from five patients.

    Science.gov (United States)

    Ledezma, E; Apitz-Castro, R

    1985-10-01

    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.

  7. Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent

    Energy Technology Data Exchange (ETDEWEB)

    Bienz, D.; Clemetson, K.J.

    1989-01-05

    Glycoprotein Ia (GP Ia) is a relatively minor component of human blood platelets thought to be a receptor involved in collagen-induced platelet activation. However, some difficulties exist with the definition of this glycoprotein. The expression of GP Ia on resting (prostacyclin analogue-treated) and thrombin-activated platelets was compared by surface labeling with /sup 125/I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/(/sup 3/H)NaBH/sub 4/. Analysis on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that GP Ia is very poorly labeled in resting platelets. After activation a new spot (GP Ia*) appears with the same relative molecular mass as GP Ia under reducing conditions. GP Ia and Ia* can be clearly separated by two-dimensional nonreduced/reduced gel electrophoresis. Therefore, two glycoproteins which have been termed GP Ia exist in platelets with similar molecular weight and pI under reducing conditions. One of these (GP Ia*) is only surface-labeled when platelets are activated, indicating that it is only exposed on the surface of activated platelets. Supernatant from activated platelets contains this glycoprotein as well as other granule components. This glycoprotein is missing in platelets from two patients with collagen-response defects.

  8. 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

    2003-12-01

    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.

  9. Envelope glycoprotein of arenaviruses.

    Science.gov (United States)

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

    2012-10-17

    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

    2012-10-01

    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. Simultaneous Pathoproteomic Evaluation of the Dystrophin-Glycoprotein Complex and Secondary Changes in the mdx-4cv Mouse Model of Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Sandra Murphy

    2015-06-01

    Full Text Available In skeletal muscle, the dystrophin-glycoprotein complex forms a membrane-associated assembly of relatively low abundance, making its detailed proteomic characterization in normal versus dystrophic tissues technically challenging. To overcome this analytical problem, we have enriched the muscle membrane fraction by a minimal differential centrifugation step followed by the comprehensive label-free mass spectrometric analysis of microsomal membrane preparations. This organelle proteomic approach successfully identified dystrophin and its binding partners in normal versus dystrophic hind limb muscles. The introduction of a simple pre-fractionation step enabled the simultaneous proteomic comparison of the reduction in the dystrophin-glycoprotein complex and secondary changes in the mdx-4cv mouse model of dystrophinopathy in a single analytical run. The proteomic screening of the microsomal fraction from dystrophic hind limb muscle identified the full-length dystrophin isoform Dp427 as the most drastically reduced protein in dystrophinopathy, demonstrating the remarkable analytical power of comparative muscle proteomics. Secondary pathoproteomic expression patterns were established for 281 proteins, including dystrophin-associated proteins and components involved in metabolism, signalling, contraction, ion-regulation, protein folding, the extracellular matrix and the cytoskeleton. Key findings were verified by immunoblotting. Increased levels of the sarcolemmal Na+/K+-ATPase in dystrophic leg muscles were also confirmed by immunofluorescence microscopy. Thus, the reduction of sample complexity in organelle-focused proteomics can be advantageous for the profiling of supramolecular protein complexes in highly intricate systems, such as skeletal muscle tissue.

  12. A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets.

    Science.gov (United States)

    Tsuji, M; Ezumi, Y; Arai, M; Takayama, H

    1997-09-19

    The mechanism by which occupancy of collagen receptors is coupled to platelet activation has been uncertain. Our group previously demonstrated that glycoprotein (GP) VI, an uncharacterized platelet membrane protein, is specifically required for collagen-platelet interaction leading to activation of protein-tyrosine kinase Syk. Since collagen stimulation of platelets has recently been found to induce tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, a signal-generating subunit of FcR, we further investigated the relationships between FcR gamma-chain and GPVI in human platelets. Our present study revealed the following. FcR gamma-chain was physically and stably associated with GPVI in human platelets; both FcR gamma-chain and GPVI were proportionally absent in GPVI-deficient platelets; GPVI cross-linking or collagen stimulation of platelets resulted in tyrosine phosphorylation of GPVI-associated FcR gamma-chain accompanied by Syk association and activation. These findings strongly suggest that the associated complex of GPVI and FcR gamma-chain is a collagen receptor featuring the signaling through immune receptors.

  13. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-16

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

  14. Glycoprotein synthesis

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-02

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

  15. Glycoprotein synthesis

    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.

    2009-07-14

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

  16. Human pregnancy-specific glycoprotein 1a (PSG1a) induces alternative activation in human and mouse monocytes and suppresses the accessory cell-dependent T cell proliferation.

    Science.gov (United States)

    Motrán, Claudia Cristina; Díaz, Fernando López; Gruppi, Adriana; Slavin, Daniela; Chatton, Bruno; Bocco, José Luis

    2002-09-01

    It has been proposed that pregnancy-specific factors induce the suppression of a specific arm of the maternal response accompanied by activation of the nonspecific, innate immune system. The aim of this study was to determine whether pregnancy-specific glycoprotein 1a (PSG1a), the major variant of PSG polypeptides, is able to modulate the monocyte/macrophage (Mo) metabolism to regulate T cell activation and proliferation. Using the recombinant form of this glycoprotein (rec-PSG1a), expressed in mammalian cells with a vaccinia-based expression vector, we have demonstrated that human PSG1a induces arginase activity in peripheral blood human Mo and human and murine Mo cell lines. In addition, rec-PSG1a is able to induce alternative activation because it up-regulates the arginase activity and inhibits the nitric oxide production in Mo activated by lipopolysaccharides. We also observed that rec-PSG1a is an important accessory cells-dependent T cell suppressor factor that causes partial growth arrest at the S/G2/M phase of the cell cycle. Additionally, an impaired T cell proliferative response induced by mitogens and specific antigen was observed in BALB/c mice upon in vivo expression of PSG1a. Our results suggest that PSG1a function contributes to the immunomodulation during pregnancy, having opposite effects on maternal innate and adaptative systems.

  17. HIV-1 gp120 envelope glycoprotein determinants for cytokine burst in human monocytes

    Science.gov (United States)

    Coutu, Mathieu; Prévost, Jérémie; Brassard, Nathalie; Peres, Adam; Stegen, Camille; Madrenas, Joaquín; Kaufmann, Daniel E.; Finzi, Andrés

    2017-01-01

    The first step of HIV infection involves the interaction of the gp120 envelope glycoprotein to its receptor CD4, mainly expressed on CD4+ T cells. Besides its role on HIV-1 entry, the gp120 has been shown to be involved in the production of IL-1, IL-6, CCL20 and other innate response cytokines by bystander, uninfected CD4+ T cells and monocytes. However, the gp120 determinants involved in these functions are not completely understood. Whether signalling leading to cytokine production is due to CD4 or other receptors is still unclear. Enhanced chemokine receptor binding and subsequent clustering receptors may lead to cytokine production. By using a comprehensive panel of gp120 mutants, here we show that CD4 binding is mandatory for cytokine outburst in monocytes. Our data suggest that targeting monocytes in HIV-infected patients might decrease systemic inflammation and the potential tissue injury associated with the production of inflammatory cytokines. Understanding how gp120 mediates a cytokine burst in monocytes might help develop new approaches to improve the chronic inflammation that persists in these patients despite effective suppression of viremia by antiretroviral therapy. PMID:28346521

  18. Reversion of P-Glycoprotein-Mediated Multidrug Resistance in Human Leukemic Cell Line by Diallyl Trisulfide

    Directory of Open Access Journals (Sweden)

    Qing Xia

    2012-01-01

    Full Text Available Multidrug resistance (MDR is the major obstacle in chemotherapy, which involves multiple signaling pathways. Diallyl trisulfide (DATS is the main sulfuric compound in garlic. In the present study, we aimed to explore whether DATS could overcome P-glycoprotein-(P-gp-mediated MDR in K562/A02 cells, and to investigate whether NF-κB suppression is involved in DATS-induced reversal of MDR. MTT assay revealed that cotreatment with DATS increased the response of K562/A02 cells to adriamycin (the resistance reversal fold was 3.79 without toxic side effects. DATS could enhance the intracellular concentration of adriamycin by inhibiting the function and expression of P-gp, as shown by flow cytometry, RT-PCR, and western blot. In addition, DATS resulted in more K562/A02 cell apoptosis, accompanied by increased expression of caspase-3. The expression of NF-κB/p65 (downregulation was significantly linked to the drug-resistance mechanism of DATS, whereas the expression of IκBα was not affected by DATS. Our findings demonstrated that DATS can serve as a novel, nontoxic modulator of MDR, and can reverse the MDR of K562/A02 cells in vitro by increasing intracellular adriamycin concentration and inducing apoptosis. More importantly, we proved for the first time that the suppression of NF-κB possibly involves the molecular mechanism in the course of reversion by DATS.

  19. Comparison of Methods for the Purification of Alpha-1 Acid Glycoprotein from Human Plasma

    Directory of Open Access Journals (Sweden)

    Teresa R. McCurdy

    2011-01-01

    Full Text Available Alpha-1 acid glycoprotein (AGP is a highly glycosylated, negatively charged plasma protein suggested to have anti-inflammatory and/or immunomodulatory activities. Purification of AGP could be simplified if methods that exploit its high solubility under chemically harsh conditions could be demonstrated to leave the protein in its native conformation. Procedures involving exposure of AGP to hot phenol or sulphosalicylic acid (SSA were compared to solely chromatographic methods. Hot phenol-purified AGP was more rapidly cleared from mice in vivo following intravenous injection than chromatographically purified AGP. In contrast, SSA-purified AGP demonstrated an identical in vivo clearance profile and circular dichroism spectrum to chromatographically purified AGP. Similarly, no differences in susceptibility to enzymatic deglycosylation or reactivity with Sambucus nigra lectin were detected between AGP purified via the two methods. Incorporation of the SSA step in the purification scheme for AGP eliminated the need for a large (4 mL resin/mL of plasma initial chromatographic step and simplified its purification without causing any detectable distortion in the conformation of the protein. Confirmation that this procedure is nondenaturing will simplify AGP purification and investigation of its possible biological roles in laboratory animals.

  20. Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

    Science.gov (United States)

    2008-12-23

    contributors Abstract Background: Sera from convalescent Lassa fever patients often contains antibodies to Lassa virus (LASV) glycoprotein 1 (GP1...uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever . To this end, mammalian expression systems were engineered for...stages of Lassa fever . Published: 23 December 2008 Virology Journal 2008, 5:161 doi:10.1186/1743-422X-5-161 Received: 14 December 2008 Accepted: 23

  1. Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

    Science.gov (United States)

    2008-12-23

    contributors Abstract Background: Sera from convalescent Lassa fever patients often contains antibodies to Lassa virus (LASV) glycoprotein 1 (GP1...uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever . To this end, mammalian expression systems were engineered for...stages of Lassa fever . Published: 23 December 2008 Virology Journal 2008, 5:161 doi:10.1186/1743-422X-5-161 Received: 14 December 2008 Accepted: 23

  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

    1999-01-01

    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...

  3. Drug-binding energetics of human α-1-acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

    Science.gov (United States)

    Huang, Johnny X.; Cooper, Matthew A.; Baker, Mark A.; Azad, Mohammad A.K.; Nation, Roger L.; Li, Jian; Velkov, Tony

    2012-01-01

    This study utilizes sensitive, modern isothermal titration calorimetric (ITC) methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α-1-acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug-AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug-binding thermodynamics were characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed enthalpy-entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (ΔH°) and favorable (positive) entropic (ΔS°) contributions to the Gibbs free energy (ΔG°). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug-serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side-chain sub-domains within the multi-lobed AGP ligand binding cavity. PMID:23192962

  4. The Transmission Interfaces Contribute Asymmetrically to the Assembly and Activity of Human P-glycoprotein.

    Science.gov (United States)

    Loo, Tip W; Clarke, David M

    2015-07-01

    P-glycoprotein (P-gp; ABCB1) is an ABC drug pump that protects us from toxic compounds. It is clinically important because it confers multidrug resistance. The homologous halves of P-gp each contain a transmembrane (TM) domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Each NBD is connected to the TMDs by a transmission interface involving a pair of intracellular loops (ICLs) that form ball-and-socket joints. P-gp is different from CFTR (ABCC7) in that deleting NBD2 causes misprocessing of only P-gp. Therefore, NBD2 might be critical for stabilizing ICLs 2 and 3 that form a tetrahelix bundle at the NBD2 interface. Here we report that the NBD1 and NBD2 transmission interfaces in P-gp are asymmetric. Point mutations to 25 of 60 ICL2/ICL3 residues at the NBD2 transmission interface severely reduced P-gp assembly while changes to the equivalent residues in ICL1/ICL4 at the NBD1 interface had little effect. The hydrophobic nature at the transmission interfaces was also different. Mutation of Phe-1086 or Tyr-1087 to arginine at the NBD2 socket blocked activity or assembly while the equivalent mutations at the NBD1 socket had only modest effects. The results suggest that the NBD transmission interfaces are asymmetric. In contrast to the ICL2/3-NBD2 interface, the ICL1/4-NBD1 transmission interface is more hydrophilic and insensitive to mutations. Therefore the ICL2/3-NBD2 transmission interface forms a precise hydrophobic connection that acts as a linchpin for assembly and trafficking of P-gp.

  5. In Vitro Inhibitory Effects of Scutellarin on Six Human/Rat Cytochrome P450 Enzymes and P-glycoprotein

    Directory of Open Access Journals (Sweden)

    Yong-Long Han

    2014-05-01

    Full Text Available Inhibition of cytochrome P450 (CYP and P-glycoprotein (P-gp are regarded as the most frequent and clinically important pharmacokinetic causes among the various possible factors for drug-drug interactions. Scutellarin is a flavonoid which is widely used for the treatment of cardiovascular diseases. In this study, the in vitro inhibitory effects of scutellarin on six major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 and six rat CYPs (CYP1A2, CYP2C7, CYP2C11, CYP2C79, CYP2D4, and CYP3A2 activities were examined by using liquid chromatography-tandem mass spectrometry. Meanwhile, the inhibitory effects of scutellarin on P-gp activity were examined on a human metastatic malignant melanoma cell line WM-266-4 by calcein-AM fluorometry screening assay. Results demonstrated that scutellarin showed negligible inhibitory effects on the six major CYP isoenzymes in human/rat liver microsomes with almost all of the IC50 values exceeding 100 μM, whereas it showed values of 63.8 μM for CYP2C19 in human liver microsomes, and 63.1 and 85.6 μM for CYP2C7 and CYP2C79 in rat liver microsomes, respectively. Scutellarin also showed weak inhibitory effect on P-gp. In conclusion, this study demonstrates that scutellarin is unlikely to cause any clinically significant herb-drug interactions in humans when co-administered with substrates of the six CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 and P-gp.

  6. Herpes simplex virus type 2 glycoprotein H interacts with integrin αvβ3 to facilitate viral entry and calcium signaling in human genital tract epithelial cells.

    Science.gov (United States)

    Cheshenko, Natalia; Trepanier, Janie B; González, Pablo A; Eugenin, Eliseo A; Jacobs, William R; Herold, Betsy C

    2014-09-01

    Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These

  7. Effect of multidrug resistance 1/P-glycoprotein on the hypoxia-induced multidrug resistance of human laryngeal cancer cells.

    Science.gov (United States)

    Li, Dawei; Zhou, Liang; Huang, Jiameng; Xiao, Xiyan

    2016-08-01

    In a previous study, it was demonstrated that hypoxia upregulated the multidrug resistance (MDR) of laryngeal cancer cells to chemotherapeutic drugs, with multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp) expression also being upregulated. The present study aimed to investigate the role and mechanism of MDR1/P-gp on hypoxia-induced MDR in human laryngeal carcinoma cells. The sensitivity of laryngeal cancer cells to multiple drugs and cisplatin-induced apoptosis was determined by CCK-8 assay and Annexin-V/propidium iodide staining analysis, respectively. The accumulation of rhodamine 123 (Rh123) in the cells served as an estimate of drug accumulation and was evaluated by flow cytometry (FCM). MDR1/P-gp expression was inhibited using interference RNA, and the expression of the MDR1 gene was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. As a result, the sensitivity to multiple chemotherapeutic agents and the apoptosis rate of the hypoxic laryngeal carcinoma cells increased following a decrease in MDR1/P-gp expression (PP-gp markedly increased intracellular Rh123 accumulation (PP-gp serves an important role in regulating hypoxia-induced MDR in human laryngeal carcinoma cells through a decrease in intracellular drug accumulation.

  8. High-throughput 1,536-well fluorescence polarization assays for α(1-acid glycoprotein and human serum albumin binding.

    Directory of Open Access Journals (Sweden)

    Adam Yasgar

    Full Text Available Two major plasma proteins in humans are primarily responsible for drug binding, the α(1-acid-glycoprotein (AGP and human serum albumin (HSA. The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC(1280 collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets.

  9. Collagen-stimulated activation of Syk but not c-Src is severely compromised in human platelets lacking membrane glycoprotein VI.

    Science.gov (United States)

    Ichinohe, T; Takayama, H; Ezumi, Y; Arai, M; Yamamoto, N; Takahashi, H; Okuma, M

    1997-01-03

    Activation of circulating platelets by subendothelial collagen is an essential event in vascular hemostasis. In human platelets, two membrane glycoprotein (GP) abnormalities, integrin alpha2 beta1 deficiency and GPVI deficiency, have been reported to result in severe hyporesponsiveness to fibrillar collagen. Although it has been well established that integrin alpha2 beta1, also known as the GPIa-IIa complex, functions as a primary platelet adhesion receptor for collagen, the mechanism by which GPVI contributes to collagen-platelet interaction has been ill defined to date. However, our recent observation that GPVI cross-linking couples to cyclic AMP-insensitive activation of c-Src and Syk tyrosine kinases suggested a potential role for GPVI in regulating protein-tyrosine phosphorylation by collagen (Ichinohe, T., Takayama, H., Ezumi, Y., Yanagi, S., Yamamura, H., and Okuma, M. (1995) J. Biol. Chem. 270, 28029-28036). To further investigate this hypothesis, here we examined the collagen-induced protein-tyrosine phosphorylation in GPVI-deficient platelets expressing normal amounts of alpha2 beta1. In response to collagen, these platelets exhibited alpha2 beta1-dependent c-Src activation accompanied by tyrosine phosphorylation of several substrates including cortactin. In contrast, severe defects were observed in collagen-stimulated Syk activation and tyrosine phosphorylation of phospholipase C-gamma2, Vav, and focal adhesion kinase, implicating a specific requirement of GPVI for recruiting these molecules to signaling cascades evoked by collagen-platelet interaction.

  10. Innate immunity glycoprotein gp-340 variants may modulate human susceptibility to dental caries

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    Johansson Ingegerd

    2007-06-01

    Full Text Available Abstract Background Bacterial adhesion is an important determinant of colonization and infection, including dental caries. The salivary scavenger receptor cysteine-rich glycoprotein gp-340, which mediates adhesion of Streptococcus mutans (implicated in caries, harbours three major size variants, designated gp-340 I to III, each specific to an individual saliva. Here we have examined the association of the gp-340 I to III polymorphisms with caries experience and adhesion of S. mutans. Methods A case-referent study was performed in 12-year-old Swedish children with high (n = 19 or low (n = 19 caries experiences. We measured the gp-340 I to III saliva phenotypes and correlated those with multiple outcome measures for caries experience and saliva adhesion of S. mutans using the partial least squares (PLS multivariate projection technique. In addition, we used traditional statistics and 2-year caries increment to verify the established PLS associations, and bacterial adhesion to purified gp-340 I to III proteins to support possible mechanisms. Results All except one subject were typed as gp-340 I to III (10, 23 and 4, respectively. The gp-340 I phenotype correlated positively with caries experience (VIP = 1.37 and saliva adhesion of S. mutans Ingbritt (VIP = 1.47. The gp-340 II and III phenotypes tended to behave in the opposite way. Moreover, the gp-340 I phenotype tended to show an increased 2-year caries increment compared to phenotypes II/III. Purified gp-340 I protein mediated markedly higher adhesion of S. mutans strains Ingbritt and NG8 and Lactococcus lactis expressing AgI/II adhesins (SpaP or PAc compared to gp-340 II and III proteins. In addition, the gp-340 I protein appeared over represented in subjects positive for Db, an allelic acidic PRP variant associated with caries, and subjects positive for both gp-340 I and Db tended to experience more caries than those negative for both proteins. Conclusion Gp-340 I behaves as a caries

  11. Despite WT1 binding sites in the promoter region of human and mouse nucleoporin glycoprotein 210, WT1 does not influence expression of GP210

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    Licht Jonathan D

    2004-12-01

    Full Text Available Abstract Background Glycoprotein 210 (GP210 is a transmembrane component of the nuclear pore complex of metazoans, with a short carboxyterminus protruding towards the cytoplasm. Its function is unknown, but it is considered to be a major structural component of metazoan nuclear pores. Yet, our previous findings showed pronounced differences in expression levels in embryonic mouse tissues and cell lines. In order to identify factors regulating GP210, the genomic organization of human GP210 was analyzed in silico. Results The human gene was mapped to chromosome 3 and consists of 40 exons spread over 102 kb. The deduced 1887 amino acid showed a high degree of alignment homology to previously reported orthologues. Experimentally we defined two transcription initiation sites, 18 and 29 bp upstream of the ATG start codon. The promoter region is characterized by a CpG island and several consensus binding motifs for gene regulatory transcription factors, including clustered sites associated with Sp1 and the Wilms' tumor suppressor gene zinc finger protein (WT1. In addition, distal to the translation start we found a (GTn repetitive sequence, an element known for its ability to bind WT1. Homologies for these motifs could be identified in the corresponding mouse genomic region. However, experimental tetracycline dependent induction of WT1 in SAOS osteosarcoma cells did not influence GP210 transcription. Conclusion Although mouse GP210 was identified as an early response gene during induced metanephric kidney development, and WT1 binding sites were identified in the promoter region of the human GP210 gene, experimental modulation of WT1 expression did not influence expression of GP210. Therefore, WT1 is probably not regulating GP210 expression. Instead, we suggest that the identified Sp binding sites are involved.

  12. Approaching human language with complex networks

    Science.gov (United States)

    Cong, Jin; Liu, Haitao

    2014-12-01

    The interest in modeling and analyzing human language with complex networks is on the rise in recent years and a considerable body of research in this area has already been accumulated. We survey three major lines of linguistic research from the complex network approach: 1) characterization of human language as a multi-level system with complex network analysis; 2) linguistic typological research with the application of linguistic networks and their quantitative measures; and 3) relationships between the system-level complexity of human language (determined by the topology of linguistic networks) and microscopic linguistic (e.g., syntactic) features (as the traditional concern of linguistics). We show that the models and quantitative tools of complex networks, when exploited properly, can constitute an operational methodology for linguistic inquiry, which contributes to the understanding of human language and the development of linguistics. We conclude our review with suggestions for future linguistic research from the complex network approach: 1) relationships between the system-level complexity of human language and microscopic linguistic features; 2) expansion of research scope from the global properties to other levels of granularity of linguistic networks; and 3) combination of linguistic network analysis with other quantitative studies of language (such as quantitative linguistics).

  13. P-glycoprotein-170 inhibition significantly reduces cortisol and ciclosporin efflux from human intestinal epithelial cells and T lymphocytes.

    Science.gov (United States)

    Farrell, R J; Menconi, M J; Keates, A C; Kelly, C P

    2002-05-01

    To assess the role of P-glycoprotein-170 (P-gp) in transporting cortisol and ciclosporin from human intestinal epithelium and T lymphocytes. The effect of P-gp inhibitors (verapamil, 0-100 microM; PSC 833, 0-20 microM) on the intracellular accumulation of 3H-cortisol and 3H-ciclosporin was studied in confluent layers of human Caco-2 cells (n=6), a P-gp-dependent absorptive intestinal epithelial cell phenotype, and moderately resistant MDRhigh CEM/VBL 100 T cells (n=6). The transport of 3H-vinblastine, a strong multidrug resistance (MDR) substrate, and 3H-progesterone, a poor MDR substrate, was also studied. Caco-2 cells had a 2.4-, 6.6-, 6.7- and 1.03-fold higher net basal to apical transport (efflux) of 3H-cortisol, 3H-ciclosporin, 3H-vinblastine and 3H-progesterone, respectively. PSC 833 (20 microM) reduced cortisol efflux by 69% (0.23 +/- 0.04 to 0.07 +/- 0.01 pmol/cm2/h, P 0.1 microM) and verapamil (> 1 microM) restored the intracellular level of 3H-cortisol and 3H-ciclosporin in MDRhigh CEM/VBL 100 T cells to that of MDRlow CEM cells with little change in accumulation in the MDRlow parental cell line. P-gp inhibitors significantly increase intracellular cortisol and ciclosporin levels in human intestinal epithelium and T lymphocytes in a dose-dependent manner, demonstrating a potential mechanism for overcoming poor response to immunosuppressant therapy in refractory inflammatory bowel disease.

  14. Investigation of age-related decline of microfibril-associated glycoprotein-1 in human skin through immunohistochemistry study.

    Science.gov (United States)

    Zheng, Qian; Chen, Siming; Chen, Ying; Lyga, John; Wyborski, Russell; Santhanam, Uma

    2013-01-01

    During aging, the reduction of elastic and collagen fibers in dermis can lead to skin atrophy, fragility, and aged appearance, such as increased facial wrinkling and sagging. Microfibril-associated glycoprotein-1 (MAGP-1) is an extracellular matrix protein critical for elastic fiber assembly. It integrates and stabilizes the microfibril and elastin matrix network that helps the skin to endure mechanical stretch and recoil. However, the observation of MAGP-1 during skin aging and its function in the dermis has not been established. To better understand age-related changes in the dermis, we investigated MAGP-1 during skin aging and photoaging, using a combination of in vitro and in vivo studies. Gene expression by microarray was performed using human skin biopsies from young and aged female donors. In addition, immunofluorescence analysis on the MAGP-1 protein was performed in dermal fibroblast cultures and in human skin biopsies. Specific antibodies against MAGP-1 and fibrillin-1 were used to examine protein expression and extracellular matrix structure in the dermis via biopsies from donors of multiple age groups. A reduction of the MAGP-1 gene and protein levels were observed in human skin with increasing age and photoexposure, indicating a loss of the functional MAGP-1 fiber network and a lack of structural support in the dermis. Loss of MAGP-1 around the hair follicle/pore areas was also observed, suggesting a possible correlation between MAGP-1 loss and enlarged pores in aged skin. Our findings demonstrate that a critical "pre-elasticity" component, MAGP-1, declines with aging and photoaging. Such changes may contribute to age-related loss of dermal integrity and perifollicular structural support, which may lead to skin fragility, sagging, and enlarged pores.

  15. CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization.

    Science.gov (United States)

    Sundling, Christopher; Schön, Karin; Mörner, Andreas; Forsell, Mattias N E; Wyatt, Richard T; Thorstensson, Rigmor; Karlsson Hedestam, Gunilla B; Lycke, Nils Y

    2008-12-01

    Strategies to induce potent and broad antibody responses against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) at both systemic and mucosal sites represent a central goal for HIV-1 vaccine development. Here, we show that the non-toxic CTA1-DD adjuvant promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal (i.n.) immunizations with trimeric or monomeric forms of HIV-1 Env in mice and in non-human primates. Env-specific IgG subclasses in the serum of immunized mice reflected a balanced Th1/Th2 type of response. Strikingly, i.n. immunizations with Env and the CTA1-DD adjuvant induced substantial levels of mucosal anti-Env IgA in bronchial alveolar lavage and also detectable levels in vaginal secretions. By contrast, parenteral immunizations of Env formulated in Ribi did not stimulate mucosal IgA responses, while the two adjuvants induced a similar distribution of Env-specific IgG-subclasses in serum. A single parenteral boost with Env in Ribi adjuvant into mice previously primed i.n. with Env and CTA1-DD, augmented the serum anti-Env IgG levels to similar magnitudes as those observed after three intraperitoneal immunizations with Env in Ribi. The augmenting potency of CTA1-DD was similar to that of LTK63 or CpG oligodeoxynucleotides (ODN). However, in contrast to CpG ODN, the effect of CTA1-DD and LTK63 appeared to be independent of MyD88 and toll-like receptor signalling. This is the first demonstration that CTA1-DD augments specific immune responses also in non-human primates, suggesting that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for humoral and cell-mediated immunity against HIV-1 Env.

  16. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    Science.gov (United States)

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

    2009-01-01

    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.

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

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    Ian Loke

    2015-08-01

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

  18. Inhibition of human immunodeficiency virus (HIV) infection in vitro by anticarbohydrate monoclonal antibodies: peripheral glycosylation of HIV envelope glycoprotein gp120 may be a target for virus neutralization

    DEFF Research Database (Denmark)

    Hansen, J E; Clausen, H; Nielsen, C

    1990-01-01

    Carbohydrate structures are often involved in the initial adhesion of pathogens to target cells. In the present study, a panel of anticarbohydrate monoclonal antibodies (MAbs) was tested for their ability to inhibit in vitro human immunodeficiency virus infectivity. MAbs against three different N...... carbohydrate structures expressed by the viral envelope glycoprotein gp120, indicating that glycans of the viral envelope are possible targets for immunotherapy or vaccine development or both....

  19. Structural basis for penetration of the glycan shield of hepatitis C virus E2 glycoprotein by a broadly neutralizing human antibody.

    Science.gov (United States)

    Li, Yili; Pierce, Brian G; Wang, Qian; Keck, Zhen-Yong; Fuerst, Thomas R; Foung, Steven K H; Mariuzza, Roy A

    2015-04-17

    Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2(412-423)) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2(412-423) epitope to 2.0 Å resolution. The conformation of E2(412-423) bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2(412-423) complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2(412-423) epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

  20. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

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    Sofie Ignoul

    and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal versus overexpressed (early and recycling endosomal ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II, and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes.

  1. Complexities in human herpesvirus-6A and -6B binding to host cells

    DEFF Research Database (Denmark)

    Pedersen, Simon Metz; Höllsberg, Per

    2006-01-01

    Human herpesvirus-6A and -6B uses the cellular receptor CD46 for fusion and infection of the host cell. The viral glycoprotein complex gH-gL from HHV-6A binds to the short consensus repeat 2 and 3 in CD46. Although all the major isoforms of CD46 bind the virus, certain isoforms may have higher...... affinity than others for the virus. Within recent years, elucidation of the viral complex has identified additional HHV-6A and -6B specific glycoproteins. Thus, gH-gL associates with a gQ1-gQ2 dimer to form a heterotetrameric complex. In addition, a novel complex consisting of gH-gL-gO has been described...... that does not bind CD46. Accumulating evidence suggests that an additional HHV-6A and -6B receptor exists. The previous simple picture of HHV-6A/B-host cell contact therefore includes more layers of complexities on both the viral and the host cell side of the interaction....

  2. Repertoire of Epitopes Recognized by Serum IgG from Humans Vaccinated with Herpes Simplex Virus 2 Glycoprotein D

    Science.gov (United States)

    Huang, Zhen-Yu; Cairns, Tina M.; Gallagher, John R.; Lou, Huan; Ponce-de-Leon, Manuel; Belshe, Robert B.; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366:34–43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. IMPORTANCE Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2

  3. Michaelis-Menten kinetic analysis of drugs of abuse to estimate their affinity to human P-glycoprotein.

    Science.gov (United States)

    Meyer, Markus R; Orschiedt, Tina; Maurer, Hans H

    2013-02-27

    The pharmacokinetics of various important drugs are known to be significantly influenced by the human ABC transporter P-glycoprotein (P-gp), which may lead to clinically relevant drug-drug interactions. In contrast to therapeutic drugs, emerging drugs of abuse (DOA) are sold and consumed without any safety pharmacology testing. Only some studies on their metabolism were published, but none about their affinity to the transporter systems. Therefore, 47 DOAs from various classes were tested for their P-gp affinity using human P-gp (hP-gp) to predict possible drug-drug interactions. DOAs were initially screened for general hP-gp affinity and further characterized by modeling classic Michaelis-Menten kinetics and assessing their K(m) and V(max) values. Among the tested drugs, 12 showed a stimulation of ATPase activity. The most intensive stimulating DOAs were further investigated and compared with the known P-gp model substrates sertraline and verapamil. ATPase stimulation kinetics could be modeled for the entactogen 3,4-methylenedioxy-α-ethylphenethylamine (3,4-BDB), the hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), the abused alkaloid glaucine, the opioid-like drugs N-iso-propyl-1,2-diphenylethylamine (NPDPA), and N-(1-phenylcyclohexyl)-3-ethoxypropanamine (PCEPA), with K(m) and V(max) values within the same range as for verapamil or sertraline. As a consequence interactions with other drugs being P-gp substrates might be considered to be very likely and further studies should be encouraged. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  4. Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoids.

    Science.gov (United States)

    Ramesh, Radha; Kozhaya, Lina; McKevitt, Kelly; Djuretic, Ivana M; Carlson, Thaddeus J; Quintero, Maria A; McCauley, Jacob L; Abreu, Maria T; Unutmaz, Derya; Sundrud, Mark S

    2014-01-13

    IL-17A-expressing CD4(+) T cells (Th17 cells) are generally regarded as key effectors of autoimmune inflammation. However, not all Th17 cells are pro-inflammatory. Pathogenic Th17 cells that induce autoimmunity in mice are distinguished from nonpathogenic Th17 cells by a unique transcriptional signature, including high Il23r expression, and these cells require Il23r for their inflammatory function. In contrast, defining features of human pro-inflammatory Th17 cells are unknown. We show that pro-inflammatory human Th17 cells are restricted to a subset of CCR6(+)CXCR3(hi)CCR4(lo)CCR10(-)CD161(+) cells that transiently express c-Kit and stably express P-glycoprotein (P-gp)/multi-drug resistance type 1 (MDR1). In contrast to MDR1(-) Th1 or Th17 cells, MDR1(+) Th17 cells produce both Th17 (IL-17A, IL-17F, and IL-22) and Th1 (IFN-γ) cytokines upon TCR stimulation and do not express IL-10 or other anti-inflammatory molecules. These cells also display a transcriptional signature akin to pathogenic mouse Th17 cells and show heightened functional responses to IL-23 stimulation. In vivo, MDR1(+) Th17 cells are enriched and activated in the gut of Crohn's disease patients. Furthermore, MDR1(+) Th17 cells are refractory to several glucocorticoids used to treat clinical autoimmune disease. Thus, MDR1(+) Th17 cells may be important mediators of chronic inflammation, particularly in clinical settings of steroid resistant inflammatory disease.

  5. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

    Directory of Open Access Journals (Sweden)

    Weibin Zha

    Full Text Available BACKGROUND: HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages. METHODOLOGY AND PRINCIPAL FINDINGS: Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp. CONCLUSION AND SIGNIFICANCE: HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

  6. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    Science.gov (United States)

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals.

  7. Molecular Characterization of the Interactions between Vascular Selectins and Glycoprotein Ligands on Human Hematopoietic Stem/Progenitor Cells

    KAUST Repository

    Abusamra, Dina

    2016-12-01

    The human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E

  8. Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

    Science.gov (United States)

    Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

    2017-05-15

    The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C(Δ170)). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation.IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect

  9. Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

    KAUST Repository

    Merzaban, Jasmeen S.

    2011-06-09

    Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin-/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved byWestern blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand- 1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL."E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL\\'s contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. © 2011 by The American Society of Hematology.

  10. Antibodies to phosphatidylserine/prothrombin complex in suspected antiphospholipid syndrome in the absence of antibodies to cardiolipin or Beta-2-glycoprotein I.

    Science.gov (United States)

    Sanfelippo, M J; Joshi, A; Schwartz, Sl; Meister, J A; Goldberg, J W

    2013-11-01

    Antibodies to phosphatidylserine/prothrombin (aPS/PT) complex were measured in 728 serum specimens from patients suspected of having antiphospholipid syndrome (APS), but without diagnostic elevations in the levels of antibodies to cardiolipin or Beta-2 Glycoprotein 1 (β2-GP1). Of the 728 specimens, 41 had elevated levels of aPS/PT. Thrombotic events occurred in 11 of the 22 patients with accessible medical histories. Six of the patients with accessible medical records also had laboratory evidence of the lupus anticoagulant. The identification of aPS/PT in patients without evidence of antibodies to cardiolipin, β2-GP1, or the lupus anticoagulant can contribute to the identification of APS in patients that may go undetected with current testing methods.

