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Sample records for glycoprotein affect gp120-gp41

  1. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility.

    Science.gov (United States)

    Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Kwon, Young Do; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E; Schief, William R; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D

    2010-01-19

    The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded beta-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate--and structurally plastic--layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated beta-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a beta-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.

  2. Internalization and Axonal Transport of the HIV Glycoprotein gp120

    Science.gov (United States)

    Berth, Sarah; Caicedo, Hector Hugo; Sarma, Tulika; Morfini, Gerardo

    2015-01-01

    The HIV glycoprotein gp120, a neurotoxic HIV glycoprotein that is overproduced and shed by HIV-infected macrophages, is associated with neurological complications of HIV such as distal sensory polyneuropathy, but interactions of gp120 in the peripheral nervous system remain to be characterized. Here, we demonstrate internalization of extracellular gp120 in a manner partially independent of binding to its coreceptor CXCR4 by F11 neuroblastoma cells and cultured dorsal root ganglion neurons. Immunocytochemical and pharmacological experiments indicate that gp120 does not undergo trafficking through the endolysosomal pathway. Instead, gp120 is mainly internalized through lipid rafts in a cholesterol-dependent manner, with a minor fraction being internalized by fluid phase pinocytosis. Experiments using compartmentalized microfluidic chambers further indicate that, after internalization, endocytosed gp120 selectively undergoes retrograde but not anterograde axonal transport from axons to neuronal cell bodies. Collectively, these studies illuminate mechanisms of gp120 internalization and axonal transport in peripheral nervous system neurons, providing a novel framework for mechanisms for gp120 neurotoxicity. PMID:25636314

  3. N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

    International Nuclear Information System (INIS)

    Dey, Antu K.; David, Kathryn B.; Ray, Neelanjana; Ketas, Thomas J.; Klasse, Per J.; Doms, Robert W.; Moore, John P.

    2008-01-01

    The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B

  4. Determining the Structure of an Unliganded and Fully Glycosylated SIV gp120 Envelope Glycoprotein

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    Chen, Bing; Vogan, Erik M.; Gong, Haiyun; Skehel, John J.; Wiley, Don C.; Harrison, Stephen C. (Harvard-Med); (NIMR)

    2010-07-13

    HIV/SIV envelope glycoproteins mediate the first steps in viral infection. They are trimers of a membrane-anchored polypeptide chain, cleaved into two fragments known as gp120 and gp41. The structure of HIV gp120 bound with receptor (CD4) has been known for some time. We have now determined the structure of a fully glycosylated SIV gp120 envelope glycoprotein in an unliganded conformation by X-ray crystallography at 4.0 {angstrom} resolution. We describe here our experimental and computational approaches, which may be relevant to other resolution-limited crystallographic problems. Key issues were attention to details of beam geometry mandated by small, weakly diffracting crystals, and choice of strategies for phase improvement, starting with two isomorphous derivatives and including multicrystal averaging. We validated the structure by analyzing composite omit maps, averaged among three distinct crystal lattices, and by calculating model-based, SeMet anomalous difference maps. There are at least four ordered sugars on many of the thirteen oligosaccharides.

  5. A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120

    International Nuclear Information System (INIS)

    Beddows, Simon; Franti, Michael; Dey, Antu K.; Kirschner, Marc; Iyer, Sai Prasad N.; Fisch, Danielle C.; Ketas, Thomas; Yuste, Eloisa; Desrosiers, Ronald C.; Klasse, Per Johan; Maddon, Paul J.; Olson, William C.; Moore, John P.

    2007-01-01

    The human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein (Env) complex, a homotrimer containing gp120 surface glycoprotein and gp41 transmembrane glycoprotein subunits, mediates the binding and fusion of the virus with susceptible target cells. The Env complex is the target for neutralizing antibodies (NAbs) and is the basis for vaccines intended to induce NAbs. Early generation vaccines based on monomeric gp120 subunits did not confer protection from infection; one alternative approach is therefore to make and evaluate soluble forms of the trimeric Env complex. We have directly compared the immunogenicity in rabbits of two forms of soluble trimeric Env and monomeric gp120 based on the sequence of HIV-1 JR-FL . Both protein-only and DNA-prime, protein-boost immunization formats were evaluated, DNA-priming having little or no influence on the outcome. One form of trimeric Env was made by disrupting the gp120-gp41 cleavage site by mutagenesis (gp140 UNC ), the other contains an intramolecular disulfide bond to stabilize the cleaved gp120 and gp41 moieties (SOSIP.R6 gp140). Among the three immunogens, SOSIP.R6 gp140 most frequently elicited neutralizing antibodies against the homologous, neutralization-resistant strain, HIV-1 JR-FL . All three proteins induced NAbs against more sensitive strains, but the breadth of activity against heterologous primary isolates was limited. When antibodies able to neutralize HIV-1 JR-FL were detected, antigen depletion studies showed they were not directed at the V3 region but were targeted at other, undefined gp120 and also non-gp120 epitopes