  11. Successful post-exposure prophylaxis of Ebola infected non-human primates using Ebola glycoprotein-specific equine IgG

    Science.gov (United States)

    Pyankov, Oleg V.; Setoh, Yin Xiang; Bodnev, Sergey A.; Edmonds, Judith H.; Pyankova, Olga G.; Pyankov, Stepan A.; Pali, Gabor; Belford, Shane; Lu, Louis; La, Mylinh; Lovrecz, George; Volchkova, Valentina A.; Chappell, Keith J.; Watterson, Daniel; Marsh, Glenn; Young, Paul R.; Agafonov, Alexander A.; Farmer, Jillann F.; Volchkov, Victor E.; Suhrbier, Andreas; Khromykh, Alexander A.

    2017-01-01

    Herein we describe production of purified equine IgG obtained from horses immunized with plasmid DNA followed by boosting with Kunjin replicon virus-like particles both encoding a modified Ebola glycoprotein. Administration of the equine IgG over 5 days to cynomolgus macaques infected 24 hours previously with a lethal dose of Ebola virus suppressed viral loads by more than 5 logs and protected animals from mortality. Animals generated their own Ebola glycoprotein-specific IgG responses 9–15 days after infection, with circulating virus undetectable by day 15–17. Such equine IgG may find utility as a post-exposure prophylactic for Ebola infection and provides a low cost, scalable alternative to monoclonal antibodies, with extensive human safety data and WHO-standardized international manufacturing capability available in both high and low income countries. PMID:28155869

  12. Cross-Reactive Human IgM-Derived Monoclonal Antibodies that Bind to HIV-1 Envelope Glycoproteins

    Directory of Open Access Journals (Sweden)

    Barton F. Haynes

    2010-02-01

    Full Text Available Elicitation of antibodies with potent and broad neutralizing activity against HIV by immunization remains a challenge. Several monoclonal antibodies (mAbs isolated from humans with HIV-1 infection exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. All known broadly neutralizing mAbs (bnmAbs are immunoglobulin (Ig Gs (IgGs and highly somatically hypermutated which could impede their elicitation. Ig Ms (IgMs are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to IgGs. Here we describe the identification and characterization of several human IgM-derived mAbs against HIV-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. These antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, Envs of HIV-1 isolates from different clades. They enhanced or did not neutralize infection by some of the HIV-1 primary isolates using CCR5 as a coreceptor but neutralized all CXCR4 isolates tested although weakly. One of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. It bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the CD4 binding site. These results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. Further studies will show whether such a strategy plays a role in HIV infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. The newly identified mAbs could be used as reagents to further

  13. A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein.

    Science.gov (United States)

    Chan, Annie Hoi Yi; Tan, Hwee Cheng; Chow, Angelia Yee; Lim, Angeline Pei Chiew; Lok, Shee Mei; Moreland, Nicole J; Vasudevan, Subhash G; MacAry, Paul A; Ooi, Eng Eong; Hanson, Brendon J

    2012-01-01

    Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.

  14. Interaction of the recently approved anticancer drug nintedanib with human acute phase reactant α 1-acid glycoprotein

    Science.gov (United States)

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

    2016-07-01

    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.

  15. Human placental extract mediated inhibition of proteinase K: implications of heparin and glycoproteins in wound physiology.

    Science.gov (United States)

    Sharma, Kanika; Mukherjee, Chaitali; Roy, Siddhartha; De, Debashree; Bhattacharyya, Debasish

    2014-09-01

    Efficient debridement of the wound bed following the removal of microbial load prevents its progression into a chronic wound. Bacterial infection and excessive proteolysis characterize impaired healing and therefore, their inhibition might restore the disturbed equilibrium in the healing process. Human placental extract exhibits reversible, non-competitive inhibition towards Proteinase K, a microbial protease, by stabilizing it against auto-digestion. Scattering and fluorescence studies followed by biochemical analysis indicated the involvement of a glycan moiety. Surface plasmon resonance demonstrated specific interaction of heparin with Proteinase K having Kd in μM range. Further, Proteinase K contains sequence motifs similar to other heparin-binding proteins. Molecular docking revealed presence of clefts suitable for binding of heparin-derived oligosaccharides. Comprehensive analysis of this inhibitory property of placental extract partly explains its efficacy in curing wounds with common bacterial infections.

  16. Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

    Directory of Open Access Journals (Sweden)

    Pierre Becquart

    Full Text Available Ebola virus (EBOV is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP, Nucleoprotein (NP, and matrix Viral Protein (VP40 and VP35. For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms and the late memory phase (7-12 years post-infection. We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects. We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

  17. Identification of continuous human B-cell epitopes in the envelope glycoprotein of dengue virus type 3 (DENV-3.

    Directory of Open Access Journals (Sweden)

    Andréa N M Rangel da Silva

    Full Text Available BACKGROUND: Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. METHODOLOGY/PRINCIPAL FINDINGS: Synthetic peptides were used to identify B-cell epitopes in the envelope (E glycoprotein of dengue virus type 3 (DENV-3. Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa positions: 51-65, 71-90, 131-170, 196-210 and 246-260 were identified by employing an enzyme- linked immunosorbent assay (ELISA, using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51-65, 27 and 28 (aa131-150 also reacted with dengue 1 (DENV-1 and dengue 2 (DENV-2 patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51-65, 131-170, 196-210 and 246-260 elicited the highest antibody response and epitopes131-170, 196-210 and 246-260, elicited IFN-gamma production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. CONCLUSIONS/SIGNIFICANCE: Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine.

  18. Human Error Mechanisms in Complex Work Environments

    DEFF Research Database (Denmark)

    Rasmussen, Jens

    1988-01-01

    will account for most of the action errors observed. In addition, error mechanisms appear to be intimately related to the development of high skill and know-how in a complex work context. This relationship between errors and human adaptation is discussed in detail for individuals and organisations...

  19. Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane.

    OpenAIRE

    Jabbar, M A; Nayak, D P

    1990-01-01

    The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays a major role in the down-regulation of its receptor, CD4. Using a transient-expression system, we investigated the interaction of the HIV-1 envelope glycoprotein with CD4 during their movement through the intracellular membrane traffic. In singly transfected cells, the envelope glyprotein gp160 was synthesized, glycosylated, and localized predominantly in the endoplasmic reticulum. Only a minor fraction of gp160 wa...

  20. Evidence for differences in the binding of drugs to the two main genetic variants of human alpha 1-acid glycoprotein.

    Science.gov (United States)

    Herve, F; Gomas, E; Duche, J C; Tillement, J P

    1993-09-01

    1. Human alpha 1-acid glycoprotein (AAG), a plasma transport protein, has three main genetic variants. F1. S and A. Native commercial AAG (a mixture of almost equal proportions of these three variants) has been separated by chromatography into variants which correspond to the proteins of the two genes which code for AAG in humans: the A variant and a mixture of the F1 and S variants (60% F1 and 40% S). Their binding properties towards imipramine, warfarin and mifepristone were studied by equilibrium dialysis. 2. The F1S variant mixture strongly bound warfarin and mifepristone with an affinity of 1.89 and 2.06 x 10(6) l mol-1, respectively, but had a low affinity for imipramine. Conversely, the A variant strongly bound imipramine with an affinity of 0.98 x 10(6) l mol-1. The low degree of binding of warfarin and mifepristone to the A variant sample was explained by the presence of protein contaminants in this sample. These results indicate specific drug transport roles for each variant, with respect to its separate genetic origin. 3. Control binding experiments performed with (unfractionated) commercial AAG and with AAG isolated from individuals with either the F1/A or S/A phenotypes, agreed with these findings. The results for the binding of warfarin and mifepristone by the AAG samples were similar to those obtained with the F1S mixture: the mean high-affinity association constant of the AAG samples for each drug was of the same order as that of the F1S mixture: the decrease in the number of binding sites of the AAG samples, as compared with the F1S mixture, was explained by the smaller proportion of variants F1 and/or S in these samples. Conversely, results of the imipramine binding study with the AAG samples concurred with those for the binding of this basic drug by the A variant, with respect to the proportion of the A variant in these samples.

  1. Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein.

    Science.gov (United States)

    Yeggoni, Daniel Pushpa Raju; Manidhar, Darla Mark; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

    2016-09-01

    Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be KUmb-1=3.1 ± .01 × 10(4) M(-1), KUmb-2 = 7 ± .01 × 10(4) M(-1), which corresponds to -6.1 and -6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of KUmb-1-AGP=3.1 ± .01 × 10(3) M(-1) and KUmb-2-AGP = 4.6 ± .01 × 10(3) M(-1). Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster's theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1-HSA and Umb-2-HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.

  2. Expression of peanut agglutinin-binding mucin-type glycoprotein in human esophageal squamous cell carcinoma as a marker

    Directory of Open Access Journals (Sweden)

    Balakrishnan Ramathilakam

    2003-11-01

    Full Text Available Abstract Background The TF (Thomson – Friedenreich blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen by Peanut agglutinin (PNA and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. Results We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. Conclusion The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple

  3. Interactions of human P-glycoprotein transport substrates and inhibitors at the drug binding domain: Functional and molecular docking analyses.

    Science.gov (United States)

    Kadioglu, Onat; Saeed, Mohamed E M; Valoti, Massimo; Frosini, Maria; Sgaragli, Giampietro; Efferth, Thomas

    2016-03-15

    Rhodamine 123 (R123) transport substrate sensitizes P-glycoprotein (P-gp) to inhibition by compound 2c (cis-cis) N,N-bis(cyclohexanolamine)aryl ester isomer in a concentration-dependent manner in human MDR1-gene transfected mouse T-lymphoma L5178 cells as shown previously. By contrast, epirubicin (EPI) concentration changes left unaltered 2c IC50 values of EPI efflux. To clarify this discrepancy, defined molecular docking (DMD) analyses of 12 N,N-bis(cyclohexanolamine)aryl esters, the highly flexible aryl ester analog 4, and several P-gp substrate/non-substrate inhibitors were performed on human P-gp drug- or nucleotide-binding domains (DBD or NBD). DMD measurements yielded lowest binding energy (LBE, kcal/mol) values (mean ± SD) ranging from -11.8 ± 0.54 (valspodar) to -3.98 ± 0.01 (4). Lys234, Ser952 and Tyr953 residues formed H-bonds with most of the compounds. Only 2c docked also at ATP binding site (LBE value of -6.9 ± 0.30 kcal/mol). Inhibition of P-gp-mediated R123 efflux by 12 N,N-bis(cyclohexanolamine)aryl esters and 4 significantly correlated with LBE values. DMD analysis of EPI, (3)H-1EPI, (3)H-2EPI, (14)C-1EPI, (14)C-2EPI, R123 and 2c before and after previous docking of each of them indicated that pre-docking of either 2c or EPI significantly reduced LBE of both EPI and R123, and that of both (3)H-2EPI and (14)C-2EPI, respectively. Since the clusters of DBD amino acid residues interacting with EPI were different, if EPI docked alone or after pre-docking of EPI or 2c, the existence of alternative secondary binding site for EPI on P-gp is credible. In conclusion, 2c may allocate the drug-binding pocket and reduce strong binding of EPI and R123 in agreement with P-gp inhibition experiments, where 2c reduced efflux of EPI and R123.

  4. Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes.

    Science.gov (United States)

    Alam, S M K; Jasti, S; Kshirsagar, S K; Tannetta, D S; Dragovic, R A; Redman, C W; Sargent, I L; Hodes, H C; Nauser, T L; Fortes, T; Filler, A M; Behan, K; Martin, D R; Fields, T A; Petroff, B K; Petroff, M G

    2017-01-01

    Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

  5. Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Wetsel, R A; Tack, B F

    1986-01-01

    We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide ....... Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element...

  6. Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study.

    Science.gov (United States)

    Morgeaux, Sylvie; Poirier, Bertrand; Ragan, C Ian; Wilkinson, Dianna; Arabin, Ulrich; Guinet-Morlot, Françoise; Levis, Robin; Meyer, Heidi; Riou, Patrice; Shaid, Shahjahan; Volokhov, Dmitriy; Tordo, Noël; Chapsal, Jean-Michel

    2017-02-07

    Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

  7. Antibodies Against β2-Glycoprotein I Complexed With an Oxidised Lipoprotein Relate to Intima Thickening of Carotid Arteries in Primary Antiphospholipid Syndrome

    Directory of Open Access Journals (Sweden)

    P. R. J. Ames

    2006-01-01

    Full Text Available To explore whether antibodies against β2-glycoprotein I (β2GPI complexed to 7-ketocholesteryl-9-carboxynonanoate (oxLig-1 and to oxidised low-density lipoproteins (oxLDL relate to paraoxonase activity (PONa and/or intima media thickness (IMT of carotid arteries in primary antiphospholipid syndrome (PAPS. As many as 29 thrombotic patients with PAPS, 10 subjects with idiopathic antiphospholipid antibodies (aPL without thrombosis, 17 thrombotic patients with inherited thrombophilia and 23 healthy controls were investigated. The following were measured in all participants: β2GPI−oxLDL complexes, IgG anti-β2GPI−oxLig-1, IgG anti-β2GPI−oxLDL antibodies (ELISA, PONa, (para-nitrophenol method, IMT of common carotid (CC artery, carotid bifurcation (B, internal carotid (IC by high resolution sonography. β2GPI−oxLDL complex was highest in the control group (p < 0.01, whereas, IgG anti-β2GPI−oxLig1 and IgG anti-β2GPI−oxLDL were highest in PAPS (p < 0.0001. In healthy controls, β2GPI−oxLDL complexes positively correlated to IMT of the IC (p = 0.007 and negatively to PONa after correction for age (p < 0.03. PONa inversely correlated with age (p = 0.008. In PAPS, IgG anti-2GPI−oxLig-1 independently predicted PONa (p = 0.02 and IMT of B (p = 0.003, CC, (p = 0.03 and of IC (p = 0.04. In PAPS, PONa inversely correlated to the IMT of B, CC and IC (p = 0.01, 0.02 and 0.003, respectively. IgG anti-2GPI−oxLig-1 may be involved in PAPS related atherogenesis via decreased PON activity.

  8. An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

    KAUST Repository

    Ali, Amal J.

    2017-05-03

    Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

  9. Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

    Science.gov (United States)

    Noack, Andreas; Noack, Sandra; Hoffmann, Andrea; Maalouf, Katia; Buettner, Manuela; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Alms, Dana; Römermann, Kerstin; Naim, Hassan Y; Löscher, Wolfgang

    2014-01-01

    P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid

  10. Molecular model of the outward facing state of the human P-glycoprotein (ABCB1, and comparison to a model of the human MRP5 (ABCC5

    Directory of Open Access Journals (Sweden)

    Sager Georg

    2007-09-01

    Full Text Available Abstract Background Multidrug resistance is a particular limitation to cancer chemotherapy, antibiotic treatment and HIV medication. The ABC (ATP binding cassette transporters human P-glycoprotein (ABCB1 and the human MRP5 (ABCC5 are involved in multidrug resistance. Results In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model. The electrostatic potential surface (EPS of the ABCB1 substrate translocation chamber, which transports cationic amphiphilic and lipophilic substrates, was neutral with negative and weakly positive areas. In contrast, EPS of the ABCC5 substrate translocation chamber, which transports organic anions, was generally positive. Positive-negative ratios of amino acids in the TMDs of ABCB1 and ABCC5 were also analyzed, and the positive-negative ratio of charged amino acids was higher in the ABCC5 TMDs than in the ABCB1 TMDs. In the ABCB1 model residues Leu65 (transmembrane helix 1 (TMH1, Ile306 (TMH5, Ile340 (TMH6 and Phe343 (TMH6 may form a binding site, and this is in accordance with previous site directed mutagenesis studies. Conclusion The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5. The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5.

  11. Prediction of exposed domains of envelope glycoprotein in Indian HIV-1 isolates and experimental confirmation of their immunogenicity in humans

    Directory of Open Access Journals (Sweden)

    Mohabatkar H.

    2004-01-01

    Full Text Available We describe the impact of subtype differences on the seroreactivity of linear antigenic epitopes in envelope glycoprotein of HIV-1 isolates from different geographical locations. By computer analysis, we predicted potential antigenic sites of envelope glycoprotein (gp120 and gp4l of this virus. For this purpose, after fetching sequences of proteins of interest from data banks, values of hydrophilicity, flexibility, accessibility, inverted hydrophobicity, and secondary structure were considered. We identified several potential antigenic epitopes in a B subtype strain of envelope glycoprotein of HIV-1 (IIIB. Solid- phase peptide synthesis methods of Merrifield and Fmoc chemistry were used for synthesizing peptides. These synthetic peptides corresponded mainly to the C2, V3 and CD4 binding sites of gp120 and some parts of the ectodomain of gp41. The reactivity of these peptides was tested by ELISA against different HIV-1-positive sera from different locations in India. For two of these predicted epitopes, the corresponding Indian consensus sequences (LAIERYLKQQLLGWG and DIIGDIRQAHCNISEDKWNET (subtype C were also synthesized and their reactivity was tested by ELISA. These peptides also distinguished HIV-1-positive sera of Indians with C subtype infections from sera from HIV-negative subjects.

  12. The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex.

    Science.gov (United States)

    Suzuki, Kaori; Tatebe, Nanami; Kojima, Sayuri; Hamano, Ayumi; Orita, Misaki; Yonezawa, Naoto

    2015-01-01

    The zona pellucida (ZP) surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding.

  13. Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins.

    Science.gov (United States)

    Kullolli, Majlinda; Hancock, William S; Hincapie, Marina

    2008-08-01

    We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.

  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

    2011-01-01

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

    Science.gov (United States)

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

    2011-01-01

    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

  16. Resistant mechanisms of anthracyclines--pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues.

    Science.gov (United States)

    Kubota, T; Furukawa, T; Tanino, H; Suto, A; Otan, Y; Watanabe, M; Ikeda, T; Kitajima, M

    2001-01-01

    Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing "permeability" of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdr1 gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of "ATP-Binding Cassette transporter"(ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc.htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express P-gp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear.

  17. Effects of the I559P gp41 change on the conformation and function of the human immunodeficiency virus (HIV-1 membrane envelope glycoprotein trimer.

    Directory of Open Access Journals (Sweden)

    Nirmin Alsahafi

    Full Text Available The mature human immunodeficiency virus (HIV-1 envelope glycoprotein (Env trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env

  18. Complex Loci in human and mouse genomes.

    Science.gov (United States)

    Engström, Pär G; Suzuki, Harukazu; Ninomiya, Noriko; Akalin, Altuna; Sessa, Luca; Lavorgna, Giovanni; Brozzi, Alessandro; Luzi, Lucilla; Tan, Sin Lam; Yang, Liang; Kunarso, Galih; Ng, Edwin Lian-Chong; Batalov, Serge; Wahlestedt, Claes; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Wells, Christine; Bajic, Vladimir B; Orlando, Valerio; Reid, James F; Lenhard, Boris; Lipovich, Leonard

    2006-04-01

    Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.

  19. Complex Loci in human and mouse genomes.

    Directory of Open Access Journals (Sweden)

    Pär G Engström

    2006-04-01

    Full Text Available Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs, along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.

  20. Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein.

    Science.gov (United States)

    Falconar, Andrew K I

    2008-07-01

    The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

  1. The complex co-translational processing of glycoprotein GP5 of type 1 porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Thaa, Bastian; Kaufer, Susanne; Neumann, Sara A; Peibst, Bernadett; Nauwynck, Hans; Krause, Eberhard; Veit, Michael

    2017-08-12

    GP5 and M, the major membrane proteins of porcine reproductive and respiratory syndrome virus (PRRSV), are the driving force for virus budding and a target for antibodies. We studied co-translational processing of GP5 from an European PRRSV-1 strain. Using mass spectrometry, we show that in virus particles of a Lelystad variant, the signal peptide of GP5 was absent due to cleavage between glycine-34 and asparagine-35. This cleavage site removes an epitope for a neutralizing monoclonal antibody, but leaves intact another epitope recognized by neutralizing pig sera. Upon ectopic expression of this GP5 in cells, signal peptide cleavage was however inefficient. Complete cleavage occurred when cysteine-24 was changed to proline or an unused glycosylation site involving asparagine-35 was mutated. Insertion of proline at position 24 also caused carbohydrate attachment to asparagine-35. Glycosylation sites introduced downstream of residue 35 were used, but did not inhibit signal peptide processing. Co-expression of the M protein rescued this processing defect in GP5, suggesting a novel function of M towards GP5. We speculate that a complex interplay of the co-translational modifications of GP5 affect the N-terminal structure of the mature proteins and hence its antigenicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. 婴幼儿人巨细胞病毒感染的临床表现和糖蛋白B基因分型%Clinical manifestations of human cytomegalovirus infection of infants and genotype of glycoprotein B

    Institute of Scientific and Technical Information of China (English)

    陆晓东; 单小云; 袁青; 朱以军; 郑雅萍; 徐瑞龙

    2011-01-01

    Objective: To understand the clinical manifestations of human cytomegalovirus (HCMV) activate infection of infants and its relationship with genotype of glycoprotein B. Methods: ELISA method was used to detect 51 infants with positive HCMV diagnosed by HCMV - IgM, and the different clinical symptoms were analyzed. Genotyping of HCMV glycoprotein B was performed among 43 infants by nested PCR and restriction fragment length polymorphism (RFLP) . Results: Among 51 infants with HCMV infection, 25. 49% of them were systemic infection and 74. 51% of them were single organ infection, the proportions of HCMV inclusion disease and hepatitis were 25.49% and 21.57%, respectively. The results of genotyping of HCMV glycoprotein B among 43 infants: 20 infants with glycoprotein B Ⅰ genotype, 7 infants with glycoprotein B Ⅱ genotype, 9 infants with glycoprotein B Ⅲ genotype, 4 infants with glycoprotein B Ⅰ genotype and glycoprotein B Ⅱ genotype, 2 infants with glycoprotein B Ⅰ genotype and glycoprotein B Ⅲ genotype, one infant with glycoprotein B Ⅱ genotype and glycoprotein B Ⅲ genotype, no infant was found with glycoprotein B Ⅳ genotype; glycoprotein B Ⅰ genotype accounted for 46. 51%.Conclusion: The clinical manifestations of infantile HCMV infection are various; glycoprotein B Ⅰ genotype is in the majority among HCMV infected infants.%目的:了解婴幼儿人巨细胞病毒(HCMV)活动性感染的临床表现,以及与糖蛋白B(gB)基因型的关系.方法:ELISA法检测HCMV-lgM确定的HCMV阳性的婴幼儿51例,对其不同临床症状进行分析.对其中43例患儿使用套式PCR(nPCR)法加限制性长度多态性分析(RFLP)进行HCMV gB基因分型.结果:在51例HCMV感染患儿中全身性感染和单脏器感染分别占25.49%和74.51%,HCMV包涵体病和肝炎分别占25.49%和21.57%.43例患儿HCMV gB的基因分型结果为,gBI型20例,gBⅡ型7例,gBⅢ型9例,gB Ⅰ、Ⅱ混合型4例,gBⅠ、Ⅲ混合型2

  3. Assessment of the quality and structural integrity of a complex glycoprotein mixture following extraction from the formulated biopharmaceutical drug product.

    Science.gov (United States)

    Liu, Cuihua; Dong, Shiming; Xu, Xiao-Jin; Yin, Yan; Shriver, Zachary; Capila, Ishan; Myette, James; Venkataraman, Ganesh

    2011-01-05

    Biological drugs represent an important and rapidly growing class of therapeutics useful in the treatment of a variety of disorders ranging from cancer to inflammation to infectious diseases. Unlike single chemical entities, the recombinant production of these drugs in living cells confers considerable structural and chemical heterogeneity to the biologically derived protein product that constitutes the active pharmaceutical ingredient (API). In mammalian based expression systems, much of this diversity is conferred through heterogeneous protein glycosylation. These post-translational modifications can have significant effects on the structure, biological function, and pharmacological properties of the API. In addition, the bulk proteins that comprise the API are further formulated through the use of multiple excipients designed to ensure product stability, solubility, and lot-to-lot consistency. Unfortunately, these matrices can interfere with commonly available analytical methods used in the thorough chemical characterization of the biological drug product. At the same time, a demonstration of the suitable extraction of the bulk drug substance in a manner and form that does not destabilize the active ingredient or introduce any structural bias with direct reference to the original drug product is both critical and necessary. Here, we use recombinant human follicle stimulating hormone (follitropin alpha for injection) from a pharmaceutical source as an example to illustrate a suitable purification strategy to effectively extract the bulk drug substance from the formulated drug product with high purity and yield. We assess the suitability of this extraction method in preserving the structural integrity and overall quality of the drug substance relative to the formulated drug product, placing a particular emphasis on glycosylation as a key product attribute. In so doing, we demonstrate that it is possible to effectively extract the active pharmaceutical ingredient

  4. Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

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    Alain Le Coupanec

    Full Text Available Human coronaviruses (HCoV are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43. In an attempt to study the role of this protein in virus spread within the central nervous system (CNS and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758, which introduces a putative furin-like cleavage (↓ site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.

  5. The Hinge Region of Bovine Zona Pellucida Glycoprotein ZP3 Is Involved in the Formation of the Sperm-Binding Active ZP3/ZP4 Complex

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    Kaori Suzuki

    2015-11-01

    Full Text Available The zona pellucida (ZP surrounds the mammalian oocyte and mediates species-selective sperm-oocyte interactions. Bovine ZP consists of glycoproteins ZP2, ZP3, and ZP4. Neither ZP3 nor ZP4 alone shows inhibitory activity for the binding of sperm to the ZP; however, this activity is seen with the ZP3/ZP4 heterocomplex. Here, we constructed a series of bovine ZP3 mutants to identify the ZP4-binding site on ZP3. Each ZP3 mutant was co-expressed with ZP4 using a baculovirus-Sf9 cell expression system and examined for interaction with ZP4 as well as inhibitory activity for sperm-ZP binding. N-terminal fragment Arg-32 to Arg-160 of ZP3 interacted with ZP4 and inhibited sperm-ZP binding, whereas fragment Arg-32 to Thr-155 showed much weaker interaction with ZP4. Mutation of N-glycosylated Asn-146 to Asp in the N-terminal fragment Arg-32 to Glu-178 of ZP3 did not interrupt the interaction of this fragment with ZP4, but it did reduce the inhibitory activity of the complex for sperm-ZP binding. In contrast, mutation of N-glycosylated Asn-124 to Asp did not significantly reduce the activity. Taken together, these results suggest that one of the ZP4 binding sites exists in the flexible hinge region of ZP3 and that the N-glycosylation in this region is involved in the sperm binding.

  6. Characterization of a Novel 99mTc-Carbonyl Complex as a Functional Probe of MDR1 P-Glycoprotein Transport Activity

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    Mary Dyszlewski

    2002-01-01

    Full Text Available Multidrug resistance (MDR mediated by overexpression of MDR1 P-glycoprotein (Pgp is one of the best characterized barriers to chemotherapy in cancer patients. Furthermore, the protective function of Pgp-mediated efflux of xenobiotics in various organs has a profound effect on the bioavailability of drugs in general. Thus, there is an expanding requirement to noninvasively interrogate Pgp transport activity in vivo. We herein report the Pgp recognition properties of a novel 99mTc(I-tricarbonyl complex, [99mTc(CO3(MIBI3] + (Tc-CO-MIBI. Tc-CO-MIBI showed 60-fold higher accumulation in drug-sensitive KB 3–1 cells compared to colchicine-selected drug-resistant KB 8-5 cells. In KB 8-5 cells, tracer enhancement was observed with the potent MDR modulator LY335979 (EC50 = 62 nM. Similar behavior was observed using drug-sensitive MCF-7 breast adenocarcinoma cells and MCF-7/MDR1 stable transfectants, confirming that Tc-CO-MIBI is specifically excluded by overexpression of MDR1 Pgp. By comparison, net accumulation in control H69 lung tumor cells was 9-fold higher than in MDR-associated protein (MRP1-expressing H69AR cells, indicating only modest transport by MRP1. Biodistribution analysis following tail vein injection of Tc-CO-MIBI showed delayed liver clearance as well as enhanced brain uptake and retention in mdr1a/1b(−/− gene deleted mice versus wild-type mice, directly demonstrating that Tc-CO-MIBI is a functional probe of Pgp transport activity in vivo.

  7. Interplay between Human Cytomegalovirus and Intrinsic/Innate Host Responses: A Complex Bidirectional Relationship

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    Giada Rossini

    2012-01-01

    Full Text Available The interaction between human cytomegalovirus (HCMV and its host is a complex process that begins with viral attachment and entry into host cells, culminating in the development of a specific adaptive response that clears the acute infection but fails to eradicate HCMV. We review the viral and cellular partners that mediate early host responses to HCMV with regard to the interaction between structural components of virions (viral glycoproteins and cellular receptors (attachment/entry receptors, toll-like receptors, and other nucleic acid sensors or intrinsic factors (PML, hDaxx, Sp100, viperin, interferon inducible protein 16, the reactions of innate immune cells (antigen presenting cells and natural killer cells, the numerous mechanisms of viral immunoevasion, and the potential exploitation of events that are associated with early phases of virus-host interplay as a therapeutic strategy.

  8. P-Glycoprotein/MDR1 Regulates Pokemon Gene Transcription Through p53 Expression in Human Breast Cancer Cells

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    Wei Xu

    2010-08-01

    Full Text Available P-glycoprotein (Pgp, encoded by the multidrug resistance 1 (MDR1 gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

  9. Modeling the human prothrombinase complex components

    Science.gov (United States)

    Orban, Tivadar

    Thrombin generation is the culminating stage of the blood coagulation process. Thrombin is obtained from prothrombin (the substrate) in a reaction catalyzed by the prothrombinase complex (the enzyme). The prothrombinase complex is composed of factor Xa (the enzyme), factor Va (the cofactor) associated in the presence of calcium ions on a negatively charged cell membrane. Factor Xa, alone, can activate prothrombin to thrombin; however, the rate of conversion is not physiologically relevant for survival. Incorporation of factor Va into prothrombinase accelerates the rate of prothrombinase activity by 300,000-fold, and provides the physiological pathway of thrombin generation. The long-term goal of the current proposal is to provide the necessary support for the advancing of studies to design potential drug candidates that may be used to avoid development of deep venous thrombosis in high-risk patients. The short-term goals of the present proposal are to (1) to propose a model of a mixed asymmetric phospholipid bilayer, (2) expand the incomplete model of human coagulation factor Va and study its interaction with the phospholipid bilayer, (3) to create a homology model of prothrombin (4) to study the dynamics of interaction between prothrombin and the phospholipid bilayer.

  10. IPEC-J2 MDR1, a Novel High-Resistance Cell Line with Functional Expression of Human P-glycoprotein (ABCB1) for Drug Screening Studies

    DEFF Research Database (Denmark)

    Saaby, Lasse; Helms, Hans Christian Cederberg; Brodin, Birger

    2016-01-01

    The P-glycoprotein (P-gp) efflux pump has been shown to affect drug distribution and absorption in various organs and to cause drug resistance in cancer therapy. The aim of this work was to develop a cell line to serve as a screening system for potential substrates of P-gp. This requires a cell...... line with high paracellular tightness, low expression of nonhuman ABC transporters, and high expression of functional human P-gp (ABCB1). The porcine intestinal epithelial cell line, IPEC-J2, was selected as a transfection host, due to its ability to form extremely high-resistance monolayers (>10,000 Ω...... for the efflux transport by substrate profiling, combined with application of P-gp and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram, and mitoxantrone were identified as P-gp substrates. Functional P-gp expression was shown to be stable through at least 10 cell passages. In conclusion...

  11. Advances in the study of human cytomegalovirus glycoprotein B%人巨细胞病毒gB糖蛋白研究进展

    Institute of Scientific and Technical Information of China (English)

    李敏环; 范骏

    2011-01-01

    The envelope glycoprotein B(gB) of human cytomegalovirus(HCMV) is required in the course of virus entry and cell-to-cell spread.In addition,it takes an important part in inducing the immune response in host.In this article,gB structure,genotypes,biological characteristics,gB-specific monoclonal antibodies and vaccines are reviewed.%人巨细胞病毒(HCMV)的包膜gB糖蛋白在病毒穿入宿主细胞、细胞间传播,以及诱导宿主的免疫应答中起着重要作用.此文就gB的结构、基因分型、生物学特性、单克隆抗体及HCMV疫苗情况进行了综述.

  12. 呼吸道合胞病毒F蛋白研究进展%The research pogress in fusion glycoprotein of human respiratory syncytial virus

    Institute of Scientific and Technical Information of China (English)

    袁媛

    2009-01-01

    The fusion glycoprotein (F) of human respiratory syncytial vires(RSV) is an important viral envelop protein which can induce protective immunity against RSV infection.The structural and functional characteristics of F protein play a central role in the understanding of infection and immunity,and in the development of vaccine.%人呼吸道合胞病毒(RSV)融合蛋白(F蛋白)为病毒表面结构蛋白,可以刺激机体产生保护性抗体和细胞免疫反应.因此对F蛋白的深入研究将有助于了解病毒感染的机理,从而为研究RSV的免疫机理、研制特定部位的亚单位或多肽疫苗提供帮助.

  13. Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein

    DEFF Research Database (Denmark)

    Keck, Zhenyong; Wang, Wenyan; Wang, Yong

    2013-01-01

    polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay......A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While...... between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions...

  14. Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

    Science.gov (United States)

    Liu, Y; Qu, F; Cao, X; Chen, G; Guo, Q; Ying, X; Guo, W; Lu, L; Ding, Z

    2012-04-01

    Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

  15. Differential sensitivity of human, avian, and equine influenza A viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants.

    Science.gov (United States)

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

    1983-12-01

    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

  16. Further investigations on the macromolecular complex in human bile

    NARCIS (Netherlands)

    Verschure, J.C.M.; Wael, J. de; Mijnlieff, P.F.

    1956-01-01

    The formation of complexes in human bile was further studied by the preparation of various synthetic complexes and extracts. These were compared for a number of properties with the natural complex of human gall bladder bile. It appeared that protein is probably and bilirubin quite definitely a const

  17. The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

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    Markus Hoffmann

    Full Text Available Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule. Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.

  18. Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex : characterization of the epitopes

    NARCIS (Netherlands)

    Burgess, J K; Lopez, J A; Berndt, M C; Dawes, I; Chesterman, C N; Chong, B H

    1998-01-01

    Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to ch

  19. Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex: Characterization of the epitopes

    NARCIS (Netherlands)

    Burgess, Janette K.; Lopez, Jose A.; Berndt, Michael C.; Dawes, Ian; Chesterman, Colin N.; Chong, Beng H.

    1998-01-01

    Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb- IIIa or both epitopes has also been reported. The objective of this study was to c

  20. Reducing the Complexity Gap: Expanding the Period of Human Nurturance

    Science.gov (United States)

    Kiel, L. Douglas

    2014-01-01

    Socio-techno-cultural reality, in the current historical era, evolves at a faster rate than do human brain or human institutions. This reality creates a "complexity gap" that reduces human and institutional capacities to adapt to the challenges of late modernity. New insights from the neurosciences may help to reduce the complexity gap.…

  1. The effect of industrial processing of salmon oil on its ability to reduce serum concentrations of oxidized low-density lipoprotein- β2-glycoprotein-I complex in a mouse model

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    Bomi Framroze

    2014-10-01

    Full Text Available Background: Circulating serum levels of oxidized low-density lipoprotein, β2-glycoprotein I complex (oxLDL-GP, have been previously correlated with adverse cardiovascular events and have been shown to be reduced by consumption of enzymatically liberated extra virgin salmon oil (EVSO. This mouse study measured the changes in the oxLDL-GP lowering effect when consuming EVSO with varying levels of EPA+DHA (eicosapentenoic acid and docosahexenoic acid as well as when consuming EVSO that was subjected to various processing treatments commonly carried out during fish oil production. Methods: Sprague Dawley mice were fed a diet containing eight different EVSO’s incorporated into a normal diet at the Human Equivalent Dose (HED of 1000 mg for 8 weeks. Serum was collected at the start and at the end of the trial and the oxLDL-GP concentrations were measured using an ELISA assay. Statistical analysis of the results was carried out using a 1-tail, paired Student t-Test. Results: In order to lower circulatory oxLDL-GP levels, the mice had to consume a minimum of 80 mg per day HED of EPA+DHA. Heat treatment of the EVSO did not affect this bioactivity but hydrolysis with acid or base and re-esterification to the triglyceride form or significant oxidation (rancidity rendered the oil inactive on this important cardio-vascular disease (CVD biomarker. Conclusions: This result shows that harsh processing conditions on fish oils can lead to the destruction of biological efficacy in spite of increasing the concentration of typical fish oil bioactive constituents such as EPA+DHA. It also lends support to the developing nutrition theory that eating highly-refined, processed or concentrated-ingredient supplements derived from functional foods may not be able to reproduce their full nutritive and health-benefiting effects

  2. Human Herpesvirus 7 Glycoprotein B (gB) , gH, gL, gO Can Mediate Cell Fusion%人类疱疹病毒7型糖蛋白gB、gH、gL、gO介导细胞融合

    Institute of Scientific and Technical Information of China (English)

    徐建; 姚堃; 窦洁; 秦健; 许文嵘; 陈云; 尹全章; 周锋

    2007-01-01

    人类疱疹病毒7型(HHV-7)的感染依赖于包膜糖蛋白在病毒生命周期的多个阶段发挥功能.这些蛋白质可以介导病毒吸附,病毒包膜和宿主细胞膜融合以及病毒在细胞间的接触传播.将表达HHV-7糖蛋白的293T细胞与HHV-7易感的SupT1细胞共培养,检测虫荧光素酶报告基因的表达,以鉴定介导膜融合的HHV-7糖蛋白.研究发现,HHV-7糖蛋白gB、gH、gL、gO能介导293T细胞与SupT1细胞的融合,且融合可被抗CD4单抗所抑制.结果表明,糖蛋白gB、gH、gL、gO对于HHV-7引发的膜融合是必需的,其中某个蛋白质或所形成的蛋白质复合物可能是CD4的配体.%Human herpesvirus 7 (HHV-7) infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteins mediate the initial attachment and fusion events that occur between the viral envelope and a host cell membrane, as well as cell to cell spread of the virus. To characterize the HHV-7 glycoproteins that can mediate cell fusion, a cell-based fusion assay was used. 293T cells expressing the HHV-7 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with SupT1 cells transfected with T7 RNA polymerase. HHV-7 glycoproteins gB, gH, gL and gO can mediate the fusion of 293T cells with SupT1 cells, and the fusion can be inhibited by anti-CD4 mAbs. Thus, the coexpression of HHV-7 gB, gO, gH and gL is sufficient and necessary for HHV-7 induced membrane fusion, and one of these glycoproteins or protein complex formed by these glycoproteins might be the ligand(s) of CD4 molecule.

  3. Expression of the UL16 glycoprotein of Human Cytomegalovirus protects the virus-infected cell from attack by natural killer cells

    Directory of Open Access Journals (Sweden)

    Browne Helena

    2003-03-01

    Full Text Available Abstract Background Human Cytomegalovirus (HCMV has acquired through evolution a number of genes to try to evade immune recognition of the virus-infected cell. Many of these mechanisms act to inhibit the MHC class I antigen presentation pathway, but any virus-infected cell which has down-regulated cell surface expression of MHC class I proteins, to avoid CTL attack, would be expected to become susceptible to lysis by Natural Killer cells. Surprisingly, however, HCMV infected fibroblasts were found to be resistant to NK cell mediated cytotoxicity. Expression of the UL16 glycoprotein could represent one mechanism to help the virus to escape from NK cell attack, as it has been shown to bind, in vitro, some of the ligands for NKG2D, the NK cell activating receptor. Here, we explored the role of UL16, in the context of a viral infection, by comparing the susceptibility to NK lysis of cells infected with HCMV and cells infected with a UL16 deletion mutant of this virus. Results Cells infected with the UL16 knockout virus were killed at substantially higher levels than cells infected with the wild-type virus. This increased killing could be correlated with a UL16-dependent reduction in surface expression of ligands for the NK cell activating receptor NKG2D. Conclusions Expression of the UL16 glycoprotein was associated with protection of HCMV-infected cells from NK cell attack. This observation could be correlated with the downregulation of cell surface expression of NKG2D ligands. These data represent a first step towards understanding the mechanism(s of action of the UL16 protein.

  4. Human cytomegalovirus prevalence and distribution of glycoprotein B, O genotypes among hospitalized children with respiratory infections in West China, 2009-2014.

    Science.gov (United States)

    Chen, Jia-Yi; Zheng, Tian-Li; Zhou, Tao; Hu, Peng-Wei; Huang, Meng-Jiao; Xu, Xin; Pei, Xiao-Fang

    2016-11-01

    Human cytomegalovirus (HCMV) is an important pathogen causing morbidity and mortality in children. HCMV prevalence in children with respiratory infections has not been investigated in West China. Previous studies have suggested that glycoproteins genotypes may be associated with different clinical presentations, but the associations were controversial. The aim of this study was to determine the prevalence of HCMV infection in children with respiratory infections, the distributions of gB, gO genotypes among these isolates and their potential predictive roles for the development of symptoms in children. A total of 1709 respiratory specimens were obtained from hospitalised children with respiratory symptoms from 2009 to 2014 for the confirmation of HCMV infection. Glycoprotein B,O genotyping was carried out by multiplex nested PCR and sequencing. The overall infection rate was 10.8%, and dominant genotypes were gB1 (74.2%) and gO1 (37.1%). Clinical characteristics differed between infants and children >1 year of age. Infants infected with HCMV had a higher frequency of fever (P < 0.001), cough (P < 0.001), rhinorrhea (P < 0.001), expectoration (P = 0.001) and diarrhoea (P = 0.005). Children <1 year age infected with gB1 had a higher rate of cough (P = 0.0192). Infants infected with HCMV had a severe clinical outcome. gB1 may negatively associate with clinical presentations and quality of life in these children. The prevalence of HCMV infection and genotype distribution emphasises the importance of HCMV screening, vaccination and control for transmission. © 2016 John Wiley & Sons Ltd.

  5. Circulating Immune Complexes of IgA Bound to Beta 2 Glycoprotein are Strongly Associated with the Occurrence of Acute Thrombotic Events

    Science.gov (United States)

    Martínez-Flores, José A.; Serrano, Manuel; Pérez, Dolores; de la Cámara A, Gómez; Lora, David; Morillas, Luis; Ayala, Rosa; Paz-Artal, Estela; Morales, José M.

    2016-01-01

    Aim: Antiphospholipid syndrome (APS) is characterized by recurrent thrombosis and/or gestational morbidity in patients with antiphospholipid autoantibodies (aPL). Over recent years, IgA anti-beta2-glycoprotein I (B2GPI) antibodies (IgA aB2GPI) have reached similar clinical relevance as IgG or IgM isotypes. We recently described the presence of immune complexes of IgA bounded to B2GPI (B2A-CIC) in the blood of patients with antecedents of APS symptomalology. However, B2A-CIC's clinical associations with thrombotic events (TEV) have not been described yet. Methods: A total of 145 individuals who were isolate positive for IgA aB2GPI were studied: 50 controls without any APS antecedent, 22 patients with recent TEV (Group-1), and 73 patients with antecedents of old TEV (Group-2). Results: Mean B2A-CIC levels and prevalence in Group-1 were 29.6 ± 4.1 AU and 81.8%, respectively, and were significantly higher than those of Group-2 and controls (p < 0.001). In a multivariable analysis, positivity of B2A-CIC was an independent variable for acute thrombosis with a 22.7 odd ratio (confidence interval 5.1 –101.6, 95%, p < 0.001). Levels of B2A-CIC dropped significantly two months after the TEV. B2A-CIC positive patients had lower platelet levels than B2A-CIC-negative patients (p < 0.001) and more prevalence of thrombocytopenia (p < 0.019). Group-1 had no significant differences in C3 and C4 levels compared with other groups. Conclusion: B2A-CIC is strongly associated with acute TEV. Patients who did not develop thrombosis and were B2A-CIC positive had lower platelet levels, which suggest a hypercoagulable state. This mechanism is unrelated to complement-fixing aPL. B2A-CIC could potentially select IgA aB2GPI-positive patients at risk of developing a thrombotic event. PMID:27063992

  6. Evodiamine synergizes with doxorubicin in the treatment of chemoresistant human breast cancer without inhibiting P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Shengpeng Wang

    Full Text Available Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO, a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose polymerase (PARP, caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs. Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp. Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.

  7. Synthesis, biological evaluation and 3D-QSAR studies of new chalcone derivatives as inhibitors of human P-glycoprotein.

    Science.gov (United States)

    Parveen, Zahida; Brunhofer, Gerda; Jabeen, Ishrat; Erker, Thomas; Chiba, Peter; Ecker, Gerhard F

    2014-04-01

    P-glycoprotein (P-gp) is an ATP-dependent multidrug resistance efflux transporter that plays an important role in anticancer drug resistance and in pharmacokinetics of medicines. Despite a large number of structurally and functionally diverse compounds, also flavonoids and chalcones have been reported as inhibitors of P-gp. The latter share some similarity with the well studied class of propafenones, but do not contain a basic nitrogen atom. Furthermore, due to their rigidity, they are suitable candidates for 3D-QSAR studies. In this study, a set of 22 new chalcone derivatives were synthesized and evaluated in a daunomycin efflux inhibition assay using the CCRF.CEM.VCR1000 cell line. The compound 10 showed the highest activity (IC50=42nM), which is one order of magnitude higher than the activity for an equilipohillic propafenone analogue. 2D- and 3D-QSAR studies indicate the importance of H-bond acceptors, methoxy groups, hydrophobic groups as well as the number of rotatable bonds as pharmacophoric features influencing P-gp inhibitory activity.

  8. Functionalized Magnetic Resonance Contrast Agent Selectively Binds to Glycoprotein IIb/IIIa on Activated Human Platelets under Flow Conditions and Is Detectable at Clinically Relevant Field Strengths

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    Constantin von zur Mühlen

    2008-03-01

    Full Text Available Recent progress in molecular magnetic resonance imaging (MRI provides the opportunity to image cells and cellular receptors using microparticles of iron oxide (MPIOs. However, imaging targets on vessel walls remains challenging owing to the quantity of contrast agents delivered to areas of interest under shear stress conditions. We evaluated ex vivo binding characteristics of a functional MRI contrast agent to ligand-induced binding sites (LIBSs on activated glycoprotein IIb/IIIa receptors of human platelets, which were lining rupture-prone atherosclerotic plaques and could therefore facilitate detection of platelet-mediated pathology in atherothrombotic disease. MPIOs were conjugated to anti-LIBS single-chain antibodies (LIBS-MPIO or control antibodies (control MPIO. Ex vivo binding to human platelet-rich clots in a dose-dependent manner was confirmed on a 3 T clinical MRI scanner and by histology (p < .05 for LIBS-MPIO vs control MPIO. By using a flow chamber setup, significant binding of LIBS-MPIO to a platelet matrix was observed under venous and arterial flow conditions, but not for control MPIO (p < .001. A newly generated MRI contrast agent detects activated human platelets at clinically relevant magnetic field strengths and binds to platelets under venous and arterial flow conditions, conveying high payloads of contrast to specific molecular targets. This may provide the opportunity to identify vulnerable, rupture-prone atherosclerotic plaques via noninvasive MRI.

  9. Benchmarking human protein complexes to investigate drug-related systems and evaluate predicted protein complexes.

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    Min Wu

    Full Text Available Protein complexes are key entities to perform cellular functions. Human diseases are also revealed to associate with some specific human protein complexes. In fact, human protein complexes are widely used for protein function annotation, inference of human protein interactome, disease gene prediction, and so on. Therefore, it is highly desired to build an up-to-date catalogue of human complexes to support the research in these applications. Protein complexes from different databases are as expected to be highly redundant. In this paper, we designed a set of concise operations to compile these redundant human complexes and built a comprehensive catalogue called CHPC2012 (Catalogue of Human Protein Complexes. CHPC2012 achieves a higher coverage for proteins and protein complexes than those individual databases. It is also verified to be a set of complexes with high quality as its co-complex protein associations have a high overlap with protein-protein interactions (PPI in various existing PPI databases. We demonstrated two distinct applications of CHPC2012, that is, investigating the relationship between protein complexes and drug-related systems and evaluating the quality of predicted protein complexes. In particular, CHPC2012 provides more insights into drug development. For instance, proteins involved in multiple complexes (the overlapping proteins are potential drug targets; the drug-complex network is utilized to investigate multi-target drugs and drug-drug interactions; and the disease-specific complex-drug networks will provide new clues for drug repositioning. With this up-to-date reference set of human protein complexes, we believe that the CHPC2012 catalogue is able to enhance the studies for protein interactions, protein functions, human diseases, drugs, and related fields of research. CHPC2012 complexes can be downloaded from http://www1.i2r.a-star.edu.sg/xlli/CHPC2012/CHPC2012.htm.

  10. Increasing BMI is associated with reduced expression of P-glycoprotein (ABCB1 gene) in the human brain with a stronger association in African Americans than Caucasians

    DEFF Research Database (Denmark)

    2016-01-01

    The efflux pump, p-glycoprotein, controls bioavailability and excretion of pharmaceutical compounds. In the blood-brain barrier, p-glycoprotein regulates the delivery of pharmaceutical substances to the brain, influencing efficacy and side effects for some drugs notably antipsychotics. Common sid...... online publication, 29 November 2016; doi:10.1038/tpj.2016.74....

  11. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier.

    Directory of Open Access Journals (Sweden)

    Yangfang Li

    Full Text Available While the blood-brain barrier (BBB protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp and breast cancer resistance protein (BCRP, two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP but not those overexpressing human P-gp (MDCKII-MDR cells had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated.

  12. Functional stability of unliganded envelope glycoprotein spikes among isolates of human immunodeficiency virus type 1 (HIV-1.

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    Nitish Agrawal

    Full Text Available The HIV-1 envelope glycoprotein (Env spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T(90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34. For select Envs (n = 10, the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T(90 (p = 0.029, though two 'outliers' were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1(ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1(JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.

  13. Neutralizing antibody responses in macaques induced by human immunodeficiency virus type 1 monovalent or trivalent envelope glycoproteins.

    Directory of Open Access Journals (Sweden)

    Gerald V Quinnan

    Full Text Available A major goal of efforts to develop a vaccine to prevent HIV-1 infection is induction of broadly cross-reactive neutralizing antibodies (bcnAb. In previous studies we have demonstrated induction of neutralizing antibodies that did cross-react among multiple primary and laboratory strains of HIV-1, but neutralized with limited potency. In the present study we tested the hypothesis that immunization with multiple HIV-1 envelope glycoproteins (Envs would result in a more potent and cross-reactive neutralizing response. One Env, CM243(N610Q, was selected on the basis of studies of the effects of single and multiple mutations of the four gp41 glycosylation sites. The other two Envs included R2 (subtype B and 14/00/4 (subtype F, both of which were obtained from donors with bcnAb. Rhesus monkeys were immunized using a prime boost regimen as in previous studies. Individual groups of monkeys were immunized with either one of the three Envs or all three. The single N610Q and N615Q mutations of CM243 Env did not disrupt protein secretion, processing into, or reactivity with mAbs, unlike other single or multiple deglycosylation mutations. In rabbit studies the N610Q mutation alone or in combination was associated with an enhanced neutralizing response against homologous and heterologous subtype E viruses. In the subsequent monkey study the response induced by the R2 Env regimen was equivalent to the trivalent regimen and superior to the other monovalent regimens against the virus panel used for testing. The 14/00/4 Env induced responses superior to CM243(N610Q. The results indicate that elimination of the glycosylation site near the gp41 loop results in enhanced immunogenicity, but that immunization of monkeys with these three distinct Envs was not more immunogenic than with one.

  14. Design, synthesis, and biological evaluation of (S)-valine thiazole-derived cyclic and noncyclic peptidomimetic oligomers as modulators of human P-glycoprotein (ABCB1).

    Science.gov (United States)

    Singh, Satyakam; Prasad, Nagarajan Rajendra; Kapoor, Khyati; Chufan, Eduardo E; Patel, Bhargav A; Ambudkar, Suresh V; Talele, Tanaji T

    2014-01-01

    Multidrug resistance caused by ATP binding cassette transporter P-glycoprotein (P-gp) through extrusion of anticancer drugs from the cells is a major cause of failure in cancer chemotherapy. Previously, selenazole-containing cyclic peptides were reported as P-gp inhibitors and were also used for co-crystallization with mouse P-gp, which has 87 % homology to human P-gp. It has been reported that human P-gp can simultaneously accommodate two to three moderately sized molecules at the drug binding pocket. Our in silico analysis, based on the homology model of human P-gp, spurred our efforts to investigate the optimal size of (S)-valine-derived thiazole units that can be accommodated at the drug binding pocket. Towards this goal, we synthesized varying lengths of linear and cyclic derivatives of (S)-valine-derived thiazole units to investigate the optimal size, lipophilicity, and structural form (linear or cyclic) of valine-derived thiazole peptides that can be accommodated in the P-gp binding pocket and affects its activity, previously an unexplored concept. Among these oligomers, lipophilic linear (13) and cyclic trimer (17) derivatives of QZ59S-SSS were found to be the most and equally potent inhibitors of human P-gp (IC50 =1.5 μM). As the cyclic trimer and linear trimer compounds are equipotent, future studies should focus on noncyclic counterparts of cyclic peptides maintaining linear trimer length. A binding model of the linear trimer 13 within the drug binding site on the homology model of human P-gp represents an opportunity for future optimization, specifically replacing valine and thiazole groups in the noncyclic form.

  15. Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

    Science.gov (United States)

    Pluchino, Kristen M; Hall, Matthew D; Moen, Janna K; Chufan, Eduardo E; Fetsch, Patricia A; Shukla, Suneet; Gill, Deborah R; Hyde, Stephen C; Xia, Di; Ambudkar, Suresh V; Gottesman, Michael M

    2016-02-23

    The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes.

  16. Qualitative and quantitative reversed-phase high performance liquid chromatographic analysis of glycoprotein hormones in the presence of a large excess of human serum albumin.

    Science.gov (United States)

    Almeida, B E; Oliveira, J E; Damiani, R; Dalmora, S L; Bartolini, P; Ribela, M T C P

    2012-04-07

    The present work describes reversed-phase high performance liquid chromatographic methodologies (RP-HPLC) for the qualitative and quantitative analysis of the human glycoprotein hormones thyrotropin (hTSH), follitropin (hFSH), choriogonadotropin (hCG) and lutropin (hLH) in the presence of a large excess (up to 250:1) of human serum albumin (HSA). Chromatographic profiles with a good separation between the hormone and HSA were obtained by using a C4 column and specific gradient elution conditions for each hormone. Parameters such as resolution factor, tailing factor and relative retention time, were determined, and are useful for the evaluation of the quality of the separation obtained between the active pharmaceutical ingredient and the excipient present in the final formulation. The potential of each method for quantification of both HSA and the hormone was also demonstrated. Besides furnishing chromatographic quantifications that can substitute for in vivo bioassays and animal use, the chromatograms also provide a direct panorama of the quality and heterogeneity of the protein of interest.

  17. Leucine-rich α2-glycoprotein is a novel biomarker of neurodegenerative disease in human cerebrospinal fluid and causes neurodegeneration in mouse cerebral cortex.

    Directory of Open Access Journals (Sweden)

    Masakazu Miyajima

    Full Text Available Leucine-rich α2-glycoprotein (LRG is a protein induced by inflammation. It contains a leucine-rich repeat (LRR structure and easily binds with other molecules. However, the function of LRG in the brain during aging and neurodegenerative diseases has not been investigated. Here, we measured human LRG (hLRG concentration in the cerebrospinal fluid (CSF and observed hLRG expression in post-mortem human cerebral cortex. We then generated transgenic (Tg mice that over-expressed mouse LRG (mLRG in the brain to examine the effects of mLRG accumulation. Finally, we examined protein-protein interactions using a protein microarray method to screen proteins with a high affinity for hLRG. The CSF concentration of hLRG increases with age and is significantly higher in patients with Parkinson's disease with dementia (PDD and progressive supranuclear palsy (PSP than in healthy elderly people, idiopathic normal pressure hydrocephalus (iNPH patients, and individuals with Alzheimer's disease (AD. Tg mice exhibited neuronal degeneration and neuronal decline. Accumulation of LRG in the brains of PDD and PSP patients is not a primary etiological factor, but it is thought to be one of the causes of neurodegeneration. It is anticipated that hLRG CSF levels will be a useful biomarker for the early diagnosis of PDD and PSP.

  18. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  19. Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

    Directory of Open Access Journals (Sweden)

    Meron Mengistu

    2015-03-01

    Full Text Available The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step

  20. Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase.

    Directory of Open Access Journals (Sweden)

    Victòria Ceperuelo-Mallafré

    Full Text Available Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes.ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR.ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG.ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.

  1. The effect of the plasticizer diethylhexyl phthalate on transport activity and expression of P-glycoprotein in parental and doxo-resistant human sarcoma cell lines.

    Science.gov (United States)

    Angelini, A; Centurione, L; Sancilio, S; Castellani, M L; Conti, P; Di Ilio, C; Porreca, E; Cuccurullo, F; Di Pietro, R

    2011-01-01

    Multidrug resistance (MDR) to cancer therapy is frequently associated with the over-expression of the multidrug transporter MDR1 gene product P-glycoprotein (Pgp) in several types of human tumours. Various chemosensitizers have been used to inhibit Pgp activity but toxicity limits their clinical application. Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that is released from polyvinyl chloride (PVC) medical devices. Therefore, cancer patients undertaking chemotherapy are exposed to a clinically important amount of DEHP through blood and blood component transfusions, apheresis products, intravenous chemotherapy, parenteral nutrition and other medical treatments. The present study was designed to investigate the effects of DEHP on transport activity and expression of Pgp in order to evaluate its potential use as a chemosensitizer in cancer therapy. Human doxorubicin (doxo) resistant sarcoma cells (MES-SA/Dx5) that over-express Pgp were treated with different doses of doxo (2, 4 and 8 μM) in the presence or absence of various concentrations of DEHP (3, 6 and 12 μM) that were clinically achievable in vivo. Our results show that co-treatment with 2, 4 and 8 μM doxo in the presence of the lowest concentration of DEHP (3 μM) enhanced significantly doxo accumulation in MES-SA/Dx5 cells and, consistently increased the sensitivity to doxo, when compared to controls receiving only doxo. In contrast, higher DEHP concentrations (6 and 12 μM) induced MES-SA/Dx5 to extrude doxo decreasing doxo cytotoxicity toward resistant cells below control values. These results are consistent with the increase in Pgp expression levels in parental MES-SA cells treated with 3, 6 and 12 μM DEHP for 24 h and compared to untreated controls. All in all, these findings suggest a potential clinical application of DEHP as a chemosensitizer to improve effectiveness of the antineoplastic drugs in MDR human tumours.

  2. ANTIPSYCHOTICS REVERSE P-GLYCOPROTEIN-MEDIATED DOXORUBICIN RESISTANCE IN HUMAN UTERINE SARCOMA MES-SA/Dx5 CELLS: A NOVEL APPROACH TO CANCER CHEMOTHERAPY.

    Science.gov (United States)

    Angelini, A; Ciofani, G; Conti, P

    2015-01-01

    Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) remains one of the major obstacles to effective cancer chemotherapy. Several chemosensitizers have been used in vivo and in vitro to reverse MDR but have exhibited several unwanted side effects. Antipsychotics are often administered to treat psychiatric disorders such as delirium, anxiety and sleep disorders in cancer patients during chemotherapy. The present in vitro study, examined the effects of two common antipsychotic compounds, haloperidol and risperidone, and a natural compound such as theobromine on reversing MDR Pgp-mediated, to evaluate their potential use as chemosensitizing agents. The human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) that overexpress Pgp (100-fold), were treated with the antipsychotic alone (1, 10 and 20 μM) or in combination with different concentrations of doxo (2, 4 and 8 μM). The accumulation and cytotoxicity of doxo (MTT assay) and cellular GSH content (GSH assay) in comparison with verapamil, a well-known Pgp inhibitor, used as reference molecule were examined. It was found that the three compounds significantly enhanced the intracellular accumulation of doxo in resistant cancer cells, when compared with cells receiving doxo alone (p 30%) in resistant cells, when compared to untreated control cells (peffective Pgp inhibitor with the lowest toxicity.

  3. Comparative evaluation of purified Taenia solium glycoproteins and crude metacestode extracts by immunoblotting for the serodiagnosis of human T. solium cysticercosis.

    Science.gov (United States)

    Rodriguez-Canul, R; Allan, J C; Fletes, C; Sutisna, I P; Kapti, I N; Craig, P S

    1997-09-01

    A lentil-lectin purified glycoprotein (LL-Gp) and a crude saline extract of Taenia solium metacestodes were compared for the immunodiagnosis of human cysticercosis by immunoblotting. The LL-Gp preparation was 95% sensitive for antibodies against a range of seven antigens with molecular masses of 50 to 13 kDa, whereas the sensitivity of the crude saline extract for the detection of antibodies against two major polypeptide molecules (26 and 8 kDa) was 91%. Specificity was 100% with both sets of diagnostic antigens. Affinity-purified antibodies against the 26-kDa molecule from the crude saline extract recognized the 24-kDa diagnostic region in the LL-Gp-purified extract and vice versa, suggesting that the antigens had common epitopes recognized by cysticercotic sera. In addition, in a preliminary community study of 115 randomly selected people from Bali (Indonesia), seroprevalence by immunoblot assay varied from 7.8% (with the crude saline antigen extract) to 9.6% (with the LL-Gp-purified extract). The results of this study demonstrate that both antigenic preparations are applicable for the immunodiagnosis of T. solium cysticercosis. The crude T. solium metacestode antigen extract was as specific as the purified LL-Gp T. solium metacestode extract and simpler to produce but slightly less sensitive.

  4. Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

    Science.gov (United States)

    Tselios, Theodore; Aggelidakis, Mihalis; Tapeinou, Anthi; Tseveleki, Vivian; Kanistras, Ioannis; Gatos, Dimitrios; Matsoukas, John

    2014-11-04

    Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

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

    Science.gov (United States)

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

    2000-09-24

    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.

  6. Rational Design and Synthesis of Altered Peptide Ligands based on Human Myelin Oligodendrocyte Glycoprotein 35–55 Epitope: Inhibition of Chronic Experimental Autoimmune Encephalomyelitis in Mice

    Directory of Open Access Journals (Sweden)

    Theodore Tselios

    2014-11-01

    Full Text Available Experimental autoimmune encephalomyelitis (EAE is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS. Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35–55 epitope of myelin oligodendrocyte glycoprotein (MOG, plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR contact residues of the human MOG35–55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35–55 peptide at the time of immunization.

  7. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  8. Human cytomegalovirus glycoprotein B genotypes in blood of AIDS patients: lack of association with either the viral DNA load in leukocytes or presence of retinitis.

    Science.gov (United States)

    Gilbert, C; Handfield, J; Toma, E; Lalonde, R; Bergeron, M G; Boivin, G

    1999-09-01

    It has been suggested that human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes could be used as a marker for viral virulence in patients with AIDS. The present study was designed to evaluate a possible association between specific gB genotypes, the presence of HCMV retinitis, and the HCMV viral load. Fifty-four blood samples were obtained from 54 HIV- and HCMV-infected patients. Twenty-seven of these patients were asymptomatic for HCMV, whereas the other 27 patients had been diagnosed recently with HCMV retinitis. HCMV gB genotyping was carried out by using restriction enzyme analysis of PCR-amplified PMNL extracts. Determination of the HCMV viral load in the same specimens was carried out using a quantitative-PCR. HCMV gB genotype 2 was found more frequently than other genotypes in PCR-amplified polymorphonuclear leukocytes (PMNL) of patients with AIDS (P < 0.05) but not more frequently in samples from patients with HCMV retinitis. No significant association was found between any HCMV gB genotypes and the viral load in blood. In conclusion, the actual HCMV gB genotyping system using PMNL provides no additional benefit over the viral load in blood for identification of HIV-infected subjects at risk of HCMV disease.

  9. Complex Human Dynamics From Mind to Societies

    CERN Document Server

    Winkowska-Nowak, Katarzyna; Brée, David

    2013-01-01

    This book, edited and authored by a closely collaborating network of social scientists and psychologists, recasts typical research topics in these fields into the language of nonlinear, dynamic and complex systems. The aim is to provide scientists with different backgrounds - physics, applied mathematics and computer sciences - with the opportunity to apply the tools of their trade to an altogether new range of possible applications. At the same time, this book will serve as a first reference for a new generation of social scientists and psychologists wishing to familiarize themselves with the new methodology and the "thinking in complexity".

  10. 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.

  11. IgG Autoantibodies against β2-Glycoprotein I Complexed with a Lipid Ligand Derived from Oxidized Low-Density Lipoprotein are Associated with Arterial Thrombosis in Antiphospholipid Syndrome

    Directory of Open Access Journals (Sweden)

    Daniel Lopez

    2003-01-01

    Full Text Available We recently reported [J. Lipid Res. 42 (2001, 697; 43 (2002, 1486; 44 (2003, 716] that β2-glycoprotein I (β2GPI forms complexes with oxidized LDL (oxLDL and autoantibodies against these complexes are present in patients with SLE and antiphospholipid syndrome (APS. The relationship of β2GPI/oxLDL complexes and IgG autoantibodies against β2GPI complexed with oxLig-1 (an oxLDL-derived ligand with clinical manifestations of APS was studied in 150 APS and SLE patients. The β2GPI/oxLDL levels of APS patients were similar to those of SLE patients without APS, but they were significantly higher than healthy individuals. There was no difference in the complex levels among the patients with arterial, venous thrombosis, or pregnancy morbidity. IgG anti-β2GPI/oxLig-1 levels of APS were significantly higher than those of SLE without APS and healthy individuals. Further, antibody levels of APS patients with arterial thrombosis were significantly higher than those patients with venous thrombosis and pregnancy morbidity. Thus, oxidation of LDL leads the complex formation with β2GPI in SLE and APS patients. In contrast, anti-β2GPI/oxLig-1 autoantibodies were generated only in APS and were strongly associated with arterial thrombosis. These results suggest that autoantibodies against β2GPI/oxLDL complexes are etiologically important in the development of atherosclerosis in APS.

  12. Modulation of P-glycoprotein by Stemona alkaloids in human multidrug resistance leukemic cells and structural relationships.

    Science.gov (United States)

    Umsumarng, Sonthaya; Pitchakarn, Pornsiri; Yodkeeree, Supachai; Punfa, Wanisa; Mapoung, Sariya; Ramli, Rosdayati Alino; Pyne, Stephen G; Limtrakul, Pornngarm

    2017-10-15

    Multidrug resistance (MDR) is a major reason for the failure of chemotherapy in the treatment of cancer patients. P-gp over-expression in MDR cancer cells is a multifactorial phenomenon with biochemical resistance mechanisms. Stemofoline (STF), isolated from Stemona bukillii, has been reported to be an MDR reversing compound. This study investigated whether other Stemona alkaloids that had been purified from Stemonaceae plants exerted MDR modulation activity. MTT assay was performed to determine the MDR reversing property of the alkaloids. Modulation of P-gp function by these compounds was investigated using cell cycle analysis and P-gp fluorescent substrate accumulation assays. P-gp expression was determined by Western blot analysis. We preliminarily examined the safety of these compounds in normal human fibroblasts and human peripheral blood mononuclear cells (PBMCs) using the MTT assay, and in red blood cells (human and rat) through in vitro hemolysis assays. Three of the eight alkaloids tested, isostemofoline (ISTF), 11Z -didehydrostemofoline (11Z-DSTF) and 11E-didehydrostemofoline (11E-DSTF), enhanced the chemotherapeutic sensitivity of MDR leukemic K562/Adr cells, which overexpressed P-gp. The P-gp functional studies showed that these three alkaloids increased the accumulation of P-gp substrates, calcein-AM (C-AM) and rhodamine123 (Rho 123) in K562/Adr cells, while this effect was not seen in drug sensitive parental K562 cells. Whereas, the alkaloids did not alter P-gp expression as was determined by Western blotting analysis. The alkaloids reversed MDR via the inhibition of P-gp function. For pharmaceutical safety testing, the alkaloids were found to be not toxic to normal human fibroblasts and PBMCs. Moreover, the effective compounds did not induce hemolysis in either human or rat erythrocytes. These compounds may be introduced as potential candidate molecules for treating cancers exhibiting P-gp-mediated MDR. Copyright © 2017 Elsevier GmbH. All rights

  13. Raman, SERS, and induced circular dichroism techniques as a probe of pharmaceuticals in their interactions with the human serum albumin and p-glycoprotein

    Science.gov (United States)

    Fleury, Fabrice; Ianoul, Anatoli I.; Baggetto, Loris; Jardillier, Jean-Claude; Alix, Alain J.; Nabiev, Igor R.

    1999-04-01

    Camptothecin (CPT) derivatives are the well known inhibitors of the human DNA topoisomerase (topo) I. Two of them, irinotecan and topotecan, are just in the clinics; 9-amino- CPT is on the stage II of clinical trials, and the active search for new derivatives is now in progress. Stability of the CPT derivatives on their way to the target and resistance of cancer cells to these drugs present the crucial problem of the chemotherapy. Human serum albumin (HSA) is the mediator of transport and metabolism of numerous pharmaceuticals in the blood and P-glycoprotein (P- gp) plays a crucial role of the mediator of the multidrug resistance (MDR) of the cancer cells. This paper present the result of analysis of molecular interactions of some drugs of CPT family with the HSA and P-gp. Induced circular dichroism (CD) and Raman techniques have been applied for monitoring molecular interaction of drugs with HSA as well as to identify the conformational transition of the protein induced by the drug binding. Drug molecular determinants responsible for interaction have been identified and their binding sites within the HSA have been localized. New cancer cells lines exhibiting an extremely high level of MDR resistance have been established and were shown to contain the P-gp overproduced in the quantities of 35 percent from the all membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental membrane proteins. The membrane fractions of these cells with the controls presented by the membranes of the parental sensitive cells may be used as a model system for spectroscopic analysis of the specific pharmaceuticals/P-gp interactions.

  14. Complex Systems and Human Performance Modeling

    Science.gov (United States)

    2013-12-01

    constitute a cognitive architecture or decomposing the work flows and resource constraints that characterize human-system interactions, the modeler...also explored the generation of so-called “ fractal ” series from simple task network models where task times are the calculated by way of a moving

  15. Purification and characterization of a native zinc-binding high molecular weight multiprotein complex from human seminal plasma.

    Science.gov (United States)

    Yadav, Vikash Kumar; Kumar, Vijay; Chhikara, Nirmal; Kumar, Sanjay; Manral, Pallavi; Kashav, Tara; Saini, Savita; Srinivasan, A; Singh, Sarman; Singh, Tej P; Yadav, Savita

    2011-05-01

    The seminal plasma comprises secretions from various accessory sex glands. During fertilization spermatozoa undergo complex sequences of precisely timed events that are regulated by the activation of different intracellular signaling pathways. The precision and efficacy of these pathways are often influenced by the assembly and interactions of multiprotein complexes, thereby directing the flow of regulatory information. Our knowledge about these protein complexes present in human seminal plasma (HuSP) is limited. Here we report the identification and characterization of a native high molecular weight zinc-binding multiprotein complex from HuSP by utilizing 2-DE followed by MS. Twenty-six proteins representing isoforms and/or fragments of 11 different proteins were found to be assembled in this complex. Prostate-specific antigen, zinc α2-glycoprotein, prostatic acid phosphatase, and prolactin inducible protein were the major proteins of this complex. Dynamic light scattering experiments revealed changes in aggregation pattern accompanied with deviation from physiological pH and in presence of SDS. However, no significant changes were observed in the presence of physiological ligands such as zinc and fructose. The present study will be useful and contribute to guide the future studies performed for elucidation of biological significance of this native complex in HuSP.

  16. Protein phosphatase complex PP5/PPP2R3C dephosphorylates P-glycoprotein/ABCB1 and down-regulates the expression and function.

    Science.gov (United States)

    Katayama, Kazuhiro; Yamaguchi, Miho; Noguchi, Kohji; Sugimoto, Yoshikazu

    2014-04-01

    P-glycoprotein (P-gp)/ABCB1 is a key molecule of multidrug resistance in cancer. Protein phosphatase (PP) 2A, regulatory subunit B, gamma (PPP2R3C), which is a regulatory subunit of PP2A and PP5, was identified as a binding candidate to P-gp. Immunoprecipitation-western blotting revealed that PP5 and PPP2R3C were coprecipitated with P-gp, while PP2A was not. PP5/PPP2R3C dephosphorylated protein kinase A/protein kinase C-phosphorylation of P-gp. Knockdown of PP5 and/or PPP2R3C increased P-gp expression and lowered the sensitivity to vincristine and doxorubicin. Consequently, our results indicate that PP5/PPP2R3C negatively regulates P-gp expression and function.

  17. Comparative analysis of the fusion efficiency elicited by the envelope glycoprotein V1-V5 regions derived from human immunodeficiency virus type 1 transmitted perinatally.

    Science.gov (United States)

    Guo, Hongyan; Abrahamyan, Levon G; Liu, Chang; Waltke, Mackenzie; Geng, Yunqi; Chen, Qimin; Wood, Charles; Kong, Xiaohong

    2012-12-01

    Understanding the properties of viruses preferentially establishing infection during perinatal transmission of human immunodeficiency virus type 1 (HIV-1) is critical for the development of effective measures to prevent transmission. A previous study demonstrated that the newly transmitted viruses (in infants) of chronically infected mother-infant pairs (MIPs) were fitter in terms of growth, which was imparted by their envelope (Env) glycoprotein V1-V5 regions, than those in the corresponding chronically infected mothers. In order to investigate whether the higher fitness of transmitted viruses was conferred by their higher entry efficiency directed by the V1-V5 regions during perinatal transmission, the fusogenicity of Env containing V1-V5 regions derived from transmitted and non-tranmsmitted viruses of five chronically infected MIPs and two acutely infected MIPs was analysed using two different cell-cell fusion assays. The results showed that, in one chronically infected MIP, a higher fusion efficiency was induced by the infant Env V1-V5 compared with that of the corresponding mother. Moreover, the V4-V5 regions played an important role in discriminating the transmitted and non-transmitted viruses in this pair. However, neither a consistent pattern nor significant differences in fusogenicity mediated by the V1-V5 regions between maternal and infant variants was observed in the other MIPs. This study suggests that there is no consistent and significant correlation between viral fitness selection and entry efficiency directed by the V1-V5 regions during perinatal transmission. Other factors such as the route and timing of transmission may also be involved.

  18. Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytes.

    Science.gov (United States)

    Spiegl-Kreinecker, Sabine; Buchroithner, Johanna; Elbling, Leonilla; Steiner, Elisabeth; Wurm, Gabriele; Bodenteich, Angelika; Fischer, Johannes; Micksche, Michael; Berger, Walter

    2002-03-01

    The poor prognosis of glioma patients is partly based on the minor success obtained from chemotherapeutic treatments. Resistance mechanisms at the tumor cell level may be, in addition to the blood-brain barrier, involved in the intrinsic chemo-insensitivity of brain tumors. We investigated the expression of the drug-transporter proteins P-glycoprotein (P-gp) and multidrug-resistance protein 1 (MRP1) in cell lines (N = 24) and primary cell cultures (N = 36) from neuroectodermal tumors, as well as in brain tumor extracts (N = 18) and normal human astrocytes (N = 1). We found that a considerable expression of P-gp was relatively rare in glioma cells, in contrast to MRP1, which was constitutively overexpressed in cells derived from astrocytomas as well as glioblastomas. Also, normal astrocytes cultured in vitro expressed high amounts of MRPI but no detectable P-gp. Meningioma cells frequently co-expressed P-gp and MRP1, while, most of the neuroblastoma cell lines express higher P-gp but lower MRP1 levels as compared to the other tumor types. Both, a drug-exporting and a chemoprotective function of P-gp as well as MRP1 could be demonstrated in selected tumor cells by a significant upregulation of cellular 3H-daunomycin accumulation and daunomycin cytotoxicity via administration of transporter antagonists. Summing up, our data suggest that P-gp contributes to cellular resistance merely in a small subgroup of gliomas, but frequently in neuroblastomas and meningiomas. In contrast, MRP1 is demonstrated to play a constitutive role in the intrinsic chemoresistance of gliomas and their normal cell counterpart.

  19. Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.

    Science.gov (United States)

    Björling, E; Broliden, K; Bernardi, D; Utter, G; Thorstensson, R; Chiodi, F; Norrby, E

    1991-01-01

    Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys. Images PMID:2068087

  20. In vitro effects of standardized extract of Bacopa monniera and its five individual active constituents on human P-glycoprotein activity.

    Science.gov (United States)

    Singh, Rajbir; Rachumallu, Ramakrishna; Bhateria, Manisha; Panduri, Jagadeesh; Bhatta, Rabi Sankar

    2015-01-01

    1. For centuries Bacopa monniera (BM) has been used as an herbal drug for the treatment of various mental ailments. A chemically standardized alcoholic extract of BM is clinically available over the counter herbal remedy for memory enhancement in children and adults. Consumption of herbal preparations has been reported to alter the function of membrane transporters, especially P-glycoprotein (P-gp), ATP-dependent drug efflux transporter responsible for the development of herb-drug interactions. 2. In the present study, we evaluated the in vitro effect of BM extract and its five individual active constituents (namely, bacopaside I, bacopaside II and bacopasaponin C, bacoside A and bacoside A3) on P-gp function using luminescent P-gp ATPase assay and Rh123 transport assay across human MDR1 gene transfected LLC-GA5-COL150 cell line. 3. It was observed that BM extract and its five individual constituents inhibited both basal activity as well as verapamil-stimulated ATPase activity, suggesting their affinity towards P-gp. Further, BM and its five active constituents inhibited the rhodamine 123 (Rh123) transport across LLC-GA5-COL150 cell monolayer with bacopaside II being the most potent inhibitor of P-gp, which decreased P-gp efflux ratio of Rh123 by fourfold in comparison to control. 4. Our finding may prove beneficial in predicting the potential herb-drug interactions of BM on concomitant medication with P-gp substrate drugs in clinical settings.

  1. Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells.

    Science.gov (United States)

    McInerney, Mitchell P; Pan, Yijun; Short, Jennifer L; Nicolazzo, Joseph A

    2017-01-05

    An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.

  2. Understanding complexity in the human brain.

    Science.gov (United States)

    Bassett, Danielle S; Gazzaniga, Michael S

    2011-05-01

    Although the ultimate aim of neuroscientific enquiry is to gain an understanding of the brain and how its workings relate to the mind, the majority of current efforts are largely focused on small questions using increasingly detailed data. However, it might be possible to successfully address the larger question of mind-brain mechanisms if the cumulative findings from these neuroscientific studies are coupled with complementary approaches from physics and philosophy. The brain, we argue, can be understood as a complex system or network, in which mental states emerge from the interaction between multiple physical and functional levels. Achieving further conceptual progress will crucially depend on broad-scale discussions regarding the properties of cognition and the tools that are currently available or must be developed in order to study mind-brain mechanisms.

  3. 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: erica@scripps.edu [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)

    2009-11-01

    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

  4. Reversion effects of curcumin on multidrug resistance of MNNG/HOS human osteosarcoma cells in vitro and in vivo through regulation of P-glycoprotein

    Institute of Scientific and Technical Information of China (English)

    SI Meng; ZHAO Jie; LI Xin; TIAN Ji-guang; LI Yong-gang; LI Jian-min

    2013-01-01

    Background P-glycoprotein (P-gp) encoded by ATP-binding cassette sub-family B member 1 (ABCB1) gene is a kind of ATP-dependent drug transporter,which plays important roles in multidrug resistance (MDR) of human cancers,such as osteosarcoma.Curcumin is a natural phenolic coloring compound originating from the rhizomes of Curcuma longa,which is proved to possess antitumor biological activities including reversion of MDR.However,the effect and molecular mechanisms of curcumin to osteosarcoma MDR remain unclear.Methods We established a human osteosarcoma drug-resistant cell line MNNG/HOS/MTX by pulse exposure to methotrexate (MTX) and verified that the new cell lines were cross-resistant to other anticancer agents.Then,according to the cytotoxicity assay,we reversed MDR of MNNG/HOS/MTX by 30 μmol/L curcumin,and detected the mechanisms of curcumin reversing MDR through Real-time PCR,Western blotting assay,and Rhodamine123 (Rh123)transport test.Finally,we evaluated the effect of curcumin reversing MDR in vivo by MNNG/HOS/MTX cells xenograft-nude mice model.Results MNNG/HOS/MTX was proved to be a human osteosarcoma MDR cell line.MTT tumor chemosensitivity test indicates that 30 μmol/L curcumin attenuates the half maximal inhibitory concentration (IC50) and resistance index (RI)to MTX,diamminedichloroplatinum (DDP),adriamycin (ADM),ifosfamide (IFO),and epirubicin (EPI) in MNNG/HOS/MTX cells (P <0.05).Real-time PCR and Western blotting assays demonstrated that curcumin down-regulated P-gp expression of MNNG/HOS/MTX cells.Rh123 transport test showed that curcumin inhibited the transport function of P-gp in vitro.In vivo studies showed that curcumin displayed the features of sensitizing antitumor drugs and inhibiting the proliferation,invasion,and metastasis of osteosarcoma MDR cells.Conclusion Down-regulation of P-gp and inhibition of the function of P-gp efflux pump may contribute to MDR reversion induced by curcumin in vitro and in vivo.

  5. Interaction profiling identifies the human nuclear exosome targeting complex

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Christensen, Marianne Spangsberg; Kristiansen, Maiken Søndergaard

    2011-01-01

    of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded...... to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model....

  6. N-glycoprofiling analysis in a simple glycoprotein model: a comparison between recombinant and pituitary glycosylated human prolactin.

    Science.gov (United States)

    Capone, Marcos V N; Suzuki, Miriam F; Oliveira, João E; Damiani, Renata; Soares, Carlos R J; Bartolini, Paolo

    2015-05-20

    Human prolactin (hPRL) is a polypeptide hormone occurring in the non-glycosylated (NG-hPRL) and glycosylated (G-hPRL) forms, with MM of approximately 23 and 25kDa, respectively. It has a single, partially occupied N-glycosylation site located at Asn-31, which makes it a particularly simple and interesting model for glycosylation studies. The bioactivity of G-hPRL is lower than that of NG-hPRL (by ca. 4-fold) and its physiological function is not clear. However, carbohydrate moieties generally play important roles in the biosynthesis, secretion, biological activity, and plasma survival of glycohormones and can vary depending on the host cell. The main objective of this study was to determine the N-glycan structures present in native, pituitary G-hPRL and compare them with those present in the recombinant hormone. To obtain recombinant G-hPRL, genetically modified Chinese hamster ovary cells (CHO), adapted to growth in suspension, were treated with cycloheximide, thus increasing the glycosylation site occupancy from 5.5% to 38.3%, thereby facilitating G-hPRL purification. CHO cell-derived G-hPRL (CHO-G-hPRL) was compared to pituitary G-hPRL (pit-G-hPRL) especially with regard to N-glycoprofiling. Among the main differences found in the pituitary sample were an extremely low presence of sialylated (1.7%) and a high percentage of sulfated (74.0%) and of fucosylated (90.5%) glycans. A ∼6-fold lower in vitro bioactivity and a higher clearance rate in mice were also found for pit-G-hPRL versus CHO-G-hPRL. N-Glycan profiling proved to be a useful and accurate methodology also for MM and carbohydrate content determination for the two G-hPRL preparations, in good agreement with the values obtained directly via MALDI-TOF-MS.

  7. HIV-1 envelope glycoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

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

  8. Immunization of rabbits with highly purified, soluble, trimeric human immunodeficiency virus type 1 envelope glycoprotein induces a vigorous B cell response and broadly cross-reactive neutralization.

    Directory of Open Access Journals (Sweden)

    Gerald V Quinnan

    Full Text Available Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env in an adjuvant containing monophosphoryl lipid A (MPL and QS21 (AS02A. Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4, gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L, also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D or monomer (gp140-L(M. Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN human monoclonal antibodies (mAbs similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction.

  9. Decreased P-glycoprotein (P-gp/MDR1) expression in inflamed human intestinal epithelium is independent of PXR protein levels

    NARCIS (Netherlands)

    Blokzijl, Hans; Vander Borght, Sara; Bok, Lisette I. H.; Libbrecht, Louis; Geuken, Mariska; Van den Heuvel, Fiona A. J.; Dijkstra, Gerard; Roskams, Tonia A. D.; Moshage, Han; Jansen, Peter L. M.; Faber, Klaas Nico

    2007-01-01

    Background: Altered P-glycoprotein expression (P-gp/MDR1) and/or function may contribute to the pathogenesis of gastrointestinal inflammatory disorders. Low intestinal mRNA levels of the pregnane X receptor (PXR) have been linked to low MDR1 mRNA levels in patients with ulcerative colitis (UC). Here

  10. Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.

    Science.gov (United States)

    Temesvari, L; Rodriguez-Paris, J; Bush, J; Steck, T L; Cardelli, J

    1994-10-14

    Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.

  11. Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl-Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation.

    Science.gov (United States)

    Zaazaa, Hala E; Salama, Nahla N; Abd El Halim, Lobna M; Salem, Maissa Y; Abd El Fattah, Laila E

    2016-01-01

    Rapid and simple isocratic high-performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of αl -acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (ά) and the stereochemical resolution factor (Rs ) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S-hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1-10 µg mL(-1) and 0.5-5 µg mL(-1) for S-hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S-hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process.

  12. Systematic analysis of human protein complexes identifies chromosome segregation proteins.

    Science.gov (United States)

    Hutchins, James R A; Toyoda, Yusuke; Hegemann, Björn; Poser, Ina; Hériché, Jean-Karim; Sykora, Martina M; Augsburg, Martina; Hudecz, Otto; Buschhorn, Bettina A; Bulkescher, Jutta; Conrad, Christian; Comartin, David; Schleiffer, Alexander; Sarov, Mihail; Pozniakovsky, Andrei; Slabicki, Mikolaj Michal; Schloissnig, Siegfried; Steinmacher, Ines; Leuschner, Marit; Ssykor, Andrea; Lawo, Steffen; Pelletier, Laurence; Stark, Holger; Nasmyth, Kim; Ellenberg, Jan; Durbin, Richard; Buchholz, Frank; Mechtler, Karl; Hyman, Anthony A; Peters, Jan-Michael

    2010-04-30

    Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.

  13. Kinetic validation of the models for P-glycoprotein ATP hydrolysis and vanadate-induced trapping. Proposal for additional steps.

    Directory of Open Access Journals (Sweden)

    Miguel Ramón Lugo

    Full Text Available P-Glycoprotein, a member of the ATP-binding cassette (ABC superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the

  14. Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins.

    Science.gov (United States)

    Cova, Marta; Oliveira-Silva, Rui; Ferreira, José Alexandre; Ferreira, Rita; Amado, Francisco; Daniel-da-Silva, Ana Luísa; Vitorino, Rui

    2015-01-01

    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.

  15. 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)

    2009-03-16

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which 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 a trimeric variant of the Epstein-Barr virus glycoprotein B.

    Science.gov (United States)

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

    2009-02-24

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-03-25

    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

  18. Copper(II) complexes encapsulated in human red blood cells.

    Science.gov (United States)

    Bonomo, R P; De Flora, A; Rizzarelli, E; Santoro, A M; Tabbí, G; Tonetti, M

    1995-09-01

    Copper(II) complexes were encapsulated in human red blood cells in order to test their possible use as antioxidant drugs by virtue of their labile character. ESR spectroscopy was used to verify whether encapsulation in red blood cells leads to the modification of such complexes. With copper(II) complexes bound to dipeptides or tripeptides, an interaction with hemoglobin was found to be present, the hemoglobin having a strong coordinative site formed by four nitrogen donor atoms. Instead, with copper(II) complexes with TAD or PheANN3, which have the greatest stability. ESR spectra always showed the original species. Only the copper(II) complex with GHL gave rise to a complicated behavior, which contained signals from iron(III) species probably coming from oxidative processes. Encapsulation of all copper(II) complexes in erythrocytes caused a slight oxidative stress, compared to the unloaded and to the native cells. However, no significant differences were observed in the major metabolic properties (GSH, glycolytic rate, hexose monophosphate shunt, Ca(2+)-ATPase) of erythrocytes loaded with different copper(II) complexes, with the exception of methemoglobin levels, which were markedly increased in the case of [Cu(GHL)H-1] compared to [Cu(TAD)]. This latter finding suggests that methemoglobin formation can be affected by the type of complex used for encapsulation, depending on the direct interaction of the copper(II) complex with hemoglobin.

  19. Peak Oil and the Everyday Complexity of Human Progress Narratives

    Directory of Open Access Journals (Sweden)

    John C. Pruit

    2012-11-01

    Full Text Available The “big” story of human progress has polarizing tendencies featuring the binary options of progress or decline. I consider human progress narratives in the context of everyday life. Analysis of the “little” stories from two narrative environments focusing on peak oil offers a more complex picture of the meaning and contours of the narrative. I consider the impact of differential blog site commitments to peak oil perspectives and identify five narrative types culled from two narrative dimensions. I argue that the lived experience complicates human progress narratives, which is no longer an either/or proposition.

  20. Effect of Xiaoyu Zhixue Tablet(消瘀止血片)on the Expression of Platelet Membrane Glycoprotein Ⅰ b/Ⅸ/Ⅴ Complex in Patients with Chronic Renal Failure

    Institute of Scientific and Technical Information of China (English)

    QIN You; SHEN Lin; LU Fu-rong; SHI Wei; LIU Jian-guo

    2008-01-01

    Objective:To investigate the effect of Xiaoyu Zhixue Tablet(消瘀止血片,XYZXT)on the expression of platelet membrane glycoprotein(GP)Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α in patients with chronic renal failure(CRF)in early metaphase.Methods:Fifty-one patients with CRF in early metaphase(treated group)were treated with XYZXT,3 months as the course of treatment for 2 courses.The previous therapies remained unchanged.Flow cytometry was used to assess the expression of platelet GP Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α in patients with CRF,and turbidity method was used to determine the platelet maximum aggregation rate(MAR),meanwhile the renal function was measured.The final data were compared with those before the treatment,and with those in the normal control group (3) healthy subjects).Results:Compared with the normal control group,expressions of GP Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α,and platelet MAR in CRF patients were significantly lower(P=0.007,P=0.001,P=0.009)before the treatment;after the treatment with XYZXT,the above indexes in CRF patients were remarkably increased(P=0.033,P=0.026,P=0.045),but still Iower than those in the normal control group,however,it was not statistically significant.Conclusion:(1)The expression of GP Ⅰ b/Ⅸ/Ⅴ complex in CRF patients of early metaphase was decreased,which lead to platelet aggregation dysfunction.This might be one of the reasons for the hemorrhagic trend in CRF.(2)XYZXT was able to upgrade expressions of GP Ⅰ b/Ⅸ/Ⅴ complex and GP Ⅰ b α in CRF patients,improve platelet function and down-regulate platelet activation in patients with CRF.

  1. Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent M sub r approx 120,000

    Energy Technology Data Exchange (ETDEWEB)

    Viitala, J.; Carlsson, S.R.; Siebert, P.D.; Fukuda, M. (La Jolla Cancer Research Foundation, CA (USA))

    1988-06-01

    Although several lysosomal membrane glycoproteins have been characterized by using specific antibodies, none of the studies so far elucidated the amino acid sequence of a lysosomal membrane glycoprotein. Here we describe cDNA clones encoding for one of the lysosome-associated membrane proteins with apparent M{sub r} {approx} 120,000, lamp A. The amino acid sequence based on the fully coded cDNA shows that as many as 18 potential N-glycosylation sites can be found in the total of 385 amino acid residues. The results obtained by endoglycosidase F digestion support the conclusion that this glycoprotein contains 18 N-glycans. These N-glycosylation sites are clustered in two domains; one contains 10 and the other contains 8 N-glycosylation sites. These domains are separated by a (proline-serine)-rich region that has a distinct homology to the IgA hinge structure. The first N-glycosylated domain is elongated to a potential leader peptide toward the NH{sub 2}-terminal end. The second N-glycosylated domain, on the other hand, is connected to a putative transmembrane portion consisting of hydrophobic amino acids. This segment, in turn, is elongated to a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end.

  2. In situ structural analysis of the human nuclear pore complex.

    Science.gov (United States)

    von Appen, Alexander; Kosinski, Jan; Sparks, Lenore; Ori, Alessandro; DiGuilio, Amanda L; Vollmer, Benjamin; Mackmull, Marie-Therese; Banterle, Niccolo; Parca, Luca; Kastritis, Panagiotis; Buczak, Katarzyna; Mosalaganti, Shyamal; Hagen, Wim; Andres-Pons, Amparo; Lemke, Edward A; Bork, Peer; Antonin, Wolfram; Glavy, Joseph S; Bui, Khanh Huy; Beck, Martin

    2015-10-01

    Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

  3. A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard-Soulier syndrome abolishes surface expression of the GPIb-V-IX complex.

    Science.gov (United States)

    Lanza, François; De La Salle, Corinne; Baas, Marie-Jeanne; Schwartz, Agnès; Boval, Bernadette; Cazenave, Jean-Pierre; Caen, Jacques P

    2002-07-01

    This paper describes the molecular defect of the second case of Bernard-Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib-V-IX complex and revealed small amounts of intracellular GPIbalpha, GPIbbeta and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard-Soulier syndrome. The change occurred in a prototypic alpha-helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co-transfection of GPIXPro7 with normal GPIbalpha and GPIbbeta into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbalpha and GPIbbeta, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb-IX complex.

  4. Complex human activities recognition using interval temporal syntactic model

    Institute of Scientific and Technical Information of China (English)

    夏利民; 韩芬; 王军

    2016-01-01

    A novel method based on interval temporal syntactic model was proposed to recognize human activities in video flow. The method is composed of two parts: feature extract and activities recognition. Trajectory shape descriptor, speeded up robust features (SURF) and histograms of optical flow (HOF) were proposed to represent human activities, which provide more exhaustive information to describe human activities on shape, structure and motion. In the process of recognition, a probabilistic latent semantic analysis model (PLSA) was used to recognize sample activities at the first step. Then, an interval temporal syntactic model, which combines the syntactic model with the interval algebra to model the temporal dependencies of activities explicitly, was introduced to recognize the complex activities with a time relationship. Experiments results show the effectiveness of the proposed method in comparison with other state-of-the-art methods on the public databases for the recognition of complex activities.

  5. Emergence of dynamical complexity related to human heart rate variability

    Science.gov (United States)

    Chang, Mei-Chu; Peng, C.-K.; Stanley, H. Eugene

    2014-12-01

    We apply the refined composite multiscale entropy (MSE) method to a one-dimensional directed small-world network composed of nodes whose states are binary and whose dynamics obey the majority rule. We find that the resulting fluctuating signal becomes dynamically complex. This dynamical complexity is caused (i) by the presence of both short-range connections and long-range shortcuts and (ii) by how well the system can adapt to the noisy environment. By tuning the adaptability of the environment and the long-range shortcuts we can increase or decrease the dynamical complexity, thereby modeling trends found in the MSE of a healthy human heart rate in different physiological states. When the shortcut and adaptability values increase, the complexity in the system dynamics becomes uncorrelated.

  6. Dynamic analysis of the human brain with complex cerebral sulci.

    Science.gov (United States)

    Tseng, Jung-Ge; Huang, Bo-Wun; Ou, Yi-Wen; Yen, Ke-Tien; Wu, Yi-Te

    2016-07-03

    The brain is one of the most vulnerable organs inside the human body. Head accidents often appear in daily life and are easy to cause different level of brain damage inside the skull. Once the brain suffered intense locomotive impact, external injuries, falls, or other accidents, it will result in different degrees of concussion. This study employs finite element analysis to compare the dynamic characteristics between the geometric models of an assumed simple brain tissue and a brain tissue with complex cerebral sulci. It is aimed to understand the free vibration of the internal brain tissue and then to protect the brain from injury caused by external influences. Reverse engineering method is used for a Classic 5-Part Brain (C18) model produced by 3B Scientific Corporation. 3D optical scanner is employed to scan the human brain structure model with complex cerebral sulci and imported into 3D graphics software to construct a solid brain model to simulate the real complex brain tissue. Obtaining the normal mode analysis by inputting the material properties of the true human brain into finite element analysis software, and then to compare the simplified and the complex of brain models.

  7. Reversal of multidrug resistance phenotype in human breast cancer cells using doxorubicin-liposome-microbubble complexes assisted by ultrasound.

    Science.gov (United States)

    Deng, Zhiting; Yan, Fei; Jin, Qiaofeng; Li, Fei; Wu, Junru; Liu, Xin; Zheng, Hairong

    2014-01-28

    The circumvention of multidrug resistance (MDR) plays a critically important role in the success of chemotherapy. The aim of this work is to investigate the effectiveness and possible mechanisms of the reversal of MDR phenotype in human breast cancer cells by using doxorubicin-liposome-microbubble complexes (DLMC) assisted by ultrasound (US). DLMC is fabricated through conjugating doxorubicin (DOX)-liposome (DL) to the surface of microbubbles (MBs) via the biotin-avidin linkage. The resulting drug-loaded complexes are then characterized and incubated with MCF-7/ADR human breast cancer cells and followed by US exposure. Our results show the more rapid cellular uptake, evident enhancement of nuclear accumulation and less drug efflux in the resistant cells treated by DLMC+US than those treated by DL, DL+verapamil under the same US treatment or DLMC without US. The enhanced drug delivery and cellular uptake also associated with the increase of cytotoxicity against MCF-7/ADR cells, lower MCF-7/ADR cell viability and higher apoptotic cells. Mechanism investigations further disclose a significant increase of reactive oxygen species (ROS) level, enhanced DNA damage and obvious reduction of P-glycoprotein expression in the resistant cells treated with DLMC+US compared with the control cases of cells treated by DLMC, DL+US or DL+verapamil+US. In conclusion, our study demonstrates that DLMC in combination with US may provide an effective delivery of drug to sensitize cells to circumvent MDR and to enhance the therapeutic index of the chemotherapy.

  8. 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

    Science.gov (United States)

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

    2016-08-01

    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

  9. Converging Strategies in Expression of Human Complex Retroviruses

    Directory of Open Access Journals (Sweden)

    Ilaria Cavallari

    2011-08-01

    Full Text Available The discovery of human retroviruses in the early 1980s revealed the existence of viral-encoded non-structural genes that were not evident in previously described animal retroviruses. Based on the absence or presence of these additional genes retroviruses were classified as ‘simple’ and ‘complex’, respectively. Expression of most of these extra genes is achieved through the generation of alternatively spliced mRNAs. The present review summarizes the genetic organization and expression strategies of human complex retroviruses and highlights the converging mechanisms controlling their life cycles.

  10. Human mitochondrial complex I assembles through the combination of evolutionary conserved modules: a framework to interpret complex I deficiencies.

    NARCIS (Netherlands)

    Ugalde, C.; Vogel, R.O.; Huijbens, R.J.F.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.; Nijtmans, L.G.J.

    2004-01-01

    With 46 subunits, human mitochondrial complex I is the largest enzyme of the oxidative phosphorylation system. We have studied the assembly of complex I in cultured human cells. This will provide essential information about the nature of complex I deficiencies and will enhance our understanding of

  11. Long Non-Coding RNAs and Complex Human Diseases

    Directory of Open Access Journals (Sweden)

    Changning Liu

    2013-09-01

    Full Text Available Long non-coding RNAs (lncRNAs are a heterogeneous class of RNAs that are generally defined as non-protein-coding transcripts longer than 200 nucleotides. Recently, an increasing number of studies have shown that lncRNAs can be involved in various critical biological processes, such as chromatin remodeling, gene transcription, and protein transport and trafficking. Moreover, lncRNAs are dysregulated in a number of complex human diseases, including coronary artery diseases, autoimmune diseases, neurological disorders, and various cancers, which indicates their important roles in these diseases. Here, we reviewed the current understanding of lncRNAs, including their definition and subclassification, regulatory functions, and potential roles in different types of complex human diseases.

  12. Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency.

    Science.gov (United States)

    Koopman, Werner J H; Visch, Henk-Jan; Verkaart, Sjoerd; van den Heuvel, Lambertus W P J; Smeitink, Jan A M; Willems, Peter H G M

    2005-10-01

    Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

  13. Complex-tone pitch representations in the human auditory system

    DEFF Research Database (Denmark)

    Bianchi, Federica

    ) listeners and the effect of musical training for pitch discrimination of complex tones with resolved and unresolved harmonics. Concerning the first topic, behavioral and modeling results in listeners with sensorineural hearing loss (SNHL) indicated that temporal envelope cues of complex tones...... for the individual pitch-discrimination abilities, the musically trained listeners still allocated lower processing effort than did the non-musicians to perform the task at the same performance level. This finding suggests an enhanced pitch representation along the auditory system in musicians, possibly as a result......Understanding how the human auditory system processes the physical properties of an acoustical stimulus to give rise to a pitch percept is a fascinating aspect of hearing research. Since most natural sounds are harmonic complex tones, this work focused on the nature of pitch-relevant cues...

  14. How to Save Human Lives with Complexity Science

    CERN Document Server

    Helbing, Dirk; Chadefaux, Thomas; Donnay, Karsten; Blanke, Ulf; Woolley-Meza, Olivia; Moussaid, Mehdi; Johansson, Anders; Krause, Jens; Schutte, Sebastian; Perc, Matjaz

    2014-01-01

    We discuss models and data of crowd disasters, crime, terrorism, war and disease spreading to show that conventional recipes, such as deterrence strategies, are not effective and sufficient to contain them. The failure of many conventional approaches results from their neglection of feedback loops, instabilities and/or cascade effects, due to which equilibrium models do often not provide a good picture of the actual system behavior. However, the complex and often counter-intuitive behavior of social systems and their macro-level collective dynamics can be understood by means of complexity science, which enables one to address the aforementioned problems more successfully. We highlight that a suitable system design and management can help to stop undesirable cascade effects and to enable favorable kinds of self-organization in the system. In such a way, complexity science can help to save human lives.

  15. Pedigree models for complex human traits involving the mitochrondrial genome

    Energy Technology Data Exchange (ETDEWEB)

    Schork, N.J.; Guo, S.W. (Univ. of Michigan, Ann Arbor, MI (United States))

    1993-12-01

    Recent biochemical and molecular-genetic discoveries concerning variations in human mtDNA have suggested a role for mtDNA mutations in a number of human traits and disorders. Although the importance of these discoveries cannot be emphasized enough, the complex natures of mitochondrial biogenesis, mutant mtDNA phenotype expression, and the maternal inheritance pattern exhibited by mtDNA transmission make it difficult to develop models that can be used routinely in pedigree analyses to quantify and test hypotheses about the role of mtDNA in the expression of a trait. In the present paper, the authors describe complexities inherent in mitochondrial biogenesis and genetic transmission and show how these complexities can be incorporated into appropriate mathematical models. The authors offer a variety of likelihood-based models which account for the complexities discussed. The derivation of the models is meant to stimulate the construction of statistical tests for putative mtDNA contribution to a trait. Results of simulation studies which make use of the proposed models are described. The results of the simulation studies suggest that, although pedigree models of mtDNA effects can be reliable, success in mapping chromosomal determinants of a trait does not preclude the possibility that mtDNA determinants exist for the trait as well. Shortcomings inherent in the proposed models are described in an effort to expose areas in need of additional research. 58 refs., 5 figs., 2 tabs.

  16. Human copy number variation and complex genetic disease.

    Science.gov (United States)

    Girirajan, Santhosh; Campbell, Catarina D; Eichler, Evan E

    2011-01-01

    Copy number variants (CNVs) play an important role in human disease and population diversity. Advancements in technology have allowed for the analysis of CNVs in thousands of individuals with disease in addition to thousands of controls. These studies have identified rare CNVs associated with neuropsychiatric diseases such as autism, schizophrenia, and intellectual disability. In addition, copy number polymorphisms (CNPs) are present at higher frequencies in the population, show high diversity in copy number, sequence, and structure, and have been associated with multiple phenotypes, primarily related to immune or environmental response. However, the landscape of copy number variation still remains largely unexplored, especially for smaller CNVs and those embedded within complex regions of the human genome. An integrated approach including characterization of single nucleotide variants and CNVs in a large number of individuals with disease and normal genomes holds the promise of thoroughly elucidating the genetic basis of human disease and diversity.

  17. Stability of glycoprotein gene sequences of herpes simplex virus type 2 from primary to recurrent human infection, and diversity of the sequences among patients attending an STD clinic

    Science.gov (United States)

    2014-01-01

    Background Herpes simplex virus type 2 (HSV-2) is sexually transmitted, leading to blisters and ulcers in the genito-anal region. After primary infection the virus is present in a latent state in neurons in sensory ganglia. Reactivation and production of new viral particles can cause asymptomatic viral shedding or new lesions. Establishment of latency, maintenance and reactivation involve silencing of genes, continuous suppression of gene activities and finally gene activation and synthesis of viral DNA. The purpose of the present work was to study the genetic stability of the virus during these events. Methods HSV-2 was collected from 5 patients with true primary and recurrent infections, and the genes encoding glycoproteins B,G,E and I were sequenced. Results No nucleotide substitution was observed in any patient, indicating genetic stability. However, since the total number of nucleotides in these genes is only a small part of the total genome, we cannot rule out variation in other regions. Conclusions Although infections of cell cultures and animal models are useful for studies of herpes simplex virus, it is important to know how the virus behaves in the natural host. We observed that several glycoprotein gene sequences are stable from primary to recurrent infection. However, the virus isolates from the different patients were genetically different. PMID:24502528

  18. Functional Role of P-Glycoprotein and Binding Protein Effect on the Placental Transfer of Lopinavir/Ritonavir in the Ex Vivo Human Perfusion Model

    Directory of Open Access Journals (Sweden)

    Pierre-Francois Ceccaldi

    2009-01-01

    Methods. Cotyledons were perfused with lopinavir, ritonavir, and the internal control antipyrin, at various albumin concentrations (10, 30, 40 g/L. After the control phase of each experiment, the P-glycoprotein inhibitor ciclosporin A was added at middle perfusion (45 minutes. Fetal Transfer Rate (FTR and Clearance Index (CLI were compared between the 2 phases. Results. In the control phase, the clearance index of lopinavir decreased from 0.401 ± 0.058 to 0.007 ± 0.027, as albumin concentrations increased from 10 g/L to higher concentrations (30, 40 g/L. When adding ciclosporin A at physiological albumin concentrations, the clearance index of lopinavir increased significantly 10.3 fold (95% of CI difference [−0.156, −0.002], P=.046 and became positive for ritonavir. Conclusions. Even at high albumin concentrations, inhibition of placental P-glycoprotein increased placental transfer of lopinavir, suggesting that this efflux pump actively reduces placental transfer of the drug. This mechanism may play a role in fetal exposure to maternal antiretroviral therapy.

  19. Environmental layout complexity affects neural activity during navigation in humans.

    Science.gov (United States)

    Slone, Edward; Burles, Ford; Iaria, Giuseppe

    2016-05-01

    Navigating large-scale surroundings is a fundamental ability. In humans, it is commonly assumed that navigational performance is affected by individual differences, such as age, sex, and cognitive strategies adopted for orientation. We recently showed that the layout of the environment itself also influences how well people are able to find their way within it, yet it remains unclear whether differences in environmental complexity are associated with changes in brain activity during navigation. We used functional magnetic resonance imaging to investigate how the brain responds to a change in environmental complexity by asking participants to perform a navigation task in two large-scale virtual environments that differed solely in interconnection density, a measure of complexity defined as the average number of directional choices at decision points. The results showed that navigation in the simpler, less interconnected environment was faster and more accurate relative to the complex environment, and such performance was associated with increased activity in a number of brain areas (i.e. precuneus, retrosplenial cortex, and hippocampus) known to be involved in mental imagery, navigation, and memory. These findings provide novel evidence that environmental complexity not only affects navigational behaviour, but also modulates activity in brain regions that are important for successful orientation and navigation.

  20. Expression of external glycoprotein gp105 and transmembrane glycoprotein gp36 of human immunodeficiency virus type 2 in insect cell by Bac-to-Bac system%用细菌/杆状病毒系统在昆虫细胞中表达HIV-2 外膜糖蛋白gp105和跨膜糖蛋白gp36

    Institute of Scientific and Technical Information of China (English)

    张应玖; 金宁一; 金善荣; 王宏伟; 沈家骢

    2001-01-01

    目的 用细菌/杆状病毒(BactoBac)表达系统在昆虫细胞Sf9中表达HIV-2外膜糖蛋白gp105和跨膜糖蛋白gp36,为研制艾滋病疫苗和诊断试剂奠定基础。方法 分别将HIV-2外膜蛋白gp105和跨膜蛋白gp36全基因序列克隆到杆状病毒转座载体pFastBacHTa和pFastBacHTb中多角体启动子下游,构建成重组转座载体pFastBacHTa-gp105和pFastBacHTb-gp36,利用细菌/杆状病毒(BactoBac)表达系统筛选重组杆状病毒,并在昆虫细胞Sf9中表达HIV-2的gp105和gp36。结果 SDS-PAGE分析结果表明,gp105基因表达产物为一66000u糖蛋白,gp36基因则表达一41000u糖蛋白,与天然产物一致。Westernblot结果显示:两者均具有抗原特异性。结论 gp105和gp36在昆虫细胞中得到了高效表达,并均被糖基化;gp105接近天然产物,gp36与天然产物一致。%Objective To develop effective vaccine and diagnostic agents ofAIDS, the external glycoprotein gp105 and transmembrane glycoprotein gp36 of human immunodeficiency virus type 2 were expressed in insect cells Spodoptera frugiperda (Sf9) using baculovirus expression system : Bac-to-Bac system. Methods The recombinant transfer vector pFast Bac HTa-gp105 and pFast Bac HTb-gp36 were constructed by cloning HIV-2 gp105 gene and gp36 gene into the end of polyhedrin promoter of baculovirus transfer vector pFast Bac HTa and pFast Bac HTb respectively. HIV-2 external glycoprotein gp105 and transmembrane glycoprotein gp36 were expressed in insect cells Spodoptera frugiperda (Sf9) by transposing these two foreign genes to shuttle vector bacmid respectively using Bac-to-Bac system. Results The resulting products determined by SDS-PAGE showed that recombinant baculovirusescontaining gp105 gene expressed a 66 kDa glycoprotein, whereas those contai-ning gp36 gene generated a 41 000u glycoprotein, the apparent molecular weight of which is equivalent to that of natural gp36. Western blot indicated that

  1. 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

    2013-01-01

    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.

  2. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions.

    Directory of Open Access Journals (Sweden)

    Adam L Vanarsdall

    2016-04-01

    Full Text Available Human cytomegalovirus (HCMV is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16-50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1 it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

  3. Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions.

    Science.gov (United States)

    Vanarsdall, Adam L; Howard, Paul W; Wisner, Todd W; Johnson, David C

    2016-04-01

    Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16-50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

  4. Human Serum Albumin Complexed with Myristate and AZT

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Lili; Yang, Feng; Chen, Liqing; Meehan, Edward J.; Huang, Mingdong (UGA); (UAH)

    2008-06-16

    3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.

  5. Proposal for analysis of human talent from complex thought

    Directory of Open Access Journals (Sweden)

    Abel Del Río Cortina

    2015-12-01

    Full Text Available In this paper, it is expose a displaying scheme of the actions of the individual in the context of the productive world*, considering a series of demonstrations framed in the learning process, this, with respect to the complexity of the human development derived from the interactions of the individual, the family, the community, the labor environment, and society in general from the perspective of volitional, cognitive, and procedural dimensions. The proposed visualization, is conceived as a relational map that includes six pillars of human interaction immersed in the above dimensions, being them, know-to be, know-to know, Know-to live, Know-to create, know-to manage, and know-to communicate, being all these reflected as a synergic structure made manifest in the know-how, from the interplay of values, emotional skills or soft skills, attitudes, knowledge, and finally, ways of proceeding. All of the above, in order to generate an approach to the relational complexity of the human talent development in society. * The productive world, in this document, is conceived as the one in which people get articulated in order to live in family, community, entrepreneurial organization, a diverse kind of institutions, and society in general.

  6. Rendezvous Integration Complexities of NASA Human Flight Vehicles

    Science.gov (United States)

    Brazzel, Jack P.; Goodman, John L.

    2009-01-01

    Propellant-optimal trajectories, relative sensors and navigation, and docking/capture mechanisms are rendezvous disciplines that receive much attention in the technical literature. However, other areas must be considered. These include absolute navigation, maneuver targeting, attitude control, power generation, software development and verification, redundancy management, thermal control, avionics integration, robotics, communications, lighting, human factors, crew timeline, procedure development, orbital debris risk mitigation, structures, plume impingement, logistics, and in some cases extravehicular activity. While current and future spaceflight programs will introduce new technologies and operations concepts, the complexity of integrating multiple systems on multiple spacecraft will remain. The systems integration task may become more difficult as increasingly complex software is used to meet current and future automation, autonomy, and robotic operation requirements.

  7. Genetics of human episodic memory: dealing with complexity.

    Science.gov (United States)

    Papassotiropoulos, Andreas; de Quervain, Dominique J-F

    2011-09-01

    Episodic memory is a polygenic behavioral trait with substantial heritability estimates. Despite its complexity, recent empirical evidence supports the notion that behavioral genetic studies of episodic memory might successfully identify trait-associated molecules and pathways. The development of high-throughput genotyping methods, of elaborated statistical analyses and of phenotypic assessment methods at the neural systems level will facilitate the reliable identification of novel memory-related genes. Importantly, a necessary crosstalk between behavioral genetic studies and investigation of causality by molecular genetic studies will ultimately pave the way towards the identification of biologically important, and hopefully druggable, genes and molecular pathways related to human episodic memory.

  8. Preeclampsia (PE is a complex illness of the human gestation

    Directory of Open Access Journals (Sweden)

    Laura Gil Urbano

    2002-12-01

    Full Text Available Preeclampsia (PE is a complex illness of the human gestation. It is responsible for a high perinatal morbimortality.It is the illness of the multiple theories; in one environmentaland genetic factors are associated to explain it. To find genes candidates to be associated with PE, two methodology types are used: association and binding studies. In this paper we presented the foundation of both studies that explain the main genes candidates to involve in the genesis of this illness,like that ones that code for the methylenetetrahydrofolatereductase, lipoprotein lipase and endothelial nitric oxidesintase, Leiden‘s V factor, angiotensinogen, HLA-G, alphatumoral necrosis factor.

  9. Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry

    DEFF Research Database (Denmark)

    Calvano, Cosima D; Zambonin, Carlo G; Jensen, Ole Nørregaard

    2008-01-01

    glycosylation profiles are associated with certain human ailments. Glycoprotein analysis by mass spectrometry of biological samples, such as blood serum, is hampered by sample complexity and the low concentration of the potentially informative glycopeptides and -proteins. We assessed the utility of lectin...... of 63 glycosylation sites in 38 proteins were identified by both methods, demonstrating distinct differences and complementarity. Serial application of custom-made microcolumns of mixed, immobilized lectins proved efficient for recovery and analysis of glycopeptides from serum samples of breast cancer...

  10. Understanding Complex Human Ecosystems: The Case of Ecotourism on Bonaire

    Directory of Open Access Journals (Sweden)

    Thomas Abel

    2003-12-01

    Full Text Available It is suggested that ecotourism development on the island of Bonaire can be productively understood as a perturbation of a complex human ecosystem. Inputs associated with ecotourism have fueled transformations of the island ecology and sociocultural system. The results of this study indicate that Bonaire's social and economic hierarchy is approaching a new, stable systems state following a 50-yr transition begun by government and industry that stabilized with the appearance of ecotourism development and population growth. Ecotourism can be understood to have "filled in" the middle of the production hierarchy of Bonaire. Interpreted from this perspective, population growth has completed the transformation by expanding into production niches at smaller scales in the production hierarchy. Both a consequence and a cause, ecotourism has transformed the island's social structure and demography. The theory and methods applied in this case study of interdisciplinary research in the field of human ecosystems are also presented.

  11. Human more complex than mouse at cellular level.

    Directory of Open Access Journals (Sweden)

    Alexander E Vinogradov

    Full Text Available The family of transcription factors with the C2H2 zinc finger domain is expanding in the evolution of vertebrates, reaching its highest numbers in the mammals. The question arises: whether an increased amount of these transcription factors is related to embryogenesis, nervous system, pathology or more of them are expressed in individual cells? Among mammals, the primates have a more complex anatomical structure than the rodents (e.g., brain. In this work, I show that a greater number of C2H2-ZF genes are expressed in the human cells than in the mouse cells. The effect is especially pronounced for C2H2-ZF genes accompanied with the KRAB domain. The relative difference between the numbers of C2H2-ZF(-KRAB genes in the human and mouse cellular transcriptomes even exceeds their difference in the genomes (i.e. a greater subset of existing in the genome genes is expressed in the human cellular transcriptomes compared to the mouse transcriptomes. The evolutionary turnover of C2H2-ZF(-KRAB genes acts in the direction of the revealed phenomenon, i.e. gene duplication and loss enhances the difference in the relative number of C2H2-ZF(-KRAB genes between human and mouse cellular transcriptomes. A higher amount of these genes is expressed in the brain and embryonic cells (compared with other tissues, whereas a lower amount--in the cancer cells. It is specifically the C2H2-ZF transcription factors whose repertoire is poorer in the cancer and richer in the brain (other transcription factors taken together do not show this trend. These facts suggest that increase of anatomical complexity is accompanied by a more complex intracellular regulation involving these transcription factors. Malignization is associated with simplification of this regulation. These results agree with the known fact that human cells are more resistant to oncogenic transformation than mouse cells. The list of C2H2-ZF genes whose suppression might be involved in malignization is provided.

  12. Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors

    Directory of Open Access Journals (Sweden)

    Beata Olejnik

    2015-07-01

    Full Text Available The impact of seminal plasma components on the fertilization outcomes in humans is still under question. The increasing number of couples facing problems with conception raises the need for predictive biomarkers. Detailed understanding of the molecular mechanisms accompanying fertilization remains another challenge. Carbohydrate–protein recognition may be of key importance in this complex field. In this study, we analyzed the unique glycosylation pattern of seminal plasma proteins, the display of high-mannose and hybrid-type oligosaccharides, by means of their reactivity with mannose-specific Galanthus nivalis lectin. Normozoospermic infertile subjects presented decreased amounts of lectin-reactive glycoepitopes compared to fertile donors and infertile patients with abnormal semen parameters. Glycoproteins containing unveiled mannose were isolated in affinity chromatography, and 17 glycoproteins were identified in liquid chromatography-tandem mass spectrometry with electrospray ionization. The N-glycome of the isolated glycoproteins was examined in matrix-assisted laser desorption ionization mass spectrometry. Eleven out of 27 identified oligosaccharides expressed terminal mannose residues, responsible for lectin binding. We suggest that lowered content of high-mannose and hybrid type glycans in normozoospermic infertile patients may be associated with impaired sperm protection from preterm capacitation and should be considered in the search for new infertility markers.

  13. Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors

    Science.gov (United States)

    Olejnik, Beata; Jarząb, Anna; Kratz, Ewa M.; Zimmer, Mariusz; Gamian, Andrzej; Ferens-Sieczkowska, Mirosława

    2015-01-01

    The impact of seminal plasma components on the fertilization outcomes in humans is still under question. The increasing number of couples facing problems with conception raises the need for predictive biomarkers. Detailed understanding of the molecular mechanisms accompanying fertilization remains another challenge. Carbohydrate–protein recognition may be of key importance in this complex field. In this study, we analyzed the unique glycosylation pattern of seminal plasma proteins, the display of high-mannose and hybrid-type oligosaccharides, by means of their reactivity with mannose-specific Galanthus nivalis lectin. Normozoospermic infertile subjects presented decreased amounts of lectin-reactive glycoepitopes compared to fertile donors and infertile patients with abnormal semen parameters. Glycoproteins containing unveiled mannose were isolated in affinity chromatography, and 17 glycoproteins were identified in liquid chromatography-tandem mass spectrometry with electrospray ionization. The N-glycome of the isolated glycoproteins was examined in matrix-assisted laser desorption ionization mass spectrometry. Eleven out of 27 identified oligosaccharides expressed terminal mannose residues, responsible for lectin binding. We suggest that lowered content of high-mannose and hybrid type glycans in normozoospermic infertile patients may be associated with impaired sperm protection from preterm capacitation and should be considered in the search for new infertility markers. PMID:26147424

  14. Complexity of serrated sutures of a human skull

    Directory of Open Access Journals (Sweden)

    Kochenkova О.V.

    2011-09-01

    Full Text Available Objective: to reveal the variability mechanism of complexity of serrated sutures of a human skull in the correlation with cranial form. Materials and methods. Researches of 253 arches of male and female skulls of patients at the age of 1 day-105 years without signs of cranial trauma or skeletal systemic diseases with absence of morphological signs of increase of intracranial pressure. Minimal (Min and maximal (Max values, average arithmetic (M, a mistake of average arithmetic (m have been studied. For definition of reliability of average size difference parametrical and non-parametric statistical criteria were used: parametrical criterion (t-criterion of Student applied for parameters submitting to the law of normal distribution (Lakin G. R, 1990. Distinctions of average arithmetic size were considered statistically authentic from 95% (p<0,05 a level of correct judgement (Plokhinskiy N.A., 1970. Results. On the surface of the arch lambdoid and coronal sutures in male skulls and lambdoid and sagittal sutures in female were found out to be of the greatest degree of complexity. Conclusion. The increase of complexity of sutures has been observed in children and adolescents; the directed asymmetry of sutures form is absent

  15. Synchronization in human musical rhythms and mutually interacting complex systems.

    Science.gov (United States)

    Hennig, Holger

    2014-09-09

    Though the music produced by an ensemble is influenced by multiple factors, including musical genre, musician skill, and individual interpretation, rhythmic synchronization is at the foundation of musical interaction. Here, we study the statistical nature of the mutual interaction between two humans synchronizing rhythms. We find that the interbeat intervals of both laypeople and professional musicians exhibit scale-free (power law) cross-correlations. Surprisingly, the next beat to be played by one person is dependent on the entire history of the other person's interbeat intervals on timescales up to several minutes. To understand this finding, we propose a general stochastic model for mutually interacting complex systems, which suggests a physiologically motivated explanation for the occurrence of scale-free cross-correlations. We show that the observed long-term memory phenomenon in rhythmic synchronization can be imitated by fractal coupling of separately recorded or synthesized audio tracks and thus applied in electronic music. Though this study provides an understanding of fundamental characteristics of timing and synchronization at the interbrain level, the mutually interacting complex systems model may also be applied to study the dynamics of other complex systems where scale-free cross-correlations have been observed, including econophysics, physiological time series, and collective behavior of animal flocks.

  16. Bioinformatics of glycoprotein of virus strain for production of rabies vaccine for human use in China%我国人用狂犬病疫苗株病毒糖蛋白生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    李加; 曹守春; 石磊泰; 唐建蓉; 田宏; 高向东; 董关木

    2012-01-01

    Objective To sequence the glycoprotein (G) genes of virus strains 4aGV, CTN-1V and PV2061 for production of rabies vaccine for human use in China, and analyze their bioinformatics. Methods The G genes of strains 4aGV, CTN-1V and PV2061 were amplified by RT-PCR and inserted into vector Pgem-T respectively. The constructed recombinant plasmids were transformed to E. Coli DH5a, and positive clones were screened and sequenced. The glycoprotein of three strains were analyzed for homology by using DNAstar MegAlign software, and for structure and potential B cell epitope by using AntheProt5. 0 and DNAstar Protean software. Results All the three strains were rabies virus genotype I , of which the homologies of gene sequences encoding G region and ORF of G region were 76. 0% ~ 88. 4% and 84. 4% ~ 91. 5% respectively, while those of amino acid sequences were 90. 5% ~ 92. 2%. Potential signal peptide structures existed in the amino acids at first 19 sites of glycoprotein of the three strains. However, the amino acid at site 19 contained the potential signal peptide breakage sites of glycoprotein of all the three strains, while that a t site 17 contained the potential signal peptide breakage site of strain 4aGV. Potential hydrophobic regions consisting of foldaway helixes were observed in amino acids at sites 463 ~ 476, 464 ~ 475 and 463 ~ 475 of strains 4aGV, CTN-1V and PV2061 respectively, which were judged as typical transmembrane regions. The proportions of α-helix in amino acid residues of glycoprotein of strains 4aGV, CTN-1V and PV2061 were 30%, 31% and 39%, while those of β-sheets were 28%. 30% and 41%, and those of β coil were 29%, 30% and 41%, respectively. Meanwhile, the sites and numbers of potential B cell antigen epitopes of glycoprotein of the three strains were similar. Conclusion The bioinformatics of glycoprotein of three rabies vaccine virus strains were analyzed, which provided a support for perfecting the standard for quality control of virus strains for

  17. Human serum albumin complexes with chlorophyll and chlorophyllin.

    Science.gov (United States)

    Ouameur, A Ahmed; Marty, R; Tajmir-Riahi, H A

    2005-02-15

    Porphyrins and their metal derivatives are strong protein binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA and protein. The presence of several high-affinity binding sites on human serum albumin (HSA) makes it possible target for many organic and inorganic molecules. Chlorophyll a and chlorophyllin (a food-grade derivative of chlorophyll), the ubiquitous green plant pigment widely consumed by humans, are potent inhibitors of experimental carcinogenesis and interact with protein and DNA in many ways. This study was designed to examine the interaction of HSA with chlorophyll (Chl) and chlorophyllin (Chln) in aqueous solution at physiological conditions. Fourier transform infrared, UV-visible, and CD spectroscopic methods were used to determine the pigment binding mode, the binding constant, and the effects of porphyrin complexation on protein secondary structure. Spectroscopic results showed that chlorophyll and chlorophyllin are located along the polypeptide chains with no specific interaction. Stronger protein association was observed for Chl than for Chln, with overall binding constants of K(Chl) = 2.9 x 10(4)M(-1) and K(Chln) = 7.0 x 10(3)M(-1). The protein conformation was altered (infrared data) with reduction of alpha-helix from 55% (free HSA) to 41-40% and increase of beta-structure from 22% (free HSA) to 29-35% in the pigment-protein complexes. Using the CDSSTR program (CD data) also showed major reduction of alpha-helix from 66% (free HSA) to 58 and 55% upon complexation with Chl and Chln, respectively.

  18. 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.

    1985-05-01

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

  19. The complexity of human walking: a knee osteoarthritis study.

    Directory of Open Access Journals (Sweden)

    Margarita Kotti

    Full Text Available This study proposes a framework for deconstructing complex walking patterns to create a simple principal component space before checking whether the projection to this space is suitable for identifying changes from the normality. We focus on knee osteoarthritis, the most common knee joint disease and the second leading cause of disability. Knee osteoarthritis affects over 250 million people worldwide. The motivation for projecting the highly dimensional movements to a lower dimensional and simpler space is our belief that motor behaviour can be understood by identifying a simplicity via projection to a low principal component space, which may reflect upon the underlying mechanism. To study this, we recruited 180 subjects, 47 of which reported that they had knee osteoarthritis. They were asked to walk several times along a walkway equipped with two force plates that capture their ground reaction forces along 3 axes, namely vertical, anterior-posterior, and medio-lateral, at 1000 Hz. Data when the subject does not clearly strike the force plate were excluded, leaving 1-3 gait cycles per subject. To examine the complexity of human walking, we applied dimensionality reduction via Probabilistic Principal Component Analysis. The first principal component explains 34% of the variance in the data, whereas over 80% of the variance is explained by 8 principal components or more. This proves the complexity of the underlying structure of the ground reaction forces. To examine if our musculoskeletal system generates movements that are distinguishable between normal and pathological subjects in a low dimensional principal component space, we applied a Bayes classifier. For the tested cross-validated, subject-independent experimental protocol, the classification accuracy equals 82.62%. Also, a novel complexity measure is proposed, which can be used as an objective index to facilitate clinical decision making. This measure proves that knee osteoarthritis

  20. The complexity of human walking: a knee osteoarthritis study.

    Science.gov (United States)

    Kotti, Margarita; Duffell, Lynsey D; Faisal, Aldo A; McGregor, Alison H

    2014-01-01

    This study proposes a framework for deconstructing complex walking patterns to create a simple principal component space before checking whether the projection to this space is suitable for identifying changes from the normality. We focus on knee osteoarthritis, the most common knee joint disease and the second leading cause of disability. Knee osteoarthritis affects over 250 million people worldwide. The motivation for projecting the highly dimensional movements to a lower dimensional and simpler space is our belief that motor behaviour can be understood by identifying a simplicity via projection to a low principal component space, which may reflect upon the underlying mechanism. To study this, we recruited 180 subjects, 47 of which reported that they had knee osteoarthritis. They were asked to walk several times along a walkway equipped with two force plates that capture their ground reaction forces along 3 axes, namely vertical, anterior-posterior, and medio-lateral, at 1000 Hz. Data when the subject does not clearly strike the force plate were excluded, leaving 1-3 gait cycles per subject. To examine the complexity of human walking, we applied dimensionality reduction via Probabilistic Principal Component Analysis. The first principal component explains 34% of the variance in the data, whereas over 80% of the variance is explained by 8 principal components or more. This proves the complexity of the underlying structure of the ground reaction forces. To examine if our musculoskeletal system generates movements that are distinguishable between normal and pathological subjects in a low dimensional principal component space, we applied a Bayes classifier. For the tested cross-validated, subject-independent experimental protocol, the classification accuracy equals 82.62%. Also, a novel complexity measure is proposed, which can be used as an objective index to facilitate clinical decision making. This measure proves that knee osteoarthritis subjects exhibit more

  1. Loop-acting diuretics do not bind to Tamm-Horsfall urinary glycoprotein.

    Science.gov (United States)

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

    1987-09-01

    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.

  2. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5420 Alpha-1-glycoproteins immunological...

  3. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-2-glycoproteins immunological test system. 866.5425 Section 866.5425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5425 Alpha-2-glycoproteins immunological...

  4. Induction of IL-12 Production in Human Peripheral Monocytes by Trypanosoma cruzi Is Mediated by Glycosylphosphatidylinositol-Anchored Mucin-Like Glycoproteins and Potentiated by IFN-γ and CD40-CD40L Interactions

    Directory of Open Access Journals (Sweden)

    Lúcia Cristina Jamli Abel

    2014-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products—like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins—are potent inducers of proinflammatory responses (i.e., cytokines and NO production by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.

  5. A Prediction Method for P-glycoprotein-Mediated Drug-Drug Interactions at the Human Blood-Brain Barrier From Blood Concentration-Time Profiles, Validated With PET Data.

    Science.gov (United States)

    Matsuda, Akihiro; Karch, Rudolf; Bauer, Martin; Traxl, Alexander; Zeitlinger, Markus; Langer, Oliver

    2017-09-01

    The purpose of this study was to establish physiologically based pharmacokinetic models to predict in humans the brain concentration-time profiles and P-glycoprotein (Pgp)-mediated brain drug-drug interactions between the model Pgp substrate (R)-[(11)C]verapamil (VPM), the model dual Pgp/breast cancer resistance protein (BCRP) substrate [(11)C]tariquidar (TQD), and the Pgp inhibitor tariquidar. The model predictions were validated with results from positron emission tomography studies in humans. Using these physiologically based pharmacokinetic models, the differences between predicted and observed areas under the concentration-time curves (AUC) of VPM and TQD in the brain were within a 1.2-fold and 2.5-fold range, respectively. Also, brain AUC increases of VPM and TQD after Pgp inhibitor administration were predicted with 2.5-fold accuracy when in vitro inhibition constant or half-maximum inhibitory concentration values of tariquidar were used. The predicted rank order of the magnitude of AUC increases reflected the results of the clinical positron emission tomography studies. Our results suggest that the established models can predict brain exposure from the respective blood concentration-time profiles and rank the magnitude of the Pgp-mediated brain drug-drug interaction potential for both Pgp and Pgp/BCRP substrates in humans. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  6. The emergence of complex patterns in online human communication

    CERN Document Server

    Mathiesen, Joachim; Jensen, Mogens H

    2012-01-01

    Social media have become essential conduits in the worldwide exchange of ideas, opinions and consumer marketing. Complex networks are important tools for analyzing the information flow in many aspects of nature and human society. Here, we introduce a method based on networks and social media to gauge how ideas, opinions and new trends impact society. We show that correlations between different international brands, nouns or US major cities follow a universal scale free distribution. The correlations indicate a self-organizing dynamics in large social organizations where the exchange of information between individuals is highly volatile. Our method provides new fundamental insight on the propagation of opinions and the emergence of trends in online communities.

  7. Forward-time simulations of human populations with complex diseases.

    Directory of Open Access Journals (Sweden)

    Bo Peng

    2007-03-01

    Full Text Available Due to the increasing power of personal computers, as well as the availability of flexible forward-time simulation programs like simuPOP, it is now possible to simulate the evolution of complex human diseases using a forward-time approach. This approach is potentially more powerful than the coalescent approach since it allows simulations of more than one disease susceptibility locus using almost arbitrary genetic and demographic models. However, the application of such simulations has been deterred by the lack of a suitable simulation framework. For example, it is not clear when and how to introduce disease mutants-especially those under purifying selection-to an evolving population, and how to control the disease allele frequencies at the last generation. In this paper, we introduce a forward-time simulation framework that allows us to generate large multi-generation populations with complex diseases caused by unlinked disease susceptibility loci, according to specified demographic and evolutionary properties. Unrelated individuals, small or large pedigrees can be drawn from the resulting population and provide samples for a wide range of study designs and ascertainment methods. We demonstrate our simulation framework using three examples that map genes associated with affection status, a quantitative trait, and the age of onset of a hypothetical cancer, respectively. Nonadditive fitness models, population structure, and gene-gene interactions are simulated. Case-control, sibpair, and large pedigree samples are drawn from the simulated populations and are examined by a variety of gene-mapping methods.

  8. Algorithmic complexity of growth hormone release in humans.

    Science.gov (United States)

    Prank, K; Wagner, M; Brabant, G

    1997-01-01

    Most hormones are secreted in an pulsatile rather than in a constant manner. This temporal pattern of pulsatile hormone release plays an important role in the regulation of cellular function and structure. In healthy humans growth hormone (GH) secretion is characterized by distinct pulses whereas patients bearing a GH producing tumor accompanied with excessive secretion (acromegaly) exhibit a highly irregular pattern of GH release. It has been hypothesized that this highly disorderly pattern of GH release in acromegaly arises from random events in the GH-producing tumor under decreased normal control of GH secretion. Using a context-free grammar complexity measure (algorithmic complexity) in conjunction with random surrogate data sets we demonstrate that the temporal pattern of GH release in acromegaly is not significantly different from a variety of stochastic processes. In contrast, normal subjects clearly exhibit deterministic structure in their temporal patterns of GH secretion. Our results support the hypothesis that GH release in acromegaly is due to random events in the GH-producing tumorous cells which might become independent from hypothalamic regulation.

  9. Algorithmic complexity of growth hormone release in humans

    Energy Technology Data Exchange (ETDEWEB)

    Prank, K.; Wagner, M.; Brabant, G. [Medical School Hannover (Germany)

    1996-12-31

    Most hormones are secreted in an pulsatile rather than in a constant manner. This temporal pattern of pulsatile hormone release plays an important role in the regulation of cellular function and structure. In healthy humans growth hormone (GH) secretion is characterized by distinct pulses whereas patients bearing a GH producing tumor accompanied with excessive secretion (acromegaly) exhibit a highly irregular pattern of GH release. It has been hypothesized that this highly disorderly pattern of GH release in acromegaly arises from random events in the GH-producing tumor under decreased normal control of GH secretion. Using a context-free grammar complexity measure (algorithmic complexity) in conjunction with random surrogate data sets we demonstrate that the temporal pattern of GH release in acromegaly is not significantly different from a variety of stochastic processes. In contrast, normal subjects clearly exhibit deterministic structure in their temporal patterns of GH secretion. Our results support the hypothesis that GH release in acromegaly is due to random events in the GH-producing tumorous cells which might become independent from hypothalamic regulation. 17 refs., 1 fig., 2 tabs.

  10. Interrogation of multidrug resistance (MDR1) P-glycoprotein (ABCB1) expression in human pancreatic carcinoma cells: correlation of 99mTc-Sestamibi uptake with western blot analysis.

    Science.gov (United States)

    Harpstrite, Scott E; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

    2014-10-01

    Histopathological studies indicate that ∼63% of pancreatic tumors express multidrug resistance (MDR1) P-glycoprotein (Pgp) and its polymorphic variants. However, Pgp expression detected at the mRNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations and the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate the status of the functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using Tc-Sestamibi were performed and correlated with western blot analysis. Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug-sensitive KB-3-1 cells, and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug-resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Tc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp inhibitor, and correlate with western blot analysis. These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from the pancreatic duct and Tc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas.

  11. Complexity of human and ecosystem interactions in an agricultural landscape

    Science.gov (United States)

    Coupe, Richard H.; Barlow, Jeannie R.; Capel, Paul D.

    2012-01-01

    The complexity of human interaction in the commercial agricultural landscape and the resulting impacts on the ecosystem services of water quality and quantity is largely ignored by the current agricultural paradigm that maximizes crop production over other ecosystem services. Three examples at different spatial scales (local, regional, and global) are presented where human and ecosystem interactions in a commercial agricultural landscape adversely affect water quality and quantity in unintended ways in the Delta of northwestern Mississippi. In the first example, little to no regulation of groundwater use for irrigation has caused declines in groundwater levels resulting in loss of baseflow to streams and threatening future water supply. In the second example, federal policy which subsidizes corn for biofuel production has encouraged many producers to switch from cotton to corn, which requires more nutrients and water, counter to national efforts to reduce nutrient loads to the Gulf of Mexico and exacerbating groundwater level declines. The third example is the wholesale adoption of a system for weed control that relies on a single chemical, initially providing many benefits and ultimately leading to the widespread occurrence of glyphosate and its degradates in Delta streams and necessitating higher application rates of glyphosate as well as the use of other herbicides due to increasing weed resistance. Although these examples are specific to the Mississippi Delta, analogous situations exist throughout the world and point to the need for change in how we grow our food, fuel, and fiber, and manage our soil and water resources.

  12. Perception of complex motion in humans and pigeons (Columba livia).

    Science.gov (United States)

    Nankoo, Jean-François; Madan, Christopher R; Spetch, Marcia L; Wylie, Douglas R

    2014-06-01

    In the primate visual system, local motion signals are pooled to create a global motion percept. Like primates, many birds are highly dependent on vision for their survival, yet relatively little is known about motion perception in birds. We used random-dot stimuli to investigate pigeons' ability to detect complex motion (radial, rotation, and spiral) compared to humans. Our human participants had a significantly lower threshold for rotational and radial motion when compared to spiral motion. The data from the pigeons, however, showed that the pigeons were most sensitive to rotational motion and least sensitive to radial motion, while sensitivity for spiral motion was intermediate. We followed up the pigeon results with an investigation of the effect of display aperture shape for rotational motion and velocity gradient for radial motion. We found no effect of shape of the aperture on thresholds, but did observe that radial motion containing accelerating dots improved thresholds. However, this improvement did not reach the thresholds levels observed for rotational motion. In sum, our experiments demonstrate that the pooling mechanism in the pigeon motion system is most efficient for rotation.

  13. HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies.

    Science.gov (United States)

    Sliepen, Kwinten; Sanders, Rogier W

    2016-01-01

    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.

  14. Cancer Biomarker Discovery: Lectin-Based Strategies Targeting Glycoproteins

    Directory of Open Access Journals (Sweden)

    David Clark

    2012-01-01

    Full Text Available Biomarker discovery can identify molecular markers in various cancers that can be used for detection, screening, diagnosis, and monitoring of disease progression. Lectin-affinity is a technique that can be used for the enrichment of glycoproteins from a complex sample, facilitating the discovery of novel cancer biomarkers associated with a disease state.

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

    Science.gov (United States)

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

    2009-03-01

    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.

  16. Thiol-reactive drug substrates of human P-glycoprotein label the same sites to activate ATPase activity in membranes or dodecyl maltoside detergent micelles.

    Science.gov (United States)

    Loo, Tip W; Clarke, David M

    2017-07-08

    P-glycoprotein (P-gp, ABCB1) is an ABC drug pump that is clinically important because it is involved in multidrug resistance. Many studies have used purified P-gp in detergent (n-dodecyl-β-D-maltoside; DM) micelles to map the locations of the drug-binding sites. A potential problem is that DM could be a substrate and affect binding of drugs to P-gp. To test whether DM was a substrate of P-gp, we used an assay involving drug-rescue of the immature 150 kDa misprocessed P-gp mutant (L1260A) to show that DM is not substrate. By contrast, the detergents Triton X-100 or NP-35 were substrates because they rescued the L1260A P-gp mutant such that the major product was the mature 170 kDa protein. Cross-linking of mutant A80C/R741C in membranes can only be inhibited by the P-gp substrate tariquidar. We show that cross-linking A80C/R741C mutant was also inhibited by tariquidar in the presence of excess DM. This result suggests that the presence of DM did not affect the tariquidar-binding site. Similarly, the presence of DM did not alter the locations of other drug-binding sites since the thiol reactive forms of the substrates verapamil or rhodamine labeled the same sites in transmembrane segments 5 (I306C for verapamil) and 6 (F343C for rhodamine) whether P-gp was in native membranes or in detergent micelles. These results suggest that the presence of DM does not alter the locations of the P-gp drug-binding sites and that the detergent purified protein is suitable for mapping their locations using biochemical or structural assays. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

    Science.gov (United States)

    Ha, Sha; Li, Fengsheng; Troutman, Matthew C.; Freed, Daniel C.; Tang, Aimin; Loughney, John W.; Wang, I-Ming; Vlasak, Josef; Nickle, David C.; Rustandi, Richard R.; Hamm, Melissa; DePhillips, Pete A.; Zhang, Ningyan; McLellan, Jason S.; Zhu, Hua; Adler, Stuart P.; McVoy, Michael A.; An, Zhiqiang

    2017-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen—the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen—the pentameric complex. We further demonstrated that following vaccination of a replication

  18. Characterization of Lassa virus glycoprotein oligomerization and influence of cholesterol on virus replication.

    Science.gov (United States)

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

    2010-01-01

    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.

  19. Discrimination of complex human behavior by pigeons (Columba livia and humans.

    Directory of Open Access Journals (Sweden)

    Muhammad A J Qadri

    Full Text Available The cognitive and neural mechanisms for recognizing and categorizing behavior are not well understood in non-human animals. In the current experiments, pigeons and humans learned to categorize two non-repeating, complex human behaviors ("martial arts" vs. "Indian dance". Using multiple video exemplars of a digital human model, pigeons discriminated these behaviors in a go/no-go task and humans in a choice task. Experiment 1 found that pigeons already experienced with discriminating the locomotive actions of digital animals acquired the discrimination more rapidly when action information was available than when only pose information was available. Experiments 2 and 3 found this same dynamic superiority effect with naïve pigeons and human participants. Both species used the same combination of immediately available static pose information and more slowly perceived dynamic action cues to discriminate the behavioral categories. Theories based on generalized visual mechanisms, as opposed to embodied, species-specific action networks, offer a parsimonious account of how these different animals recognize behavior across and within species.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...

  1. Glycoprotein labeling with click chemistry (GLCC) and carbohydrate detection.

    Science.gov (United States)

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

    2015-08-14

    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.

  2. Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex.

    Science.gov (United States)

    Stegmann, Cora; Abdellatif, Mohamed E A; Laib Sampaio, Kerstin; Walther, Paul; Sinzger, Christian

    2017-01-01

    The glycoprotein O (gO) is betaherpesvirus specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites, containing all 13 highly conserved amino acids, were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of amino acids (aa) 181 to 186 or aa 193 to 198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa 108 or aa 249 to 254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was unique, as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably, however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent but that aa 181 to 198 and cysteines 343, 218, and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is required neither for its incorporation into virions nor for proper function in cell-free infection. Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH, and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational

  3. Reducing sample complexity of polyclonal human autoantibodies by chromatofocusing.

    Science.gov (United States)

    Hagemann, Sascha; Faude, Alexander; Rabenstein, Monika; Balzer-Geldsetzer, Monika; Nölker, Carmen; Bacher, Michael; Dodel, Richard

    2010-08-15

    Chromatofocusing was performed in order to separate a polyclonal antigen-specific mixture of human immunoglobulins (IgGs) that would then allow for further analyses of as few different IgGs as possible. Because polyclonal IgGs only differ by amino acid sequence and possible post-translational modifications but not by molecular weight, we chose chromatofocusing for protein separation by different isoelectric points. We isolated antigen-specific IgGs from commercially available intravenous immunoglobulins (IVIG) using a combination of affinity- and size exclusion-chromatography and in order to reduce the complexity of the starting material IVIG was then replaced by single-donor plasmapheresis material. Using two-dimensional gel electrophoresis (2-DE), we observed a clear decrease in the number of different light and heavy chains in the chromatofocusing peak as compared to the starting material. In parallel, we monitored slight problems with the selected peak in isoelectric focusing as the first dimension of 2-DE, displayed in by the less proper focusing of the spots. When we tested whether IgGs were binding to their specific antigen after chromatofocusing, we were able to show that they were still in native conformation. In conclusion, we showed that chromatofocusing can be used as a first step in the analysis of mixtures of very similar proteins, e.g. polyclonal IgG preparations, in order to minimize the amount of different proteins in separated fractions in a reproducible way. Copyright 2010 Elsevier B.V. All rights reserved.

  4. Model of human collective decision-making in complex environments

    Science.gov (United States)

    Carbone, Giuseppe; Giannoccaro, Ilaria

    2015-12-01

    A continuous-time Markov process is proposed to analyze how a group of humans solves a complex task, consisting in the search of the optimal set of decisions on a fitness landscape. Individuals change their opinions driven by two different forces: (i) the self-interest, which pushes them to increase their own fitness values, and (ii) the social interactions, which push individuals to reduce the diversity of their opinions in order to reach consensus. Results show that the performance of the group is strongly affected by the strength of social interactions and by the level of knowledge of the individuals. Increasing the strength of social interactions improves the performance of the team. However, too strong social interactions slow down the search of the optimal solution and worsen the performance of the group. In particular, we find that the threshold value of the social interaction strength, which leads to the emergence of a superior intelligence of the group, is just the critical threshold at which the consensus among the members sets in. We also prove that a moderate level of knowledge is already enough to guarantee high performance of the group in making decisions.

  5. Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

    Energy Technology Data Exchange (ETDEWEB)

    Ding Fei [Department of Chemistry, China Agricultural University, No. 2 Yuanmingyuan Xi Road, Haidian District, Beijing 100193 (China); Liu Wei [College of Economics and Management, China Agricultural University, Beijing 100083 (China); Diao Jianxiong [Department of Chemistry, China Agricultural University, No. 2 Yuanmingyuan Xi Road, Haidian District, Beijing 100193 (China); Yin Bin [Key Laboratory of Pesticide Chemistry and Application Technology, Ministry of Agriculture, Department of Applied Chemistry, China Agricultural University, Beijing 100193 (China); Zhang Li, E-mail: zhli.work@gmail.co [Key Laboratory of Pesticide Chemistry and Application Technology, Ministry of Agriculture, Department of Applied Chemistry, China Agricultural University, Beijing 100193 (China); Sun Ying, E-mail: sunying@cau.edu.c [Department of Chemistry, China Agricultural University, No. 2 Yuanmingyuan Xi Road, Haidian District, Beijing 100193 (China)

    2011-07-15

    Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding. - Highlights: {yields} Our study highlights for the first time how binding dynamics can predominate for the new diamide insecticide, chlorantraniliprole. {yields} Chlorantraniliprole is situated within subdomain IIIA, Sudlow's site II, which is the same as that of indole-benzodiazepine site. {yields} Biophysical and molecular modeling approaches are useful to resolve the ligand interaction with biomacromolecule. {yields} It serves as a protective device in binding and in inactivating potential toxic compounds to which the body is exposed.

  6. A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

    Directory of Open Access Journals (Sweden)

    Broder Christopher C

    2010-11-01

    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

  7. Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

    Directory of Open Access Journals (Sweden)

    M.O. Diniz

    2011-05-01

    Full Text Available Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5 expressing three proteins (E7, E6, and E5 of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose induced a strong activation of E7-specific interferon-γ (INF-γ-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

  8. Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid.

    Science.gov (United States)

    Singh, Satyakam; Prasad, Nagarajan Rajendra; Chufan, Eduardo E; Patel, Bhargav A; Wang, Yi-Jun; Chen, Zhe-Sheng; Ambudkar, Suresh V; Talele, Tanaji T

    2014-05-22

    P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [(125)I]-iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp.

  9. Molecular analysis of Brazilian strains of bovine coronavirus (BCoV) reveals a deletion within the hypervariable region of the S1 subunit of the spike glycoprotein also found in human coronavirus OC43.

    Science.gov (United States)

    Brandão, P E; Gregori, F; Richtzenhain, L J; Rosales, C A R; Villarreal, L Y B; Jerez, J A

    2006-09-01

    Bovine coronavirus (BCoV) causes enteric and respiratory dis- orders in calves and dysentery in cows. In this study, 51 stool samples of calves from 10 Brazilian dairy farms were analysed by an RT-PCR that amplifies a 488-bp fragment of the hypervariable region of the spike glycoprotein gene. Maximum parsimony genealogy with a heuristic algorithm using sequences from 15 field strains studied here and 10 sequences from GenBank and bredavirus as an outgroup virus showed the existence of two major clusters (1 and 2) in this viral species, the Brazilian strains segregating in both of them. The mean nucleotide identity between the 15 Brazilian strains was 98.34%, with a mean amino acid similarity of 98%. Strains from cluster 2 showed a deletion of 6 amino acids inside domain II of the spike protein that was also found in human coronavirus strain OC43, supporting the recent proposal of a zoonotic spill- over of BCoV. These results contribute to the molecular characterization of BCoV, to the prediction of the efficiency of immunogens, and to the definition of molecular markers useful for epidemiologic surveys on coronavirus-caused diseases.

  10. BME, a novel compound of anthraquinone, down regulated P-glycoprotein expression in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells via generation of reactive oxygen species.

    Science.gov (United States)

    Wang, Jianhong; Liu, Lu; Cen, Juan; Ji, Biansheng

    2015-09-01

    P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in tumor cells is still a main obstacle for the chemotherapeutic treatment of cancers. Thus, development of effective MDR reversing agents is an important approach in the clinic. The present study revealed that BME, a novel compound of anthraquinone, elevated intracellular accumulation of the P-gp substrates and reduced concentration resulting in 50% inhibition of cell growth (IC50) values for doxorubicin (DOX) in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. Further more, BME was also reported to down regulated P-gp expression accompanying with generation of nontoxic low level of intracellular reactive oxygen species (iROS) and activation of extracellular signal-regulated kinase (ERK)1/2 as well as c-JUN N-terminal kinase (JNK). However, treatment with N-acetyl-cysteine (NAC), U0216 and SP600125 almost abolished actions of the BME mentioned above. These results indicated that the effect of the BME on the P-gp may be involved in generation of nontoxic low level of iROS and activation of ERK1/2 or JNK, which suggested valuable clues to screen and develop P-gp reversing agents.

  11. Detailed genetic analysis of hemagglutinin-neuraminidase glycoprotein gene in human parainfluenza virus type 1 isolates from patients with acute respiratory infection between 2002 and 2009 in Yamagata prefecture, Japan

    Directory of Open Access Journals (Sweden)

    Mizuta Katsumi

    2011-12-01

    Full Text Available Abstract Background Human parainfluenza virus type 1 (HPIV1 causes various acute respiratory infections (ARI. Hemagglutinin-neuraminidase (HN glycoprotein of HPIV1 is a major antigen. However, the molecular epidemiology and genetic characteristics of such ARI are not exactly known. Recent studies suggested that a phylogenetic analysis tool, namely the maximum likelihood (ML method, may be applied to estimate the evolutionary time scale of various viruses. Thus, we conducted detailed genetic analyses including homology analysis, phylogenetic analysis (using both the neighbor joining (NJ and ML methods, and analysis of the pairwise distances of HN gene in HPIV1 isolated from patients with ARI in Yamagata prefecture, Japan. Results A few substitutions of nucleotides in the second binding site of HN gene were observed among the present isolates. The strains were classified into two major clusters in the phylogenetic tree by the NJ method. Another phylogenetic tree constructed by the ML method showed that the strains diversified in the late 1980s. No positively selected sites were found in the present strains. Moreover, the pairwise distance among the present isolates was relatively short. Conclusions The evolution of HN gene in the present HPIV1 isolates was relatively slow. The ML method may be a useful phylogenetic method to estimate the evolutionary time scale of HPIV and other viruses.

  12. Similarities between GCS and human motor cortex: complex movement coordination

    Science.gov (United States)

    Rodríguez, Jose A.; Macias, Rosa; Molgo, Jordi; Guerra, Dailos

    2014-07-01

    The "Gran Telescopio de Canarias" (GTC1) is an optical-infrared 10-meter segmented mirror telescope at the ORM observatory in Canary Islands (Spain). The GTC control system (GCS), the brain of the telescope, is is a distributed object & component oriented system based on RT-CORBA and it is responsible for the management and operation of the telescope, including its instrumentation. On the other hand, the Human motor cortex (HMC) is a region of the cerebrum responsible for the coordination of planning, control, and executing voluntary movements. If we analyze both systems, as far as the movement control of their mechanisms and body parts is concerned, we can find extraordinary similarities in their architectures. Both are structured in layers, and their functionalities are comparable from the movement conception until the movement action itself: In the GCS we can enumerate the Sequencer high level components, the Coordination libraries, the Control Kit library and the Device Driver library as the subsystems involved in the telescope movement control. If we look at the motor cortex, we can also enumerate the primary motor cortex, the secondary motor cortices, which include the posterior parietal cortex, the premotor cortex, and the supplementary motor area (SMA), the motor units, the sensory organs and the basal ganglia. From all these components/areas we will analyze in depth the several subcortical regions, of the the motor cortex, that are involved in organizing motor programs for complex movements and the GCS coordination framework, which is composed by a set of classes that allow to the high level components to transparently control a group of mechanisms simultaneously.

  13. Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants.

    Science.gov (United States)

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

    2015-01-01

    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.

  14. Exploring the potential relevance of human-specific genes to complex disease

    Directory of Open Access Journals (Sweden)

    Cooper David N

    2011-01-01

    Full Text Available Abstract Although human disease genes generally tend to be evolutionarily more ancient than non-disease genes, complex disease genes appear to be represented more frequently than Mendelian disease genes among genes of more recent evolutionary origin. It is therefore proposed that the analysis of human-specific genes might provide new insights into the genetics of complex disease. Cross-comparison with the Human Gene Mutation Database (http://www.hgmd.org revealed a number of examples of disease-causing and disease-associated mutations in putatively human-specific genes. A sizeable proportion of these were missense polymorphisms associated with complex disease. Since both human-specific genes and genes associated with complex disease have often experienced particularly rapid rates of evolutionary change, either due to weaker purifying selection or positive selection, it is proposed that a significant number of human-specific genes may play a role in complex disease.

  15. Information-Theoretic Measures Predict the Human Judgment of Rhythm Complexity.

    Science.gov (United States)

    de Fleurian, Remi; Blackwell, Tim; Ben-Tal, Oded; Müllensiefen, Daniel

    2017-04-01

    To formalize the human judgment of rhythm complexity, we used five measures from information theory and algorithmic complexity to measure the complexity of 48 artificially generated rhythmic sequences. We compared these measurements to human prediction accuracy and easiness judgments obtained from a listening experiment, in which 32 participants guessed the last beat of each sequence. We also investigated the modulating effects of musical expertise and general pattern identification ability. Entropy rate and Kolmogorov complexity were correlated with prediction accuracy, and highly correlated with easiness judgments. A logistic regression showed main effects of musical training, entropy rate, and Kolmogorov complexity, and an interaction between musical training and both entropy rate and Kolmogorov complexity. These results indicate that information-theoretic concepts capture some salient features of the human judgment of rhythm complexity, and they confirm the influence of musical expertise on complexity judgments. Copyright © 2016 Cognitive Science Society, Inc.

  16. Recent Progress in Electrochemical Biosensors for Glycoproteins.

    Science.gov (United States)

    Akiba, Uichi; Anzai, Jun-Ichi

    2016-12-01

    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.

  17. Recent Progress in Electrochemical Biosensors for Glycoproteins

    Directory of Open Access Journals (Sweden)

    Uichi Akiba

    2016-12-01

    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.

  18. Fractionation of the genetic variants of human alpha 1-acid glycoprotein in the native form by chromatography on an immobilized copper(II) affinity adsorbent. Heterogeneity of the separate variants by isoelectrofocusing and by concanavalin A affinity chromatography.

    Science.gov (United States)

    Hervé, F; Gomas, E; Duché, J C; Tillement, J P

    1993-05-19

    Fractionation of the three main genetic variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), in their native (sialylated) form, by chromatography on immobilized copper(II) affinity adsorbent was investigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had been previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different samples on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two protein peaks, irrespective of the origin of the native AAG sample used. The unbound peak 1 was found to consist of the F1, the S or both variants, depending on the phenotype of the AAG sample used in the chromatography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were found to be the same as those originally yielded by the desialylated protein. However, comparison of the interactions of native and desialylated AAG with immobilized copper(II) ions, using an affinity chromatographic method and a non-chromatographic equilibrium binding technique, respectively, showed that desialylation increased the non-specific interactions of the protein with immobilized copper(II) ions. The AAG variants were not fractionated when affinity chromatography was performed using immobilized zinc, nickel or cobalt(II) ions, instead of copper. After purification of each variant in the sialylated form (F1, S and A), their respective heterogeneity was studied by analytical isoelectrofocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants was investigated by affinity chromatography on immobilized concanavalin A.

  19. B cell line epitopes prediction of human cytomegalovirus glycoprotein B%人巨细胞病毒包膜糖蛋白B线性B细胞抗原表位的预测

    Institute of Scientific and Technical Information of China (English)

    闫晶晶; 吕茂民; 赵雄; 尹惠琼; 章金刚

    2015-01-01

    目的:通过生物信息学方法分析人巨细胞病毒( human cytomegalovirus ,HCMV)包膜糖蛋白B ( glycopro-tein B,gB)的结构及理化特性,预测gB的线性B细胞抗原表位。方法基于HCMV gB的序列,利用两种在线B细胞表位预测程序及DNAstar软件对gB进行预测;利用SWISS-MODEL服务器同源构建gB三级结构模型,进行进一步验证。结果与结论综合多项分析,预测得到HCMVgB的多个线性B细胞表位,为后续验证其优势中和表位,建立富含高效价中和抗体的原料血浆的筛选方法奠定了基础。%Objective To predict the B cell line epitopes of human cytomegalovirus glycoprotein (gB)by analyzing its structure and physicochemical properties using bioinformatics approaches .Methods Based on the sequence of the HCMV gB,the probable B cell epitopes are predicted using two online prediction programs and DNAstar software .Meanwhile,the tertiary structure of gB was constructed by homologous modeling with the assistance of SWISS -MODEL server to rule out im-possible B cell epitopes .Results and Conclusion The B cell line epitopes of gB are predicted , which provides a theoreti-cal basis for further verification of gB immunodominant epitopes and screening the source plasma with high HCMV IgG titer .

  20. Surface plasmon resonance for real-time study of lectin-carbohydrate interactions for the differentiation and identification of glycoproteins.

    Science.gov (United States)

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

    2011-06-15

    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.

  1. Worms under stress: C. elegans stress response and its relevance to complex human disease and aging

    NARCIS (Netherlands)

    Rodriguez Sanchez, M.; Snoek, L.B.; Bono, de M.; Kammenga, J.E.

    2013-01-01

    Many organisms have stress response pathways, components of which share homology with players in complex human disease pathways. Research on stress response in the nematode worm Caenorhabditis elegans has provided detailed insights into the genetic and molecular mechanisms underlying complex human d

  2. X-ray, Cryo-EM, and computationally predicted protein structures used in integrative modeling of HIV Env glycoprotein gp120 in complex with CD4 and 17b

    Directory of Open Access Journals (Sweden)

    Muhibur Rasheed

    2016-03-01

    Full Text Available We present the data used for an integrative approach to computational modeling of proteins with large variable domains, specifically applied in this context to model HIV Env glycoprotein gp120 in its CD4 and 17b bound state. The initial data involved X-ray structure PDBID:1GC1 and electron microscopy image EMD:5020. Other existing X-ray structures were used as controls to validate and hierarchically refine partial and complete computational models. A summary of the experiment protocol and data was published (Rasheed et al., 2015 [26], along with detailed analysis of the final model (PDBID:3J70 and its implications.

  3. Formation mechanism and biological activity of novel thiolated human-like collagen iron complex.

    Science.gov (United States)

    Zhu, Chenhui; Liu, Lingyun; Deng, Jianjun; Ma, Xiaoxuan; Hui, Junfeng; Fan, Daidi

    2016-03-01

    To develop an iron supplement that is effectively absorbed and utilized, thiolated human-like collagen was created to improve the iron binding capacity of human-like collagen. A thiolated human-like collagen-iron complex was prepared in a phosphate buffer, and one mole of thiolated human-like collagen-iron possessed approximately 28.83 moles of iron. The characteristics of thiolated human-like collagen-iron were investigated by ultraviolet-visible absorption spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and differential scanning calorimetry. The results showed that the thiolated human-like collagen-iron complex retained the secondary structure of human-like collagen and had greater thermodynamic stability than human-like collagen, although interactions between iron ions and human-like collagen occurred during the formation of the complex. In addition, to evaluate the bioavailability of thiolated human-like collagen-iron, an in vitro Caco-2 cell model and an in vivo iron deficiency anemia mouse model were employed. The data demonstrated that the thiolated human-like collagen-iron complex exhibited greater bioavailability and was more easily utilized than FeSO4, ferric ammonium citrate, or ferrous glycinate. These results indicated that the thiolated human-like collagen-iron complex is a potential iron supplement in the biomedical field.

  4. Enhanced dynamic complexity in the human EEG during creative thinking.

    Science.gov (United States)

    Mölle, M; Marshall, L; Lutzenberger, W; Pietrowsky, R; Fehm, H L; Born, J

    1996-04-12

    This study shows that divergent thinking, considered the general process underlying creative production, can be distinguished from convergent, analytical thought based on the dimensional complexity of ongoing electroencephalographic (EEG) activity. EEG complexity over the central and posterior cortex was higher while subjects solved tasks of divergent than convergent thinking, and also higher than during mental relaxation. Over the frontal cortex, EEG complexity was comparable during divergent thinking and mental relaxation, but reduced during convergent thinking. Results indicate that the basic process underlying the generation of novel ideas expresses itself in a strong increase in the EEG's complexity, reflecting higher degrees of freedom in the competitive interactions among cortical neuron assemblies. Frontocortical EEG complexity being comparable with that during mental relaxation, speaks for a loosened attentional control during creative thinking.

  5. Preparation of boronate-functionalized molecularly imprinted monolithic column with polydopamine coating for glycoprotein recognition and enrichment.

    Science.gov (United States)

    Lin, Zian; Wang, Juan; Tan, Xiaoqing; Sun, Lixiang; Yu, Ruifang; Yang, Huanghao; Chen, Guonan

    2013-12-06

    A novel imprinting strategy using reversible covalent complexation of glycoprotein was described for creating glycoprotein-specific recognition cavities on boronate-functionalized monolithic column. Based on it, a molecularly imprinted monolithic column was prepared by self-polymerization of dopamine (DA) on the surface of 4-vinylphenylboronic acid (VPBA)-based polymeric skeletons after reversible immobilization of horseradish peroxidase (HRP). Due to the combination of boronate affinity and surface imprinting of DA, the stable and accessible recognition sites in the as-prepared imprinted monolith could be obtained after the removal of the template, which facilitated the rebinding of the template and provided good reproducibility and lifetime of use. The recognition behaviors of proteins on the bare VPBA-based, HRP-imprinted and nonimprinted monolithic columns were evaluated in detail and the results showed that the HRP-imprinted monolith exhibited higher recognition ability toward the template than another two monolithic columns. Not only nonglycoproteins but also glycoproteins can be well separated with the HRP-imprinted monolith. In addition, the feasibility of the HRP-imprinted monolith, adopted as an in-tube solid phase microextraction (in-tube SPME), was further assessed by selective extraction and enrichment of HRP from human serum. The good results demonstrated its potential in glycoproteome analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells

    Science.gov (United States)

    Alam, Shahidul; Anugraham, Merrina; Huang, Yen-Lin; Kohler, Reto S.; Hettich, Timm; Winkelbach, Katharina; Grether, Yasmin; López, Mónica Núñez; Khasbiullina, Nailia; Bovin, Nicolai V.; Schlotterbeck, Götz; Jacob, Francis

    2017-01-01

    The (neo-) lacto series glycosphingolipids (nsGSLs) comprise of glycan epitopes that are present as blood group antigens, act as primary receptors for human pathogens and are also increasingly associated with malignant diseases. Beta-1, 3-N-acetyl-glucosaminyl-transferase 5 (B3GNT5) is suggested as the key glycosyltransferase for the biosynthesis of nsGSLs. In this study, we investigated the impact of CRISPR-Cas9 -mediated gene disruption of B3GNT5 (∆B3GNT5) on the expression of glycosphingolipids and N-glycoproteins by utilizing immunostaining and glycomics-based PGC-UHPLC-ESI-QTOF-MS/MS profiling. ∆B3GNT5 cells lost nsGSL expression coinciding with reduction of α2-6 sialylation on N-glycoproteins. In contrast, disruption of B4GALNT1, a glycosyltransferase for ganglio series GSLs did not affect α2-6 sialylation on N-glycoproteins. We further profiled all known α2-6 sialyltransferase-encoding genes and showed that the loss of α2-6 sialylation is due to silencing of ST6GAL1 expression in ∆B3GNT5 cells. These results demonstrate that nsGSLs are part of a complex network affecting N-glycosylation in ovarian cancer cells. PMID:28358117

  7. 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.

    2009-01-01

    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

  8. Human Navigational Performance in a Complex Network with Progressive Disruptions

    CERN Document Server

    Ramesh, Amitash; Iyengar, Sudarshan; Sekhar, Vinod

    2012-01-01

    The current paper is an investigation towards understanding the navigational performance of humans on a network when the "landmark" nodes are blocked. We observe that humans learn to cope up, despite the continued introduction of blockages in the network. The experiment proposed involves the task of navigating on a word network based on a puzzle called the wordmorph. We introduce blockages in the network and report an incremental improvement in performance with respect to time. We explain this phenomenon by analyzing the evolution of the knowledge in the human participants of the underlying network as more and more landmarks are removed. We hypothesize that humans learn the bare essentials to navigate unless we introduce blockages in the network which would whence enforce upon them the need to explore newer ways of navigating. We draw a parallel to human problem solving and postulate that obstacles are catalysts for humans to innovate techniques to solve a restricted variant of a familiar problem.

  9. The Impact of Evolutionary Driving Forces on Human Complex Diseases: A Population Genetics Approach

    Directory of Open Access Journals (Sweden)

    Amr T. M. Saeb

    2016-01-01

    Full Text Available Investigating the molecular evolution of human genome has paved the way to understand genetic adaptation of humans to the environmental changes and corresponding complex diseases. In this review, we discussed the historical origin of genetic diversity among human populations, the evolutionary driving forces that can affect genetic diversity among populations, and the effects of human movement into new environments and gene flow on population genetic diversity. Furthermore, we presented the role of natural selection on genetic diversity and complex diseases. Then we reviewed the disadvantageous consequences of historical selection events in modern time and their relation to the development of complex diseases. In addition, we discussed the effect of consanguinity on the incidence of complex diseases in human populations. Finally, we presented the latest information about the role of ancient genes acquired from interbreeding with ancient hominids in the development of complex diseases.

  10. Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation.

    Science.gov (United States)

    Lai, Chih-Hsin; Lai, Ying-Jung J; Chou, Feng-Pai; Chang, Hsiang-Hua D; Tseng, Chun-Che; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2016-01-01

    Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

  11. Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation.

    Directory of Open Access Journals (Sweden)

    Chih-Hsin Lai

    Full Text Available Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

  12. Expression of the Surface Glycoproteins of Human Parainfluenza Virus Type 3 by Bovine Parainfluenza Virus Type 3, a Novel Attenuated Virus Vaccine Vector

    OpenAIRE

    Haller, Aurelia A.; Miller, Tessa; Mitiku, Misrach; Coelingh, Kathleen

    2000-01-01

    Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive...

  13. P-glycoprotein-Based Loperamide-Cyclosporine Drug Interaction at the Rat Blood-Brain Barrier: Prediction from In Vitro Studies and Extrapolation to Humans

    OpenAIRE

    Hsiao, Peng; Unadkat, Jashvant D.

    2012-01-01

    We have shown that the rat can quantitatively predict the verapamil-cyclopsorine A (CsA) drug-drug interaction (DDI) at the human blood-brain barrier (BBB). In addition, the potency (EC50) of CsA to inhibit rat BBB P-gp can be predicted from in vitro studies in MDRI-transfected cells. To assess if these excellent agreements extend to other substrates, we determined the magnitude of P-gp-based DDI at the rat BBB between loperamide (Lop) or its metabolite, N-desmethyl Lop (dLop), and escalating...

  14. Mitochondrial Complex I plays an Essential Role in Human Respirasome Assembly

    OpenAIRE

    Moreno Lastres, David; Fontanesi, Flavia; García Consuegra, Inés; Martín, Miguel A.; Arenas, Joaquín; Barrientos, Antoni; Ugalde, Cristina

    2012-01-01

    The assembly and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects lead to severe human disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold fo...

  15. Alternative promoter usage of the membrane glycoprotein CD36

    Directory of Open Access Journals (Sweden)

    Whatling Carl

    2006-03-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Harriet Allison

    2014-10-01

    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.

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

    Science.gov (United States)

    Allison, Harriet; O’Reilly, Amanda J.; Sternberg, Jeremy; Field, Mark C.

    2014-01-01

    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. PMID:26167471

  18. Linguistic complex networks: Rationale, application, interpretation, and directions. Reply to comments on "Approaching human language with complex networks"

    Science.gov (United States)

    Cong, Jin; Liu, Haitao

    2014-12-01

    Amid the enthusiasm for real-world networks of the new millennium, the enquiry into linguistic networks is flourishing not only as a productive branch of the new networks science but also as a promising approach to linguistic research. Although the complex network approach constitutes a potential opportunity to make linguistics a science, the world of linguistics seems unprepared to embrace it. For one thing, linguistics has been largely unaffected by quantitative methods. Those who are accustomed to qualitative linguistic methods may find it hard to appreciate the application of quantitative properties of language such as frequency and length, not to mention quantitative properties of language modeled as networks. With this in mind, in our review [1] we restrict ourselves to the basics of complex networks and the new insights into human language with the application of complex networks. For another, while breaking new grounds and posing new challenges for linguistics, the complex network approach to human language as a new tradition of linguistic research is faced with challenges and unsolved issues of its own. It is no surprise that the comments on our review, especially their skepticism and suggestions, focus on various different aspects of the complex network approach to human language. We are grateful to all the insightful and penetrating comments, which, together with our review, mark a significant impetus to linguistic research from the complex network approach. In this reply, we would like to address four major issues of the complex network approach to human language, namely, a) its theoretical rationale, b) its application in linguistic research, c) interpretation of the results, and d) directions of future research.

  19. CD147 interacts with NDUFS6 in regulating mitochondrial complex I activity and the mitochondrial apoptotic pathway in human malignant melanoma cells.

    Science.gov (United States)

    Luo, Z; Zeng, W; Tang, W; Long, T; Zhang, J; Xie, X; Kuang, Y; Chen, M; Su, J; Chen, X

    2014-01-01

    Malignant melanoma (MM) is one of the most lethal tumors and is characterized by high invasiveness, frequent metastasis, and resistance to chemotherapy. The risk of metastatic MM is accompanied by disordered energy metabolism involving the oxidative phosphorylation (OXPHOS) process, which is largely carried out in mitochondrial complexes. Complex I is the first and largest mitochondrial enzyme complex associated with this process. CD147 is a transmembrane glycoprotein mainly expressed on the cell surface, and also appears in the cytoplasm in some tumors. We found that CD147 is often translocated to the cytoplasm in metastatic MM specimens as compared to primary MM. We also demonstrated high expression of CD147 in isolated mitochondrial fractions of A375 cells. The yeast two-hybrid (Y2H) assay identified NDUFS6 (which encodes a subunit of mitochondrial respiratory chain complex I) as a candidate that interacts with CD147 and depletion of CD147 in A375 cells significantly decreased complex I enzyme activity. We also showed that CD147 increased the viability of A375 cells exposed to berberine-induced mitochondrial damage, and protected them from apoptosis through a mitochondrial-dependent pathway. This finding was confirmed by adding exogenous Bcl-2 to A375 cell cultures. In summary, our results identify the existence of CD147 in human melanoma cell mitochondria. They indicate that CD147 appears to regulate complex I activity and apoptosis in MM by interacting with mitochondrial NDUFS6. Our findings provide new insight into the function of CD147 and identify it as a promising therapeutic target in melanoma through disruption of the energy metabolism.

  20. Attenuated human parainfluenza virus type 1 (HPIV1) expressing the respiratory syncytial virus (RSV) fusion F glycoprotein from an added gene: effects of pre-fusion stabilization and packaging of RSV F.

    Science.gov (United States)

    Liu, Xiang; Liang, Bo; Ngwuta, Joan; Liu, Xueqiao; Surman, Sonja; Lingemann, Matthias; Kwong, Peter D; Graham, Barney S; Collins, Peter L; Munir, Shirin

    2017-08-23

    Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the pre-fusion (pre-F) conformation by previously-described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane (TM) and cytoplasmic tail (CT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.Importance. RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a

  1. P-glycoprotein-based loperamide-cyclosporine drug interaction at the rat blood-brain barrier: prediction from in vitro studies and extrapolation to humans.

    Science.gov (United States)

    Hsiao, Peng; Unadkat, Jashvant D

    2012-03-05

    We have shown that the rat can quantitatively predict the verapamil-cyclopsorine A (CsA) drug-drug interaction (DDI) at the human blood-brain barrier (BBB). In addition, the potency (EC(50)) of CsA to inhibit rat BBB P-gp can be predicted from in vitro studies in MDRI-transfected cells. To assess if these excellent agreements extend to other substrates, we determined the magnitude of P-gp-based DDI at the rat BBB between loperamide (Lop) or its metabolite, N-desmethyl Lop (dLop), and escalating CsA blood concentrations. The percent increase in the brain:blood Lop concentration ratio was described by the Hill equation, E(max) = 2000%, EC(50) = 7.1 μM and γ = 3.7. The potency (EC(50)) of CsA to inhibit P-gp at the rat BBB was independent of the substrate used (verapamil, Lop, or dLop). Like the verapamil-CsA DDI, the potency (EC(50)) of CsA to inhibit rat BBB P-gp could be predicted from studies in MDRI-transfected cells. When (11)C-Lop was coadministered with a 10 mg/kg iv infusion of CsA (1) yielding ~5.6 uM CsA blood concentration to healthy volunteers, the brain distribution of (11)C-radioactivity was increased by 110%. (1) When corrected for diffusible Lop metabolite(s), this translates into an increase in (11)C-Lop brain distribution of 457%. Based on our rat data, we estimated a similar value at 5.6 μM blood CsA concentration, 588% increase in Lop brain distribution. These data support our conclusion that the rat is a promising model to predict P-gp based DDI at the human BBB.

  2. A complex genome-microRNA interplay in human mitochondria.

    Science.gov (United States)

    Shinde, Santosh; Bhadra, Utpal

    2015-01-01

    Small noncoding regulatory RNA exist in wide spectrum of organisms ranging from prokaryote bacteria to humans. In human, a systematic search for noncoding RNA is mainly limited to the nuclear and cytosolic compartments. To investigate whether endogenous small regulatory RNA are present in cell organelles, human mitochondrial genome was also explored for prediction of precursor microRNA (pre-miRNA) and mature miRNA (miRNA) sequences. Six novel miRNA were predicted from the organelle genome by bioinformatics analysis. The structures are conserved in other five mammals including chimp, orangutan, mouse, rat, and rhesus genome. Experimentally, six human miRNA are well accumulated or deposited in human mitochondria. Three of them are expressed less prominently in Northern analysis. To ascertain their presence in human skeletal muscles, total RNA was extracted from enriched mitochondria by an immunomagnetic method. The expression of six novel pre-miRNA and miRNA was confirmed by Northern blot analysis; however, low level of remaining miRNA was found by sensitive Northern analysis. Their presence is further confirmed by real time RT-PCR. The six miRNA find their multiple targets throughout the human genome in three different types of software. The luciferase assay was used to confirm that MT-RNR2 gene was the potential target of hsa-miR-mit3 and hsa-miR-mit4.

  3. Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency.

    Science.gov (United States)

    Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V

    2017-11-01

    P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. Published by Elsevier Inc.

  4. Double-transduced MDCKII cells to study human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) interplay in drug transport across the blood-brain barrier.

    Science.gov (United States)

    Poller, Birk; Wagenaar, Els; Tang, Seng Chuan; Schinkel, Alfred H

    2011-04-04

    P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) combination knockout mice display disproportionately increased brain penetration of shared substrates, including topotecan and several tyrosine kinase inhibitors, compared to mice deficient for only one transporter. To better study the interplay of both transporters also in vitro, we generated a transduced polarized MDCKII cell line stably coexpressing substantial levels of human ABCB1 and ABCG2 (MDCKII-ABCB1/ABCG2). Next, we measured concentration-dependent transepithelial transport of topotecan, sorafenib and sunitinib. By blocking either one or both of the transporters simultaneously, using specific inhibitors, we aimed to mimic the ABCB1-ABCG2 interplay at the blood-brain barrier in wild-type, single or combination knockout mice. ABCB1 and ABCG2 contributed to similar extents to topotecan transport, which was only partly saturable. For sorafenib transport, ABCG2 was the major determinant at low concentrations. However, saturation of ABCG2-mediated transport occurred at higher sorafenib concentrations, where ABCB1 was still fully active. Furthermore, sunitinib was transported equally by ABCB1 and ABCG2 at low concentrations, but ABCG2-mediated transport became saturated at lower concentrations than ABCB1-mediated transport. The relative impact of these transporters can thus be affected by the applied drug concentrations. A comparison of the in vitro observed (inverse) transport ratios and cellular accumulation of the drugs at low concentrations with in vivo brain penetration data from corresponding Abcb1a/1b⁻/⁻, Abcg2⁻/⁻ and Abcb1a/1b;Abcg2⁻/⁻ mouse strains revealed very similar qualitative patterns for each of the tested drugs. MDCKII-ABCB1/ABCG2 cells thus present a useful in vitro model to study the interplay of ABCB1 and ABCG2.

  5. Investigation of interaction of human platelet membrane components with anticoagulant drugs Abciximab and Eptifibatide

    Directory of Open Access Journals (Sweden)

    Ewa Gorodkiewicz

    2010-04-01

    Full Text Available Abciximab (Abci and eptifibatide (Epti are antiaggregate drugs which may reduce thrombotic complications inacute coronary syndromes. The aim of this work was the investigation of the interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and Abci or Epti, and the influence of these drugs on the phospholipid ratio in the plateletmembrane. The interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and antiaggregate drugs were investigatedusing the Surface Plasmon Resonance Imaging technique (SPRI. Phospholipids phosphatidylinositol (PI, phosphatidylserine(PS, phosphatidylethanolamine (PE, phosphatidylcholine (PC and sphingomyelin (SM were first immobilizedonto the gold chip surface. The phospholipid ratio in the platelet membrane was determined by the HPLC. Only PI,PS, PE and PC were determined. Human platelets treated 'in vitro' with Abci or Epti exhibit changes in the phospholipidratio in the platelet membrane. The ratio of PS decreases and PC rises. The SPRI distinctly shows interactions between phospholipidsand glycoprotein GPIIb/IIIa, and between the phospholipid-glycoprotein GPIIb/IIIa complex and Abci or Epti.The interaction between phospholipids and glycoprotein GPIIb/IIIa is growing in the sequence: PI<glycoprotein GPIIb/IIIa complex and Abci/Epti is growing in the sequence:PS

  6. Investigation of interaction of human platelet membrane components with anticoagulant drugs Abciximab and Eptifibatide.

    Directory of Open Access Journals (Sweden)

    Anna Sankiewicz

    2011-04-01

    Full Text Available Abciximab (Abci and eptifibatide (Epti are antiaggregate drugs which may reduce thrombotic complications in acute coronary syndromes. The aim of this work was the investigation of the interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and Abci or Epti, and the influence of these drugs on the phospholipid ratio in the platelet membrane. The interaction between the phospholipid-GPIIb/IIIa glycoprotein complex and antiaggregate drugs were investigated using the Surface Plasmon Resonance Imaging technique (SPRI. Phospholipids phosphatidylinositol (PI, phosphatidylserine (PS, phosphatidylethanolamine (PE, phosphatidylcholine (PC and sphingomyelin (SM were first immobilized onto the gold chip surface. The phospholipid ratio in the platelet membrane was determined by the HPLC. Only PI, PS, PE and PC were determined. Human platelets treated 'in vitro' with Abci or Epti exhibit changes in the phospholipid ratio in the platelet membrane. The ratio of PS decreases and PC rises. The SPRI distinctly shows interactions between phospholipids and glycoprotein GPIIb/IIIa, and between the phospholipid-glycoprotein GPIIb/IIIa complex and Abci or Epti. The interaction between phospholipids and glycoprotein GPIIb/IIIa is growing in the sequence: PIglycoprotein GPIIb/IIIa complex and Abci/Epti is growing in the sequence: PS

  7. Significance of respirasomes for the assembly/stability of human respiratory chain complex I.

    Science.gov (United States)

    Schägger, Hermann; de Coo, René; Bauer, Matthias F; Hofmann, Sabine; Godinot, Catherine; Brandt, Ulrich

    2004-08-27

    We showed that the human respiratory chain is organized in supramolecular assemblies of respiratory chain complexes, the respirasomes. The mitochondrial complexes I (NADH dehydrogenase) and III (cytochrome c reductase) form a stable core respirasome to which complex IV (cytochrome c oxidase) can also bind. An analysis of the state of respirasomes in patients with an isolated deficiency of single complexes provided evidence that the formation of respirasomes is essential for the assembly/stability of complex I, the major entry point of respiratory chain substrates. Genetic alterations leading to a loss of complex III prevented respirasome formation and led to the secondary loss of complex I. Therefore, primary complex III assembly deficiencies presented as combined complex III/I defects. This dependence of complex I assembly/stability on respirasome formation has important implications for the diagnosis of mitochondrial respiratory chain disorders.

  8. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host*

    Science.gov (United States)

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M.; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q.; Kilcoyne, Michelle; Hokke, Cornelis H.; Dalton, John P.; O'Neill, Sandra M.

    2016-01-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica. Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. PMID:27466253

  9. Fasciola hepatica Surface Tegument: Glycoproteins at the Interface of Parasite and Host.

    Science.gov (United States)

    Ravidà, Alessandra; Cwiklinski, Krystyna; Aldridge, Allison M; Clarke, Paul; Thompson, Roisin; Gerlach, Jared Q; Kilcoyne, Michelle; Hokke, Cornelis H; Dalton, John P; O'Neill, Sandra M

    2016-10-01

    Fasciola hepatica, commonly known as liver fluke, is a trematode that causes Fasciolosis in ruminants and humans. The outer tegumental coat of F. hepatica (FhTeg) is a complex metabolically active biological matrix that is continually exposed to the host immune system and therefore makes a good vaccine target. F. hepatica tegumental coat is highly glycosylated and helminth-derived immunogenic oligosaccharide motifs and glycoproteins are currently being investigated as novel vaccine candidates. This report presents the first systematic characterization of FhTeg glycosylation using lectin microarrays to characterize carbohydrates motifs present, and lectin histochemistry to localize these on the F. hepatica tegument. We discovered that FhTeg glycoproteins are predominantly oligomannose oligosaccharides that are expressed on the spines, suckers and tegumental coat of F. hepatica and lectin blot analysis confirmed the abundance of N- glycosylated proteins. Although some oligosaccharides are widely distributed on the fluke surface other subsets are restricted to distinct anatomical regions. We selectively enriched for FhTeg mannosylated glycoprotein subsets using lectin affinity chromatography and identified 369 proteins by mass spectrometric analysis. Among these proteins are a number of potential vaccine candidates with known immune modulatory properties including proteases, protease inhibitors, paramyosin, Venom Allergen-like II, Enolase and two proteins, nardilysin and TRIL, that have not been previously associated with F. hepatica Furthermore, we provide a comprehensive insight regarding the putative glycosylation of FhTeg components that could highlight the importance of further studies examining glycoconjugates in host-parasite interactions in the context of F. hepatica infection and the development of an effective vaccine. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency

    NARCIS (Netherlands)

    Koopman, W.J.H.; Visch, H.J.; Verkaart, S.A.J.; Heuvel, L.W. van den; Smeitink, J.A.M.; Willems, P.H.G.M.

    2005-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in

  11. C2-O-sLeX glycoproteins are E-selectin ligands that regulate invasion of human colon and hepatic carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Catherine A St Hill

    Full Text Available Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans (C2-O-sLe(X present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane expression of C2-O-sLe(X, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of alpha-2,3 fucosyltransferase (FucT-III and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1 genes, necessary for C2-O-sLe(X synthesis, by quantitative reverse-transcriptase (RT PCR. Synthesis of core 2 branched O-glycans decorated by sLe(X is dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1 and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLe(X by flow cytometry, and colon carcinoma cells possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA resulted in a 40-90% decrease in C2-O-sLe(X and a 30-50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was down-regulated in the latter cells. The sLe(X epitope was predominantly distributed on core 2 O-glycans on colon and hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLe(X carbohydrates produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic potential.

  12. 2型糖尿病患者血清β2-GPI/ox-LDL复合物水平%Clinical value of level of β2-glycoprotein I/ox-LDL complex in sera of patients with type 2 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    张春妮; 訾瑞峰; 汪俊军; 刘小传; 季茳; 周健; 李克

    2011-01-01

    Objective To measure and analyze the concentration of serum β2-glycoprotein I/oxidized low density lipoprotein ( β2-GPI/ox-LDL) complex in patients with type 2 diabetes mellitus (T2DM). Methods The concentrations of β2-GPL/ox-LDL complex were examined in 42 patients with T2DM and 41 age/sex-matched healthy controls by ELISA using rabbit anti-human β2-GPI antibody as the capture antibody, and quantitated with anti-apo (B) polyelonal antibody-enzyme conjugate. The concentrations of ox-LDL in serum were examined by ELISA, and the levels of lipids and lipoproteins in serum were determined simultaneously. Results The concentrations of serum β2-GPI/ox-LDL complex in T2DM were significantly higher than those in controls [ (0.89 ± 0. 15 ) U/mi vs (0.81 ±0.09) U/ml, P < 0.05 ]. Ox-LDL levels were also significantly elevated in the patients with DM compared with the controls [ (48.10 ± 60.40) mg/L vs (27.06 ± 12.25 ) mg/L, P < 0.05 ]. The concentrations of β2-GPI/ox-LDL positively conrelated with total cholesterol, LDL-C and ox-LDL levels in T2DM. Conclusion β2-GPI/ox-LDL complex levels significantly elevated in T2DM patients,which may be caused by oxidative stress as well as the elevated concentration of serum ox-LDL in T2DM.%目的 检测分析2型糖尿病(T2DM)患者血清β2-糖蛋白I/氧化低密度脂蛋白(β2-GPI/ox-LDL)复合物水平.方法 以抗人β2-GPI抗体为包被抗体、酶标记抗apo B为检测抗体的血清β2-GPl/ox-LDL ELISA检测法,对42例T2DM患者和41例年龄、性别匹配的健康对照者检测分析.用ELISA方法检测血清ox-LDL水平,同时对受检者进行常规血脂水平测定.结果 T2DM组β2-GPI/ox-LDL水平显著高于对照组[(0.89±0.15)U/ml vs(0.81±0.09)U/ml,P<0.05],ox-LDL浓度也明显升高[(48.10±60.40)mg/L vs(27.06±12.25)mg/L,P<0.05].相关分析显示,β2-GPI/ox-LDL水平与总胆固醇(TC)、LDL-胆固醇(LDL-c)以及ox-LDL呈显著正相关.结论 T2DM患者血清β2-GPI/ox-LDL复合物水平

  13. Complex assembly, crystallization and preliminary X-ray crystallographic analysis of the human Rod–Zwilch–ZW10 (RZZ) complex

    Energy Technology Data Exchange (ETDEWEB)

    Altenfeld, Anika; Wohlgemuth, Sabine [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); Wehenkel, Annemarie [Institut Curie, CNRS UMR 3348/INSERM U1005, Bâtiment 110, Centre Universitaire, 91405 Orsay CEDEX (France); Vetter, Ingrid R. [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); Musacchio, Andrea, E-mail: andrea.musacchio@mpi-dortmund.mpg.de [Max Planck Institute of Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund (Germany); University of Duisburg-Essen, Universitätstrasse 1, 45141 Essen (Germany)

    2015-03-20

    The 800 kDa complex of the human Rod, Zwilch and ZW10 proteins (the RZZ complex) was reconstituted in insect cells, purified, crystallized and subjected to preliminary X-ray diffraction analysis. The spindle-assembly checkpoint (SAC) monitors kinetochore–microtubule attachment during mitosis. In metazoans, the three-subunit Rod–Zwilch–ZW10 (RZZ) complex is a crucial SAC component that interacts with additional SAC-activating and SAC-silencing components, including the Mad1–Mad2 complex and cytoplasmic dynein. The RZZ complex contains two copies of each subunit and has a predicted molecular mass of ∼800 kDa. Given the low abundance of the RZZ complex in natural sources, its recombinant reconstitution was attempted by co-expression of its subunits in insect cells. The RZZ complex was purified to homogeneity and subjected to systematic crystallization attempts. Initial crystals containing the entire RZZ complex were obtained using the sitting-drop method and were subjected to optimization to improve the diffraction resolution limit. The crystals belonged to space group P3{sub 1} (No. 144) or P3{sub 2} (No. 145), with unit-cell parameters a = b = 215.45, c = 458.7 Å, α = β = 90.0, γ = 120.0°.

  14. Pneumocystis carinii glycoprotein A binds macrophage mannose receptors.

    OpenAIRE

    O'Riordan, D.M.; Standing, J E; Limper, A H

    1995-01-01

    Pneumocystis carinii causes life-threatening pneumonia in patients with impaired immunity. Recent studies suggest that alveolar macrophages interact with P. carinii through macrophage mannose receptors. However, the ligand(s) on P. carinii that is recognized by these receptors has not been fully defined. P. carinii contains a major mannose-rich surface antigen complex termed glycoprotein A (gpA). It was therefore hypothesized that gpA binds directly to macrophage mannose receptors and mediate...

  15. Colloque S&T Symposium 2008: Understanding the Human Dimension in 21st Century Conflict/Warfare: The Complexities of Human-with-Human Relationships

    Science.gov (United States)

    2008-08-01

    Colloque S&T Symposium 2008 Undestanding the Human Dimension in 21st Century Conflict/Warfare: The Complexities of Human-with-Human Relationships ...conditions for self-sustaining stability. By working towards the development of models and concepts to better understand and influence the human in...This page intentionally left blank. Colloque S&T Symposium 2008 Undestanding the Human Dimension in 21st Century Conflict

  16. HIV-1 envelope glycoprotein resistance to monoclonal antibody 2G12 is subject-specific and context-dependent in macaques and humans.

    Directory of Open Access Journals (Sweden)

    Delphine C Malherbe

    Full Text Available HIV-1 Envelope (Env protein is the sole target of neutralizing antibodies (NAbs that arise during infection to neutralize autologous variants. Under this immune pressure, HIV escape variants are continuously selected and over the course of infection Env becomes more neutralization resistant. Many common alterations are known to affect sensitivity to NAbs, including residues encoding potential N-linked glycosylation sites (PNGS. Knowledge of Env motifs associated with neutralization resistance is valuable for the design of an effective Env-based vaccine so we characterized Envs isolated longitudinally from a SHIV(SF162P4 infected macaque for sensitivity to neutralizing monoclonal antibodies (MAbs B12, 2G12, 4E10 and 2F5. The early Env, isolated from plasma at day 56 after infection, was the most sensitive and the late Env, from day 670, was the most resistant to MAbs. We identified four PNGS in these Envs that accumulated over time at positions 130, 139, 160 and 397. We determined that removal of these PNGS significantly increased neutralization sensitivity to 2G12, and conversely, we identified mutations by in silico analyses that contributed resistance to 2G12 neutralization. In order to expand our understanding of these PNGS, we analyzed Envs from clade B HIV-infected human subjects and identified additional glycan and amino acid changes that could affect neutralization by 2G12 in a context-dependent manner. Taken together, these in vitro and in silico analyses of clade B Envs revealed that 2G12 resistance is achieved by previously unrecognized PNGS substitutions in a context-dependent manner and by subject-specific pathways.

  17. Expression, purification and characterization of the human MTA2-RBBP7 complex.

    Science.gov (United States)

    Brasen, Christoffer; Dorosz, Jerzy; Wiuf, Anders; Boesen, Thomas; Mirza, Osman; Gajhede, Michael

    2017-02-04

    The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/β and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.

  18. Engineering Complex Human-Technological Work Systems: A Sensemaking Approach

    Science.gov (United States)

    2007-06-01

    by Rene Descartes , Immanuel Kant, and Ludwig Wittgenstein, and mathematically by Alfred North Whitehead and Bertrand Russell. Logical rationalism...reductionism—originally introduced by Descartes in 1673)— asserts that complex objects, phenomena, theories, and meanings can always be reduced to a...Positivism Essentialism Analytic Philosophy Social Constructivism Nominalism Autopoiesis Descartes Plato Aristotle Aquinas Bacon Locke Wittgenstein

  19. Complexity of Human Circulation Design: Tips for Students

    Science.gov (United States)

    Kurbel, Sven; Gros, Mario; Maric, Svjetlana

    2009-01-01

    Medical students are faced with a challenge to comprehend the enormous complexity of the circulatory systems. There is a gap between courses of anatomy, with detailed description of all normally present macroscopic vessels, and histology, which is focused on microscopic tissue architecture. Both courses leave arterioles, capillaries, and venules…

  20. Accessory subunits are integral for assembly and function of human mitochondrial complex I.

    Science.gov (United States)

    Stroud, David A; Surgenor, Elliot E; Formosa, Luke E; Reljic, Boris; Frazier, Ann E; Dibley, Marris G; Osellame, Laura D; Stait, Tegan; Beilharz, Traude H; Thorburn, David R; Salim, Agus; Ryan, Michael T

    2016-10-06

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

  1. Accommodating complexity and human behaviors in decision analysis.

    Energy Technology Data Exchange (ETDEWEB)

    Backus, George A.; Siirola, John Daniel; Schoenwald, David Alan; Strip, David R.; Hirsch, Gary B.; Bastian, Mark S.; Braithwaite, Karl R.; Homer, Jack [Homer Consulting

    2007-11-01

    This is the final report for a LDRD effort to address human behavior in decision support systems. One sister LDRD effort reports the extension of this work to include actual human choices and additional simulation analyses. Another provides the background for this effort and the programmatic directions for future work. This specific effort considered the feasibility of five aspects of model development required for analysis viability. To avoid the use of classified information, healthcare decisions and the system embedding them became the illustrative example for assessment.

  2. Method for analysing glycoprotein isoforms by capillary electrophoresis

    OpenAIRE

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

    2011-01-01

    [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...

  3. Beyond membrane channelopathies: alternative mechanisms underlying complex human disease

    Institute of Scientific and Technical Information of China (English)

    Konstantinos Dean BOUDOULAS; Peter J MOHLER

    2011-01-01

    Over the past fifteen years, our understanding of the molecular mechanisms underlying human disease has flourished in large part due to the discovery of gene mutations linked with membrane ion channels and transporters. In fact, ion channel defects ("channelopathies" - the focus of this review series) have been associated with a spectrum of serious human disease phenotypes including cystic fibrosis, cardiac arrhythmia, diabetes, skeletal muscle defects, and neurological disorders. However, we now know that human disease, particularly excitable cell disease, may be caused by defects in non-ion channel polypeptides including in cellular components residing well beneath the plasma membrane. For example, over the past few years, a new class of potentially fatal cardiac arrhythmias has been linked with cytoplasmic proteins that include sub-membrane adapters such as ankyrin-B (ANK2),ankyrin-G (ANK3), and alpha-1 syntrophin, membrane coat proteins including caveolin-3 (CAV3), signaling platforms including yotiao (AKAPg), and cardiac enzymes (GPD1L). The focus of this review is to detail the exciting role of lamins, yet another class of gene products that have provided elegant new insight into human disease.

  4. Alteration to the SWI/SNF complex in human cancers

    Directory of Open Access Journals (Sweden)

    Vanessa S. Gordon

    2011-12-01

    Full Text Available The SWI/SNF complex is a key catalyst for gene expression and regulates a variety of pathways, many of which have anticancer roles. Its central roles in cellular growth control, DNA repair, differentiation, cell adhesion and development are often targeted, and inactivated, during cancer development and progression. In this review, we will discuss what is known about how SWI/SNF is inactivated, and describe the potential impact of abrogating this complex. BRG1 and BRM are the catalytic subunits which are essential for SWI/SNF function, and thus, it is not surprising that they are lost in a variety of cancer types. As neither gene is mutated when lost, the mechanism of suppression, as well as the impact of potential gene activity restoration, are reviewed.

  5. Complex-tone pitch representations in the human auditory system

    DEFF Research Database (Denmark)

    Bianchi, Federica; Dau, Torsten; Santurette, Sébastien;

    , specifically those showing enhanced pitch cues (i.e., musicians) and those typically having disrupted pitch cues (i.e., hearing-impaired listeners). In particular, two main topics were addressed: the relative importance of resolved and unresolved harmonics for normal-hearing (NH) and hearing-impaired (HI......) listeners and the effect of musical training for pitch discrimination of complex tones with resolved and unresolved harmonics. Concerning the first topic, behavioral and modeling results in listeners with sensorineural hearing loss (SNHL) indicated that temporal envelope cues of complex tones...... discrimination to that of NH listeners. In the second part of this work, behavioral and objective measures of pitch discrimination were carried out in musicians and non-musicians. Musicians showed an increased pitch-discrimination performance relative to non-musicians for both resolved and unresolved harmonics...

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

    Directory of Open Access Journals (Sweden)

    Xue-Gong Fan

    2011-06-01

    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. Modeling Reduced Human Performance as a Complex Adaptive System

    Science.gov (United States)

    2003-09-01

    fittingly, the latest research paper describes these types of components as LEGOs (listener event graph objects). “The name is also a metaphor for how...Buss, A. H. and P. J. Sanchez (2002). Building Complex Models With LEGOs (Listener Event Graph Objects). Winter Simulation Conference. Buss, D. (1999...Kaarlela, C. (1997). New Gene Therapy Technique Could Eliminate Insulin Injections for many Diabetics, Jeffrey Norris and Jennifer O’Brien (415) 476-481

  8. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua;

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous......-level tagged proteins. Isolations of triple-FLAG and GFP-tagged fusion proteins involved in RNA metabolism are presented.......An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  9. Expression, purification and characterization of the human MTA2-RBBP7 complex

    DEFF Research Database (Denmark)

    Brasen, Christoffer; Dorosz, Jerzy; Wiuf, Anders

    2017-01-01

    . The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/β and the ATP-dependent helicase CDH3/4 constitutes...... the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy...... with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated...

  10. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells

    National Research Council Canada - National Science Library

    Tchasovnikarova, I. A; Timms, R. T; Matheson, N. J; Wals, K; Antrobus, R; Gottgens, B; Dougan, G; Dawson, M. A; Lehner, P. J

    2015-01-01

    ... (see the Perspective by Brummelkamp). They identified a complex of proteins in human cells they called HUSH that kept particular parts of the genome silent by changing associated histone methylation marks...

  11. Structural Biology of Proteins of the Multi-enzyme Assembly Human Pyruvate Dehydrogenase Complex

    Science.gov (United States)

    2003-01-01

    Objectives and research challenges of this effort include: 1. Need to establish Human Pyruvate Dehydrogenase Complex protein crystals; 2. Need to test value of microgravity for improving crystal quality of Human Pyruvate Dehydrogenase Complex protein crystals; 3. Need to improve flight hardware in order to control and understand the effects of microgravity on crystallization of Human Pyruvate Dehydrogenase Complex proteins; 4. Need to integrate sets of national collaborations with the restricted and specific requirements of flight experiments; 5. Need to establish a highly controlled experiment in microgravity with a rigor not yet obtained; 6. Need to communicate both the rigor of microgravity experiments and the scientific value of results obtained from microgravity experiments to the national community; and 7. Need to advance the understanding of Human Pyruvate Dehydrogenase Complex structures so that scientific and commercial advance is identified for these proteins.

  12. Concordance of gene expression in human protein complexes reveals tissue specificity and pathology

    DEFF Research Database (Denmark)

    Börnigen, Daniela; Pers, Tune Hannes; Thorrez, Lieven

    2013-01-01

    Disease-causing variants in human genes usually lead to phenotypes specific to only a few tissues. Here, we present a method for predicting tissue specificity based on quantitative deregulation of protein complexes. The underlying assumption is that the degree of coordinated expression among...... proteins in a complex within a given tissue may pinpoint tissues that will be affected by a mutation in the complex and coordinated expression may reveal the complex to be active in the tissue. We identified known disease genes and their protein complex partners in a high-quality human interactome. Each...... susceptibility gene's tissue involvement was ranked based on coordinated expression with its interaction partners in a non-disease global map of human tissue-specific expression. The approach demonstrated high overall area under the curve (0.78) and was very successfully benchmarked against a random model...

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

    Directory of Open Access Journals (Sweden)

    P. Roncada

    2010-04-01

    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.

  14. Inhibition of human aromatase complex (CYP19) by antiepileptic drugs

    DEFF Research Database (Denmark)

    Jacobsen, Naja Wessel; Halling-Sørensen, Bent; Birkved, Franziska Maria A Kramer

    2008-01-01

    transfected insect cells using dibenzylfluorescein as substrate. The drugs inhibiting CYP19 were: lamotrigine, oxcarbazepine, tiagabine, phenobarbital, phenytoin, ethosuximide, and valproate. The inhibitory effects (50% reduction in activity compared to enzymes without inhibitor present) were in the range...... with valproate and phenobarbital. When adding carbamazepine to a range of valproate concentrations no additional inhibition was seen. The data for some of the AEDs show that side effects on steroid synthesis in humans due to inhibition of aromatase should be considered....

  15. DTPA complexation of bismuth in human blood serum.

    Science.gov (United States)

    Montavon, G; Le Du, A; Champion, J; Rabung, T; Morgenstern, A

    2012-07-28

    The in vivo(212)Pb/(212)Bi generator is promising for application in targeted alpha therapy (TAT) of cancer. One main limitation of its therapeutic application is due to potential release of (212)Bi from the radioconjugate upon radioactive decay of the mother nuclide (212)Pb, potentially leading to irradiation of healthy tissue. The objective of the present work is to assess whether the chelate CHX-A''-DTPA (N-(2-aminoethyl)-trans-1,2-diaminocyclohexane-N,N',N''-pentaacetic acid) bound to a biological carrier molecule may be able to re-complex released (212)Bi under in vivo conditions to limit its translocation from the target site. CHX-A''-DTPA was bound to bovine gamma globulin (BGG) to mimic a model conjugate and the stability of the Bi-CHX-A''-DTPA-BGG conjugate was studied in blood serum by ultrafiltration. TRLFS experiments using Cm(III) as a fluorescent probe demonstrated that linking CHX-A''-DTPA to BGG does not affect the coordination properties of the ligand. Furthermore, comparable stability constants were observed between Bi(III) and free CHX-A''-DTPA, BGG-bound CHX-A''-DTPA and DTPA. The complexation constants determined between Bi(III) and the chelate molecules are sufficiently high to allow ultra trace amounts of the ligand to efficiently compete with serum transferrin controlling Bi(III) speciation in blood plasma conditions. Nevertheless, CHX-A''-DTPA is not able to complex Bi(III) generated in blood serum because of the strong competition between Bi(III) and Fe(II) for the ligand. In other words, CHX-A''-DTPA is not "selective" enough to limit Bi(iii) release in the body when applying the (212)Pb/(212)Bi in vivo generator.

  16. Genetic mapping of complex discrete human diseases by discriminant analysis

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The objective of the present study is to propose and evaluate a novel multivariate approach for genetic mapping of complex categorical diseases. This approach results from an application of standard stepwise discriminant analysis to detect linkage based on the differential marker identity-by-descent (IBD) distributions among the different groups of sib pairs. Two major advantages of this method are that it allows for simultaneously testing all markers, together with other genetic and environmental factors in a single multivariate setting and it avoids explicitly modeling the complex relationship between the affection status of sib pairs and the underlying genetic determinants. The efficiency and properties of the method are demonstrated via simulations. The proposed multivariate approach has successfully located the true position(s) under various genetic scenarios. The more important finding is that using highly densely spaced markers (1~2 cM) leads to only a marginal loss of statistical efficiency of the proposed methods in terms of gene localization and statistical power. These results have well established its utility and advantages as a fine-mapping tool. A unique property of the proposed method is the ability to map multiple linked trait loci to their precise positions due to its sequential nature, as demonstrated via simulations.

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

    Science.gov (United States)

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

    2015-06-01

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

  18. Global properties and functional complexity of human gene regulatory variation.

    Directory of Open Access Journals (Sweden)

    Daniel J Gaffney

    2013-05-01

    Full Text Available Identification and functional interpretation of gene regulatory variants is a major focus of modern genomics. The application of genetic mapping to molecular and cellular traits has enabled the detection of regulatory variation on genome-wide scales and revealed an enormous diversity of regulatory architecture in humans and other species. In this review I summarise the insights gained and questions raised by a decade of genetic mapping of gene expression variation. I discuss recent extensions of this approach using alternative molecular phenotypes that have revealed some of the biological mechanisms that drive gene expression variation between individuals. Finally, I highlight outstanding problems and future directions for development.

  19. Unraveling the complexity of lipid body organelles in human eosinophils.

    Science.gov (United States)

    Melo, Rossana C N; Weller, Peter F

    2014-11-01

    Lipid-rich organelles are common in many cell types. In cells, such as adipocytes, these organelles are termed LDs, whereas in other cells, such as leukocytes, they are called LBs. The study of leukocyte LBs has attracted attention as a result of their association with human diseases. In leukocytes, such as eosinophils, LB accumulation has been documented extensively during inflammatory conditions. In these cells, LBs are linked to the regulation of immune responses by compartmentalization of several proteins and lipids involved in the control and biosynthesis of inflammatory mediators (eicosanoids). However, it has been unclear how diverse proteins, including membrane-associated enzymes involved in eicosanoid formation, incorporate into LBs, especially if the internal content of LBs is assumed to consist solely of stores of neutral lipids, as present within adipocyte LDs. Studies of the formation, function, and ultrastructure of LBs in eosinophils have been providing insights pertinent to LBs in other leukocytes. Here, we review current knowledge of the composition and function of leukocyte LBs as provided by studies of human eosinophil LBs, including recognitions of the internal architecture of eosinophil LBs based on 3D electron tomographic analyses.

  20. EFECT OF ERYTHROMYCIN ON INTERDIGESTIVE MIGRATING MOTOR COMPLEX IN HUMANS

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To investigate the effect of erythromycin (EM) on interdigestive migrating motor complex (MMC) in healthy volunteers. Methods 20 healthy volunteers were randomly divided into 2 groups: EM group (n=11) and placebo group (n=9). The changes of MMC were observed by gastrointestinal manometry before and after oral administration of EM or placebo. Results Gastric antral MMCs that evoked by EM were similar to spontaneous MMCs. EM orally intaking decreased MMC cycle duration significantly (P<0.05). EM orally intaking decreased the percentage of phase Ⅱ duration to MMC cycle duration significantly (P<0.05). But EM orally intaking increased the percentage of phase Ⅲ duration to MMC cycle duration significantly (P<0.05). The amplitude of antral waves of phase Ⅲ increased significantly after EM orally intaking (P<0.05). Placebo orally and percentages of phase Ⅰ, phase Ⅱ, phase Ⅲ duration to MMC cycle duration. Conclusion EM has stimulating effect on gastrointestinal motor activity.

  1. The Cultural Historical Complexity of Human Personality Adaptation

    Directory of Open Access Journals (Sweden)

    Melissa E. Wynn

    2012-10-01

    Full Text Available Research on implicit intelligence has conceptualized students’ beliefs about the nature of intelligence as either fixed or malleable. This research has largely not included African American adolescents, a group for whom beliefs about intelligence have a cultural historical complexity related to both scientific racism and master narratives of race and intelligence. The purpose of this study was to investigate the nature of implicit theories of intelligence for 63 African American adolescents who are seventh and eighth graders in a public charter school. The two-way ANOVA revealed that these adolescents held a malleable view of intelligence, which did not vary by gender or grade. Exploratory correlation analysis showed some consistent relationships with achievement motivation variables found in other studies. These findings may be explained by African American cultural values and the personality characteristic adaptations that they make living within a racialized society.

  2. Network properties of complex human disease genes identified through genome-wide association studies.

    Directory of Open Access Journals (Sweden)

    Fredrik Barrenas

    Full Text Available BACKGROUND: Previous studies of network properties of human disease genes have mainly focused on monogenic diseases or cancers and have suffered from discovery bias. Here we investigated the network properties of complex disease genes identified by genome-wide association studies (GWAs, thereby eliminating discovery bias. PRINCIPAL FINDINGS: We derived a network of complex diseases (n = 54 and complex disease genes (n = 349 to explore the shared genetic architecture of complex diseases. We evaluated the centrality measures of complex disease genes in comparison with essential and monogenic disease genes in the human interactome. The complex disease network showed that diseases belonging to the same disease class do not always share common disease genes. A possible explanation could be that the variants with higher minor allele frequency and larger effect size identified using GWAs constitute disjoint parts of the allelic spectra of similar complex diseases. The complex disease gene network showed high modularity with the size of the largest component being smaller than expected from a randomized null-model. This is consistent with limited sharing of genes between diseases. Complex disease genes are less central than the essential and monogenic disease genes in the human interactome. Genes associated with the same disease, compared to genes associated with different diseases, more often tend to share a protein-protein interaction and a Gene Ontology Biological Process. CONCLUSIONS: This indicates that network neighbors of known disease genes form an important class of candidates for identifying novel genes for the same disease.

  3. Network properties of complex human disease genes identified through genome-wide association studies.

    Science.gov (United States)

    Barrenas, Fredrik; Chavali, Sreenivas; Holme, Petter; Mobini, Reza; Benson, Mikael

    2009-11-30

    Previous studies of network properties of human disease genes have mainly focused on monogenic diseases or cancers and have suffered from discovery bias. Here we investigated the network properties of complex disease genes identified by genome-wide association studies (GWAs), thereby eliminating discovery bias. We derived a network of complex diseases (n = 54) and complex disease genes (n = 349) to explore the shared genetic architecture of complex diseases. We evaluated the centrality measures of complex disease genes in comparison with essential and monogenic disease genes in the human interactome. The complex disease network showed that diseases belonging to the same disease class do not always share common disease genes. A possible explanation could be that the variants with higher minor allele frequency and larger effect size identified using GWAs constitute disjoint parts of the allelic spectra of similar complex diseases. The complex disease gene network showed high modularity with the size of the largest component being smaller than expected from a randomized null-model. This is consistent with limited sharing of genes between diseases. Complex disease genes are less central than the essential and monogenic disease genes in the human interactome. Genes associated with the same disease, compared to genes associated with different diseases, more often tend to share a protein-protein interaction and a Gene Ontology Biological Process. This indicates that network neighbors of known disease genes form an important class of candidates for identifying novel genes for the same disease.

  4. EEG correlates of spatial orientation in the human retrosplenial complex.

    Science.gov (United States)

    Lin, C-T; Chiu, T-C; Gramann, K

    2015-10-15

    Studies on spatial navigation reliably demonstrate that the retrosplenial complex (RSC) plays a pivotal role for allocentric spatial information processing by transforming egocentric and allocentric spatial information into the respective other spatial reference frame (SRF). While more and more imaging studies investigate the role of the RSC in spatial tasks, high temporal resolution measures such as electroencephalography (EEG) are missing. To investigate the function of the RSC in spatial navigation with high temporal resolution we used EEG to analyze spectral perturbations during navigation based on allocentric and egocentric SRF. Participants performed a path integration task in a clearly structured virtual environment providing allothetic information. Continuous EEG recordings were decomposed by independent component analysis (ICA) with subsequent source reconstruction of independent time source series using equivalent dipole modeling. Time-frequency transformation was used to investigate reference frame-specific orientation processes during navigation as compared to a control condition with identical visual input but no orientation task. Our results demonstrate that navigation based on an egocentric reference frame recruited a network including the parietal, motor, and occipital cortices with dominant perturbations in the alpha band and theta modulation in frontal cortex. Allocentric navigation was accompanied by performance-related desynchronization of the 8-13 Hz frequency band and synchronization in the 12-14 Hz band in the RSC. The results support the claim that the retrosplenial complex is central to translating egocentric spatial information into allocentric reference frames. Modulations in different frequencies with different time courses in the RSC further provide first evidence of two distinct neural processes reflecting translation of spatial information based on distinct reference frames and the computation of heading changes.

  5. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

    Directory of Open Access Journals (Sweden)

    Zhen-yong Keck

    2014-08-01

    Full Text Available A challenge for hepatitis C virus (HCV vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape

  6. A role for Aurora C in the chromosomal passenger complex during human preimplantation embryo development

    NARCIS (Netherlands)

    Santos, Margarida Avo; van de Werken, Christine; de Vries, Marieke; Jahr, Holger; Vromans, Martijn J. M.; Laven, Joop S. E.; Fauser, Bart C.; Kops, Geert J.; Lens, Susanne M.; Baart, Esther B.

    2011-01-01

    BACKGROUND: Human embryos generated by IVF demonstrate a high incidence of chromosomal segregation errors during the cleavage divisions. To analyse underlying molecular mechanisms, we investigated the behaviour of the chromosomal passenger complex (CPC) in human oocytes and embryos. This important m

  7. Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

    Science.gov (United States)

    Simó, Rafael; Hernández, Cristina; Sáez-López, Cristina; Soldevila, Berta; Puig-Domingo, Manel; Selva, David M

    2014-01-01

    Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight

  8. Baculovirus expression of the glycoprotein gene of Lassa virus and characterization of the recombinant protein.

    Science.gov (United States)

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

    1992-09-01

    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).

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

    Directory of Open Access Journals (Sweden)

    Shukra M. Aavula

    2011-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Shabnam Yazdian

    2014-10-01

    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.

  11. 伴刀豆凝集素修饰磁性纳米粒子富集人血清中糖蛋白及质谱鉴定%Enrichment of glycoproteins in human serum using concanavalin A-functionalized magnetic nanoparticles and identification by mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    李凤; 康经武

    2014-01-01

    发展了一种新型的磁性纳米粒子应用于人血清中特异性糖蛋白的亲和富集。制备的磁性纳米粒子具有核/壳/壳结构,即由 Fe3 O4磁性粒子/硅胶层/有机聚合物外层构成。伴刀豆凝集素 A(Con A)以共价键合的形式通过短链聚乙二醇固定在粒子表面,实现了人血清中特异性糖蛋白的高效富集。富集的蛋白经过胰蛋白酶酶解后,所得的肽段经离线的二维色谱分离,用高分辨质谱共鉴定出80种蛋白。通过 NetNGlyc 等搜索软件分析确定其中76种为糖蛋白,分析发现在血清中质量浓度仅为0.00001 g / L 的β-2-glycoprotein 1也得到了鉴定,表明我们发展的磁性纳米粒子与凝集素相结合的方式,可以高效地富集复杂体系中与主要蛋白成分含量相差12个数量级的低丰度糖蛋白。%Biomedical sciences,and in particular biomarker research,demand efficient glyco-protein enrichment platforms. Herein novel magnetic nanoparticles with an average size around 135 nm in diameter were prepared for the enrichment of glycoproteins in human serum. The prepared magnetic nanoparticles possessed uniform core / shell / shell structure which was com-posed of 8 nm magnetite internal core and double layers consisting of silica and poly glycidyl methacrylate(GMA). The latter was constructed by seed polymerization. Modified by a poly-ethylene hydrophilic linker,it made the surfaces of the magnetic nanoparticles highly hydrophil-ic so as to reduce the nonspecific adsorption of proteins. We examined affinity purification of glycoprotein in diluted human serum using our prepared magnetic nanoparticles with immobi-lization of concanavalin A( MNP@ ConA). The enriched proteins were reduced,alkylated and digested with trypsin. These peptides then were separated by offline two-dimensional chroma-tography. Protein identification was realized with nano-high performance liquid chromatogra-phy-orbitrap mass spectrometry. A total

  12. Platelet membrane glycoproteins and their function: an overview.

    Science.gov (United States)

    Kunicki, T J

    1989-07-01

    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.

  13. A Scale and Pose Invariant Algorithm for Fast Detecting Human Faces in a Complex Background

    Institute of Scientific and Technical Information of China (English)

    XING Xin; SHEN Lansun; JIA Kebin

    2001-01-01

    Human face detection is an interesting and challenging task in computer vision. A scale and pose invariant algorithm is proposed in this paper.The algorithm is able to detect human faces in a complex background in about 400ms with a detection rate of 92%. The algorithm can be used in a wide range of applications such as human-computer interface, video coding, etc.

  14. Recognition of complex human behaviours using 3D imaging for intelligent surveillance applications

    Science.gov (United States)

    Yao, Bo; Lepley, Jason J.; Peall, Robert; Butler, Michael; Hagras, Hani

    2016-10-01

    We introduce a system that exploits 3-D imaging technology as an enabler for the robust recognition of the human form. We combine this with pose and feature recognition capabilities from which we can recognise high-level human behaviours. We propose a hierarchical methodology for the recognition of complex human behaviours, based on the identification of a set of atomic behaviours, individual and sequential poses (e.g. standing, sitting, walking, drinking and eating) that provides a framework from which we adopt time-based machine learning techniques to recognise complex behaviour patterns.

  15. Invited commentary: Positive youth development and human complexity.

    Science.gov (United States)

    Larson, Reed W; Tran, Steve P

    2014-06-01

    The process of positive development for adolescents includes struggling to address a wide variety of complex, often unstated bio-psycho-social-cultural challenges. These include formulating workable values, learning self-regulation, preparation for adult work roles-and innumerable other un-tidy puzzles. Variable-based research can only scratch the surface of how youth go about these processes; nonetheless, systematic longitudinal research like this can provide valuable information about developmental pathways and directions of change. Highlights from these papers include the finding that older youth report more goals aimed at meaningful connection with others and contributing to society; yet also that moral character did not differ by age. The papers suggest that relationships adults, hope, school engagement, participation in out-of-school programs, and intentional self-regulation can serve as mediators of positive development. Yet, a striking finding was that comparatively few youth in the study manifest a pattern of change marked by the coupling of increases in positive youth development and decreases in risk/problem behavior. We believe there is much beneath the surface to be uncovered.

  16. Complex posttraumatic stress disorder and survivors of human rights violations.

    Science.gov (United States)

    McDonnell, Matthew; Robjant, Katy; Katona, Cornelius

    2013-01-01

    This article reviews recent findings on Complex Posttraumatic Stress Disorder (CPTSD) and proposes future research which would help to establish the nature of CPTSD in relation to Posttraumatic Stress Disorder (PTSD). Research on survivors of torture and war has found that CPTSD can occur when there is no history of childhood abuse. fMRI studies appear to highlight differences in neural activity in individuals exhibiting primary dissociation compared with individuals exhibiting secondary dissociation. Research has begun to show that, when symptoms of secondary dissociation are appropriately managed, exposure-based therapies are an effective treatment for individuals with CPTSD. Much research on CPTSD has emphasized its developmental basis and the disruptive effects of trauma in childhood and adolescence on subsequent emotional development. However, some studies on survivors of torture in adult life identify similar symptom patterns, despite there being no history of childhood trauma. It is argued that comparative research is required between victims of developmental trauma (such as childhood sexual abuse) and victims who experienced prolonged interpersonal trauma in adulthood (such as torture), as this could be useful in establishing the cause of CPTSD and in delineating clinically and therapeutically meaningful subtypes. It is also proposed that a focus on underlying neurobiological processes would help in developing and refining CPTSD as a construct and informing treatment.

  17. Complex formation between human prostate-specific antigen and protease inhibitors in mouse plasma.

    Science.gov (United States)

    Hekim, Can; Riipi, Tero; Zhu, Lei; Laakkonen, Pirjo; Stenman, Ulf-Håkan; Koistinen, Hannu

    2010-04-01

    When secreted from the prostate, most of prostate-specific antigen (PSA) is free and enzymatically active. Upon reaching circulation, active PSA is inactivated by complex formation with protease inhibitors. To justify the use of mouse models for evaluation of the function of PSA and for studies on therapeutic modalities based on modulation of PSA activity, it is important to know whether PSA complexation is similar in mouse and man. To characterize the circulating forms of PSA in mouse, we used subcutaneous LNCaP and 22RV1 human prostate cancer cell xenograft tumor models. We also added PSA directly to mouse serum. Free and total PSA were measured by immunoassay, and PSA complexes were extracted by immunopurification followed by SDS-PAGE, in-gel trypsin digestion and identification of signature peptides by mass spectrometry. In mice bearing xenograft tumors, 68% of the immunoreactive PSA occurred in complex, and when added to mouse serum, over 70% of PSA forms complexes that comprises alpha(2)-macroglobulin and members of the alpha(1)-antitrypsin (AAT) family. In mouse plasma, PSA forms complexes similar to those in man, but the major immunoreactive complex contains AAT rather than alpha(1)-antichymotrypsin, which is the main complex forming serpin in man. The complex formation of PSA produced by xenograft tumor models in mice is similar to that of human prostate tumors with respect to the complexation of PSA. (c) 2009 Wiley-Liss, Inc.

  18. Connectivity in the human brain dissociates entropy and complexity of auditory inputs.

    Science.gov (United States)

    Nastase, Samuel A; Iacovella, Vittorio; Davis, Ben; Hasson, Uri

    2015-03-01

    Complex systems are described according to two central dimensions: (a) the randomness of their output, quantified via entropy; and (b) their complexity, which reflects the organization of a system's generators. Whereas some approaches hold that complexity can be reduced to uncertainty or entropy, an axiom of complexity science is that signals with very high or very low entropy are generated by relatively non-complex systems, while complex systems typically generate outputs with entropy peaking between these two extremes. In understanding their environment, individuals would benefit from coding for both input entropy and complexity; entropy indexes uncertainty and can inform probabilistic coding strategies, whereas complexity reflects a concise and abstract representation of the underlying environmental configuration, which can serve independent purposes, e.g., as a template for generalization and rapid comparisons between environments. Using functional neuroimaging, we demonstrate that, in response to passively processed auditory inputs, functional integration patterns in the human brain track both the entropy and complexity of the auditory signal. Connectivity between several brain regions scaled monotonically with input entropy, suggesting sensitivity to uncertainty, whereas connectivity between other regions tracked entropy in a convex manner consistent with sensitivity to input complexity. These findings suggest that the human brain simultaneously tracks the uncertainty of sensory data and effectively models their environmental generators.

  19. Mitochondrial complex I plays an essential role in human respirasome assembly.

    Science.gov (United States)

    Moreno-Lastres, David; Fontanesi, Flavia; García-Consuegra, Inés; Martín, Miguel A; Arenas, Joaquín; Barrientos, Antoni; Ugalde, Cristina

    2012-03-01

    The biogenesis and function of the mitochondrial respiratory chain (RC) involve the organization of RC enzyme complexes in supercomplexes or respirasomes through an unknown biosynthetic process. This leads to structural interdependences between RC complexes, which are highly relevant from biological and biomedical perspectives, because RC defects often lead to severe neuromuscular disorders. We show that in human cells, respirasome biogenesis involves a complex I assembly intermediate acting as a scaffold for the combined incorporation of complexes III and IV subunits, rather than originating from the association of preassembled individual holoenzymes. The process ends with the incorporation of complex I NADH dehydrogenase catalytic module, which leads to the respirasome activation. While complexes III and IV assemble either as free holoenzymes or by incorporation of free subunits into supercomplexes, the respirasomes constitute the structural units where complex I is assembled and activated, thus explaining the significance of the respirasomes for RC function.

  20. The N2-P3 complex of the evoked potential and human performance

    Science.gov (United States)

    Odonnell, Brian F.; Cohen, Ronald A.

    1988-01-01

    The N2-P3 complex and other endogenous components of human evoked potential provide a set of tools for the investigation of human perceptual and cognitive processes. These multidimensional measures of central nervous system bioelectrical activity respond to a variety of environmental and internal factors which have been experimentally characterized. Their application to the analysis of human performance in naturalistic task environments is just beginning. Converging evidence suggests that the N2-P3 complex reflects processes of stimulus evaluation, perceptual resource allocation, and decision making that proceed in parallel, rather than in series, with response generation. Utilization of these EP components may provide insights into the central nervous system mechanisms modulating task performance unavailable from behavioral measures alone. The sensitivity of the N2-P3 complex to neuropathology, psychopathology, and pharmacological manipulation suggests that these components might provide sensitive markers for the effects of environmental stressors on the human central nervous system.

  1. Microtomography of the human tooth-alveolar bone complex

    Science.gov (United States)

    Dalstra, Michel; Cattaneo, Paolo M.; Beckmann, Felix; Sakima, Maurício T.; Lemor, Carsten; Laursen, Morten G.; Melsen, Birte

    2006-08-01

    In this study the structure of the adult human dentoalveolar process is examined using conventional and synchrotron radiation-based microtomography (SRμCT). Mandibular and maxillary segments containing two to five adjacent teeth were harvested at autopsy from 49 adult donors. These segments were embedded in blocks of methylmetacrylate and scanned using a conventional table-top μCT-scanner at a pixel size and slice thickness of 35 μm. A few segments were also scanned at a synchrotron facility at an initial pixel size of 16.4 μm, which was binned by a factor 2 to result in an effective voxel size of almost 32.8 μm. The three-dimensional reconstructions revealed how intricately the teeth are supported by the alveolar bone. Furthermore, this support is highly inhomogeneous with respect to the buccal, mesial, lingual and distal quadrants. Reflecting their various degrees of mineralization, tissues like bone, dentine, enamel and cementum, could well be identified, especially in the scans made with SRμCT. Despite comparable voxel sizes, the reconstructed data-sets obtained with conventional μCT were less detailed and somewhat fuzzy in appearance compared to the data-sets of SRμCT. However, for quantification of macroscopical features like the thickness of the alveolar wall or the presence of dehiscences/fenestrations this seemed sufficient.

  2. A review on the relation between the brain-serum concentration ratio of drugs and the influence of P-glycoprotein

    DEFF Research Database (Denmark)

    Ejsing, Thomas Broeng; Morling, Niels; Linnet, Kristian

    2007-01-01

    This overview on the brain-serum relationship for drugs illustrates the importance of the drug transporter P-glycoprotein at the blood-brain barrier. Generally, an inverse relationship exists between the magnitude of the brain-serum ratio and the influence of P-glycoprotein. Concerning...... the pharmacogenomics of P-glycoprotein, no clear effect of single nucleotide polymorphisms (SNPs) has been demonstrated in humans....

  3. Complex relationship between meiotic recombination frequency and autosomal synaptonemal complex length per cell in normal human males.

    Science.gov (United States)

    Pan, Zhenzhen; Yang, Qingling; Ye, Nan; Wang, Liu; Li, Jianhua; Yu, Dexin; Cooke, Howard J; Shi, Qinghua

    2012-03-01

    Although the relationship between meiotic recombination frequency and synaptonemal complex (SC) length has been of interest for a long time, how recombination frequency is related to SC length has not been carefully explored. To address this question, we have measured the meiotic recombination frequency as represented by MLH1 foci in 889 pachytene spermatocytes and measured the length of 19,558 autosomal SCs from 10 human males. A complex relationship between the number of MLH1 foci and total autosomal SC length per cell was observed. A positive correlation with significant correlation coefficients between the two variables was found in eight of the ten donors examined, with three donors showing weak correlation, and five showing moderate correlation. Two donors who did not show any correlation between the two variables were identified for the first time. Moreover, most cells with similar total autosomal SC length showed very different numbers of MLH1 foci both between individuals and even within an individual, and vice versa. Our data provide the first evidence for a complex relationship between the recombination frequency and total length of autosomal SCs per cell in human males.

  4. A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

    Science.gov (United States)

    Becerra-Artiles, Aniuska; Dominguez-Amorocho, Omar; Stern, Lawrence J; Calvo-Calle, J Mauricio

    2015-01-01

    Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6

  5. [Elderly human being with ostomy and environments of care: reflection on the perspective of complexity].

    Science.gov (United States)

    Barros, Edaiane Joana Lima; Santos, Silvana Sidney Costa; Lunardi, Valéria Lerch; Lunardi Filho, Wilson Danilo

    2012-01-01

    This is discussion about the relationship between elderly human beings with ostomy and their environments care, under the perspective of Complexity Edgar Morin. An axis holds the reflection: environments of care for elderly humans with ostomy. In this sense, we present three types of environment that surround the context of elderly humans with ostomy: home environment, group environment and hospital environment. This brings, as a social contribution, a new look about resizing caring of elderly humans with ostomy in their environment. It is considered that the environment hosting this human being contains a diversity of feelings, emotions, experiences; it binds multiple meanings, from the Complexity perspective, about the relationship between the environment and the caring process.

  6. Acrosome reaction: relevance of zona pellucida glycoproteins

    Institute of Scientific and Technical Information of China (English)

    Satish K Gupta; Beena Bhandari

    2011-01-01

    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.

  7. Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

    Science.gov (United States)

    Prokopenko, P G; Shkoporov, A N; Petrenko, O Yu; Efimov, B A; Negrebetskii, V V; Terent'ev, A A

    2015-11-01

    We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

  8. Structural analysis of the human SYCE2-TEX12 complex provides molecular insights into synaptonemal complex assembly.

    Science.gov (United States)

    Davies, Owen R; Maman, Joseph D; Pellegrini, Luca

    2012-07-01

    The successful completion of meiosis is essential for all sexually reproducing organisms. The synaptonemal complex (SC) is a large proteinaceous structure that holds together homologous chromosomes during meiosis, providing the structural framework for meiotic recombination and crossover formation. Errors in SC formation are associated with infertility, recurrent miscarriage and aneuploidy. The current lack of molecular information about the dynamic process of SC assembly severely restricts our understanding of its function in meiosis. Here, we provide the first biochemical and structural analysis of an SC protein component and propose a structural basis for its function in SC assembly. We show that human SC proteins SYCE2 and TEX12 form a highly stable, constitutive complex, and define the regions responsible for their homotypic and heterotypic interactions. Biophysical analysis reveals that the SYCE2-TEX12 complex is an equimolar hetero-octamer, formed from the association of an SYCE2 tetramer and two TEX12 dimers. Electron microscopy shows that biochemically reconstituted SYCE2-TEX12 complexes assemble spontaneously into filamentous structures that resemble the known physical features of the SC central element (CE). Our findings can be combined with existing biological data in a model of chromosome synapsis driven by growth of SYCE2-TEX12 higher-order structures within the CE of the SC.

  9. Hamster zona pellucida is formed by four glycoproteins: ZP1, ZP2, ZP3, and ZP4.

    Science.gov (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

    2009-02-01

    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.

  10. Human guidance of mobile robots in complex 3D environments using smart glasses

    Science.gov (United States)

    Kopinsky, Ryan; Sharma, Aneesh; Gupta, Nikhil; Ordonez, Camilo; Collins, Emmanuel; Barber, Daniel

    2016-05-01

    In order for humans to safely work alongside robots in the field, the human-robot (HR) interface, which enables bi-directional communication between human and robot, should be able to quickly and concisely express the robot's intentions and needs. While the robot operates mostly in autonomous mode, the human should be able to intervene to effectively guide the robot in complex, risky and/or highly uncertain scenarios. Using smart glasses such as Google Glass∗, we seek to develop an HR interface that aids in reducing interaction time and distractions during interaction with the robot.

  11. Cellular and biomolecular responses of human ovarian cancer cells to cytostatic dinuclear platinum(II) complexes.

    Science.gov (United States)

    Lin, Miaoxin; Wang, Xiaoyong; Zhu, Jianhui; Fan, Damin; Zhang, Yangmiao; Zhang, Junfeng; Guo, Zijian

    2011-03-01

    Polynuclear platinum(II) complexes represent a class of potential anticancer agents that have shown promising pharmacological properties in preclinical studies. The nature of cellular responses induced by these complexes, however, is poorly understood. In this research, the cellular responses of human ovarian cancer COC1 cells to dinuclear platinum(II) complexes {[cis-Pt(NH₃)₂Cl]₂L¹}(NO₃)₂ (1) and {[cis-Pt(NH₃)₂Cl]₂L²}(NO₃)₂ (2) (L¹ = α,α'-diamino-p-xylene, L² = 4,4'-methylenedianiline) has been studied using cisplatin as a reference. The effect of platinum complexes on the proliferation, death mode, mitochondrial membrane potential, and cell cycle progression has been examined by MTT assay and flow cytometry. The activation of cell cycle checkpoint kinases (CHK1/2), extracellular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) of the cells by the complexes has also been analyzed using phospho-specific flow cytometry. Complex 1 is more cytotoxic than complex 2 and cisplatin at most concentrations; complex 2 and cisplatin are comparably cytotoxic. These complexes kill the cells through an apoptotic or apoptosis-like pathway characterized by exposure of phosphatidylserine and dissipation of mitochondrial membrane potential. Complex 1 shows the strongest inductive effect on the morphological changes of the cells, followed by cisplatin and complex 2. Complexes 1 and 2 arrest the cell cycle in G2 or M phase, while cisplatin arrests the cell cycle in S phase. The influence of these complexes on CHK1/2, ERK1/2, and p38 MAPK varies with the dose of the drugs or reaction time. Activation of phospho-ERK1/2 and phospho-p38 MAPK by these complexes is closely related to the cytostatic activity. The results demonstrate that dinuclear platinum(II) complexes can induce some cellular responses different from those caused by cisplatin.

  12. 狂犬病毒糖蛋白重组的新城疫病毒诱导人胃癌 SGC 细胞自噬%The rabies virus glycoprotein recombinated Newcastle disease virus induce the human stomachal adenocarcinoma autophagy

    Institute of Scientific and Technical Information of China (English)

    王穆彬; 严玉兰; 张志坚; 赵英海; 李米; 步雪峰

    2015-01-01

    目的:观察狂犬病毒糖蛋白重组的新城疫病毒(rabies virus glycoprotein recombinated Newcastle disease vi-rus,rL-RVG)及野生型新城疫病毒(Newcastle disease virus,NDV)对胃癌 SGC 细胞自噬的影响。方法:将 SGC 细胞随机分为3组,rl-RVG 组(转染 rl-RVG)、NDV 组(转染 NDV)、对照组(转染 PBS),24 h 后观察细胞的形态变化,评价病毒对细胞的影响。用免疫双标记和免疫印迹法鉴定病毒感染是否成功;蛋白质印迹检测各组细胞自噬相关蛋白 LC3及 Beclin1的表达;透射电镜观察细胞内超微结构的变化和自噬体的形成。结果:病毒感染细胞24 h 后, rl-RVG 组及 NDV 组细胞皱缩明显,且 rl-RVG 组较 NDV 组变化明显,对照组无明显变化;rl-RVG 组和 NDV 组细胞LC3及 Beclin1蛋白表达较对照组明显升高(P <0.05),且 rl-RVG 组明显高于 NDV 组(P <0.05);rl-RVG 组及NDV 组出现的自噬体明显多于对照组,且 rl-RVG 组较 NDV 组明显增多(P <0.05)。结论:rL-RVG 较 NDV 更易引起胃癌 SGC 细胞的自噬。%Objective:To observe the autophagy of the human gastric cancer SGC infected by rabies virus glycoprotein recombinated Newcastle disease virus(rL-RVG)or wild type Newcastle disease virus (NDV).Methods:The cells were divided into three groups at random,the rL-RVG group(infected with rL-RVG),the NDV group(infected with NDV),and the control group(infected with PBS).The mor-phology of SGC cells after infected 24 hours were monitored by microscope.The infected cells were ana-lysed with immunofluorescence,the total proteins get from the cells infected with or without virus were analysed with western blotting,and the ultrastructural of SGC was observed by transmission electron mi-croscope.Results:After infected 24 hours,there was a significantly morphologic changes in the rL-RVG group and the NDV group,and the cells were shrink,the rL-RVG group changes

  13. Interactions of histidine-rich glycoprotein with immunoglobulins and proteins of the complement system.

    Science.gov (United States)

    Manderson, G A; Martin, M; Onnerfjord, P; Saxne, T; Schmidtchen, A; Mollnes, T E; Heinegård, D; Blom, A M

    2009-10-01

    This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.

  14. A Phase-Dependent Hypothesis for Locomotor Functions of Human Foot Complex

    Institute of Scientific and Technical Information of China (English)

    Lei Ren; David Howard; Lu-quan Ren; Chris Nester; Li-mei Tian

    2008-01-01

    The human foot is a very complex structure comprising numerous bones, muscles, ligaments and synovial joints. As the only component in contact with the ground, the foot complex delivers a variety of biomechanical functions during human locomotion, e.g. body support and propulsion, stability maintenance and impact absorption. These need the human foot to be rigid and damped to transmit ground reaction forces to the upper body and maintain body stability, and also to be compliant and resilient to moderate risky impacts and save energy. How does the human foot achieve these apparent conflicting functions? In this study, we propose a phase-dependent hypothesis for the overall locomotor functions of the human foot complex based on in-vivo measurements of human natural gait and simulation results of a mathematical foot model. We propse that foot functions are highly dependent on gait phase, which is a major characteristics of human locomotion. In early stance just after heel strike,the foot mainly works as a shock absorber by moderating high impacts using the viscouselastic heel pad in both. vertical and horizontal directions. In mid-stance phase(~80% of stance phase), the foot complex can be considered as a springy rocker,reserving external mechanical work using the foot arch whilst moving ground contact point forward along a curved path to maintain body stability. In late stance after heel off, the foot complex mainly serves as a force modulator like a gear box,modulating effective mechanical advantages of ankle plantiflexor muscles using metatarsal-phalangeal joints. A sound understanding of how diverse functions are implemented in a simple foot segment during human locomotion might be useful to gain insight into the overall foot locomotor functions and hence to facilitate clinical diagnosis, rehabilitation product design and humanoid robot development.

  15. GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

    Science.gov (United States)

    Tchasovnikarova, Iva A; Timms, Richard T; Matheson, Nicholas J; Wals, Kim; Antrobus, Robin; Göttgens, Berthold; Dougan, Gordon; Dawson, Mark A; Lehner, Paul J

    2015-06-26

    Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.

  16. Detailed analysis of the human mitochondrial contact site complex indicate a hierarchy of subunits.

    Science.gov (United States)

    Ott, Christine; Dorsch, Eva; Fraunholz, Martin; Straub, Sebastian; Kozjak-Pavlovic, Vera

    2015-01-01

    Mitochondrial inner membrane folds into cristae, which significantly increase its surface and are important for mitochondrial function. The stability of cristae depends on the mitochondrial contact site (MICOS) complex. In human mitochondria, the inner membrane MICOS complex interacts with the outer membrane sorting and assembly machinery (SAM) complex, to form the mitochondrial intermembrane space bridging complex (MIB). We have created knockdown cell lines of most of the MICOS and MIB components and have used them to study the importance of the individual subunits for the cristae formation and complex stability. We show that the most important subunits of the MIB complex in human mitochondria are Mic60/Mitofilin, Mic19/CHCHD3 and an outer membrane component Sam50. We provide additional proof that ApoO indeed is a subunit of the MICOS and MIB complexes and propose the name Mic23 for this protein. According to our results, Mic25/CHCHD6, Mic27/ApoOL and Mic23/ApoO appear to be periphery subunits of the MICOS complex, because their depletion does not affect cristae morphology or stability of other components.

  17. Role of sialidase in glycoprotein utilization by Tannerella forsythia.

    Science.gov (United States)

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

    2011-11-01

    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.

  18. Social-network complexity in humans is associated with the neural response to social information.

    Science.gov (United States)

    Dziura, Sarah L; Thompson, James C

    2014-11-01

    Humans have evolved to thrive in large and complex social groups, and it is likely that this increase in group complexity has come with a greater need to decode and respond to complex and uncertain communicatory signals. In this functional MRI study, we examined whether complexity of social networks in humans is related to the functioning of brain regions key to the perception of basic, nonverbal social stimuli. Greater activation to biological than to scrambled motion in the right posterior superior temporal sulcus (pSTS) and right amygdala were positively correlated with the diversity of social-network roles. In the pSTS, in particular, this association was not due to a relationship between network diversity and network size. These findings suggest that increased functioning of brain regions involved in decoding social signals might facilitate the detection and decoding of subtle signals encountered in varied social settings.

  19. Epigallocatechin-3-gallate inhibits TF and TNF-α expression induced by the anti-β2GPI/β2GPI complex in human THP-1 cells.

    Science.gov (United States)

    Wang, Ting; Zhou, Hong; Xie, Hongxiang; Mu, Yuan; Xu, Ya; Liu, Jingjing; Zhang, Xiaolei

    2014-04-01

    Epigallocatechin-3-gallate (EGCG) is the major polyphenolic component of green tea. The aim of the current study was to investigate the inhibitory effects of EGCG on anti-β2-glycoprotein I (β2GPI)/β2GPI-induced tissue factor (TF) and tumor necrosis factor-α (TNF-α) expression in the human acute monocytic leukemia cell line, THP-1, as well as the underlying mechanisms. Human THP-1 cells cultured in vitro were treated with lipopolysaccharide (LPS, 500 ng/ml) or with the anti-β2GPI (10 µg/ml)/β2GPI (100 µg/ml) complex following pre-treatment with or without EGCG (0-50 µg/ml). The expression levels of TF, TNF-α and Toll-like receptor 4 (TLR4) were measured, and the activation of mitogen-activated protein kinases (MAPKs) including p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), and the nuclear factor-κB (NF-κB) signaling pathway was determined by western blot analysis. The results revealed that the anti-β2GPI/β2GPI complex activated the THP-1 cells, resulting in the enhanced expression of the coagulation cytokine, TF, as well as that of the pro-inflammatory cytokine, TNF-α; these levels were almost comparable to those induced by LPS. Pre-treatment with EGCG decreased the TF and TNF-α levels in the THP-1 cells treated with the anti-β2GPI/β2GPI complex in a dose-dependent manner and counteracted the upregulation of TLR4 expression (mRNA and protein) which was induced by the anti-β2GPI/β2GPI complex or LPS. Furthermore, EGCG suppressed the phosphorylation of p38, ERK1/2 and JNK and blocked the activation of the NF-κB signaling pathway induced by the anti-β2GPI/β2GPI complex or LPS. In conclusion, our results indicate that EGCG decreases the anti-β2GPI/β2GPI-induced TF and TNF-α expression in THP-1 cells possibly through the inhibition of the intracellular signal transduction pathway of TLRs-MAPKs-NF-κB axis and may serve as a preventive and therapeutic agent for antiphospholipid syndrome (APS).

  20. Vitamin B-complex initiates growth and development of human embryonic brain cells in vitro.

    Science.gov (United States)

    Danielyan, K E; Abramyan, R A; Galoyan, A A; Kevorkian, G A

    2011-09-01

    We studied a combined effect of subcomponents of vitamin B complex on the growth, development, and death of human embryonic brain-derived cells (E90) cultured using a modified method of Matson. Cell death was detected by trypan blue staining. According to our results, vitamin B-complex in low-doses promote the development, maturation, and enlargement of human embryonic brain cells, on the one hand, and increases the percent of cell death, which attests to accelerated maturation and metabolism, on the other.

  1. Resolving the complexity of the human genome using single-molecule sequencing.

    Science.gov (United States)

    Chaisson, Mark J P; Huddleston, John; Dennis, Megan Y; Sudmant, Peter H; Malig, Maika; Hormozdiari, Fereydoun; Antonacci, Francesca; Surti, Urvashi; Sandstrom, Richard; Boitano, Matthew; Landolin, Jane M; Stamatoyannopoulos, John A; Hunkapiller, Michael W; Korlach, Jonas; Eichler, Evan E

    2015-01-29

    The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome--78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.

  2. The SMC5/6 complex is involved in crucial processes during human spermatogenesis.

    Science.gov (United States)

    Verver, Dideke E; Langedijk, Nathalia S M; Jordan, Philip W; Repping, Sjoerd; Hamer, Geert

    2014-07-01

    Genome integrity is crucial for safe reproduction. Therefore, chromatin structure and dynamics should be tightly regulated during germ cell development. Chromatin structure and function are in large part determined by the structural maintenance of chromosomes (SMC) protein complexes, of which SMC5/6 recently has been shown to be involved in both spermatogonial differentiation and meiosis during mouse spermatogenesis. We therefore investigated the role of this complex in human spermatogenesis. We found SMC6 to be expressed in the human testis and present in a subset of type Adark and type Apale spermatogonia, all spermatocytes, and round spermatids. During human meiosis, SMC5/6 is located at the synaptonemal complex (SC), the XY body, and at the centromeres during meiotic metaphases. However, in contrast to mouse spermatogenesis, SMC6 is not located at pericentromeric heterochromatin in human spermatogenic cells, indicating subtle but perhaps important differences in not only SMC5/6 function but maybe also in maintenance of genomic integrity at the repetitive pericentromeric regions. Nonetheless, our data clearly indicate that the SMC5/6 complex, as shown in mice, is involved in numerous crucial processes during human spermatogenesis, such as in spermatogonial development, on the SC between synapsed chromosomes, and in DNA double-strand break repair on unsynapsed chromosomes during pachynema.

  3. In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse

    OpenAIRE

    2000-01-01

    We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the...

  4. How do precision medicine and system biology response to human body's complex adaptability?

    Science.gov (United States)

    Yuan, Bing

    2016-12-01

    In the field of life sciences, although system biology and "precision medicine" introduce some complex scientifific methods and techniques, it is still based on the "analysis-reconstruction" of reductionist theory as a whole. Adaptability of complex system increase system behaviour uncertainty as well as the difficulties of precise identifification and control. It also put systems biology research into trouble. To grasp the behaviour and characteristics of organism fundamentally, systems biology has to abandon the "analysis-reconstruction" concept. In accordance with the guidelines of complexity science, systems biology should build organism model from holistic level, just like the Chinese medicine did in dealing with human body and disease. When we study the living body from the holistic level, we will fifind the adaptability of complex system is not the obstacle that increases the diffificulty of problem solving. It is the "exceptional", "right-hand man" that helping us to deal with the complexity of life more effectively.

  5. Effects of human serun albumin in some biological properties of rhodium(II complexes

    Directory of Open Access Journals (Sweden)

    Espósito Breno P.

    2000-01-01

    Full Text Available The affinities for human albumin (HSA of five rhodium(II complexes of general formula [Rh2(bridge4] (bridge = acetate, propionate, butyrate, trifluoroacetate and trifluoroacetamidate were determined by spectrophotometry. In the case of the alkylcarboxylates, an inverse correlation of affinity with their liposolubilities was observed. Diffusion of the free or protein-bound complexes into Ehrlich cells in vitro seems to be primarily governed by the hydrophobic character of the complex. The complex [Rh2(tfc4] exhibited affinity towards the protein (K = 214.1 as well as cell partition both in the absence (32.1% and presence (48.6% of HSA. The compound HSA: [Rh2(tfc4] has had its antitumoral action in tumor-bearing Balb-c mice investigated, showing that HSA can be a drug reservoir for the rhodium complex.

  6. Structure and function of airway surface layer of the human lungs & mobility of probe particles in complex fluids

    Science.gov (United States)

    Cai, Liheng

    Numerous infectious particles such as bacteria and pathogens are deposited on the airway surface of the human lungs during our daily breathing. To avoid infection the lung has evolved to develop a smart and powerful defense system called mucociliary clearance. The airway surface layer is a critical component of this mucus clearance system, which consists of two parts: (1) a mucus layer, that traps inhaled particles and transports them out of the lung by cilia-generated flow; and (2) a periciliary layer, that provides a favorable environment for ciliary beating and cell surface lubrication. For 75 years, it has been dogma that a single gel-like mucus layer, which is composed of secreted mucin glycoproteins, is transported over a "watery" periciliary layer. This one-gel model, however, does not explain fundamental features of the normal system, e.g. formation of a distinct mucus layer, nor accurately predict how the mucus clearance system fails in disease. In the first part of this thesis we propose a novel "Gel-on-Brush" model with a mucus layer (the "gel") and a "brush-like" periciliary layer, composed of mucins tethered to the luminal of airway surface, and supporting data accurately describes both the biophysical and cell biological bases for normal mucus clearance and its failure in disease. Our "Gel-on-Brush" model describes for the first time how and why mucus is efficiently cleared in health and unifies the pathogenesis of major human diseases, including cystic fibrosis and chronic obstructive pulmonary disease. It is expected that this "Gel-on-Brush" model of airway surface layer opens new directions for treatments of airway diseases. A dilemma regarding the function of mucus is that, although mucus traps any inhaled harmful particulates, it also poses a long-time problem for drug delivery: mobility of cargos carrying pharmaceutical agents is slowed down in mucus. The second part of this thesis aims to answer the question: can we theoretically understand the

  7. Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

    Directory of Open Access Journals (Sweden)

    Tin Nguyen

    Full Text Available MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.

  8. Correlation between different glycoprotein B genotypes of human cytomegalovirus and infant cytomegalovirus hepatitis%人巨细胞病毒糖蛋白B基因型与婴儿巨细胞病毒性肝炎的关系

    Institute of Scientific and Technical Information of China (English)

    郑季彦; 余钟声; 杜立中; 吴家波

    2005-01-01

    人巨细胞病毒(HCMV)感染在我国广泛存在,肝脏是婴儿期HCMV感染最主要的靶器官之一。本文应用套式PCR(nPCR)加限制性长度多态性分析(RFLP)对HCMV主要包膜糖蛋白B(glycoprotein B,gB)基因进行分型,并分析2003年10月-2005年4月我院婴儿巨细胞病毒性肝炎与gB基因型的关系。

  9. Immunolocalisation pattern of complex I-V in ageing human retina: Correlation with mitochondrial ultrastructure.

    Science.gov (United States)

    Nag, Tapas Chandra; Wadhwa, Shashi

    2016-11-01

    Earlier studies reported accumulation of mitochondrial DNA mutations in ageing and age-related macular degeneration. To know about the mitochondrial status with age, we examined immunoreactivity (IR) to markers of mitochondria (anti-mitochondrial antibody and voltage-dependent anion channel-1) and complex I-V (that mediate oxidative phosphorylation, OXPHOS) in donor human retinas (age: 19-94years; N=26; right eyes). In all samples, at all ages, IR to anti-mitochondrial antibody and voltage-dependent anion channel-1 was prominent in photoreceptor cells. Between second and seventh decade of life, strong IR to complex I-V was present in photoreceptors over macular to periph