  6. Reduction of cerebral glucose utilization by the HIV envelope glycoprotein Gp-120

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    Kimes, A.S.; London, E.D.; Szabo, G.; Raymon, L.; Tabakoff, B. (Neuropharmacology Laboratory, National Institute on Drug Abuse, Baltimore, MD (USA))

    1991-05-01

    Gp-120 is a glycoprotein constituent of the human immunodeficiency virus (HIV) envelope. The effects of gp-120 on cerebral glucose utilization in rats were studied by the quantitative 2-deoxy-D-(1-14C) glucose method. Intracerebroventricular injection of gp-120 significantly reduced glucose utilization in the lateral habenula and the suprachiasmatic nucleus and decreased the global cerebral metabolic rate for glucose. The findings suggest that gp-120 and closely related peptides can alter neuronal function, thereby contributing to the sequelae of HIV infection.

  7. Reduction of cerebral glucose utilization by the HIV envelope glycoprotein Gp-120

    International Nuclear Information System (INIS)

    Kimes, A.S.; London, E.D.; Szabo, G.; Raymon, L.; Tabakoff, B.

    1991-01-01

    Gp-120 is a glycoprotein constituent of the human immunodeficiency virus (HIV) envelope. The effects of gp-120 on cerebral glucose utilization in rats were studied by the quantitative 2-deoxy-D-[1-14C] glucose method. Intracerebroventricular injection of gp-120 significantly reduced glucose utilization in the lateral habenula and the suprachiasmatic nucleus and decreased the global cerebral metabolic rate for glucose. The findings suggest that gp-120 and closely related peptides can alter neuronal function, thereby contributing to the sequelae of HIV infection

  8. Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

    International Nuclear Information System (INIS)

    Bellamy-McIntyre, Anna K.; Baer, Severine; Ludlow, Louise; Drummer, Heidi E.; Poumbourios, Pantelis

    2010-01-01

    The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1 QH1549.13 blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation.

  9. Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.

    Science.gov (United States)

    Blattner, Claudia; Lee, Jeong Hyun; Sliepen, Kwinten; Derking, Ronald; Falkowska, Emilia; de la Peña, Alba Torrents; Cupo, Albert; Julien, Jean-Philippe; van Gils, Marit; Lee, Peter S; Peng, Wenjie; Paulson, James C; Poignard, Pascal; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Wilson, Ian A; Ward, Andrew B

    2014-05-15

    All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

    International Nuclear Information System (INIS)

    Arthur, L.O.; Pyle, S.W.; Nara, P.L.

    1987-01-01

    The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4 + and T8 + cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4 + cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo

  11. Strategies for induction of catalytic antibodies toward HIV-1 glycoprotein gp120 in autoimmune prone mice.

    Science.gov (United States)

    Durova, Oxana M; Vorobiev, Ivan I; Smirnov, Ivan V; Reshetnyak, Andrew V; Telegin, Georgy B; Shamborant, Olga G; Orlova, Nadezda A; Genkin, Dmitry D; Bacon, Andrew; Ponomarenko, Natalia A; Friboulet, Alain; Gabibov, Alexander G

    2009-11-01

    Tremendous efforts to produce an efficient vaccine for HIV infection have been unsuccessful. The ability of HIV to utilize sophisticated mechanisms to escape killing by host immune system rises dramatic problems in the development of antiviral therapeutics. The HIV infection proceeds by interaction of coat viral glycoprotein gp120 trimer with CD4(+) receptor of the lymphocyte. Thus this surface antigen may be regarded as a favorable target for immunotherapy. In the present study, we have developed three different strategies to produce gp120-specific response in autoimmune prone mice (SJL strain) as potential tools for production "catalytic vaccine". Therefore (i) reactive immunization by peptidylphosphonate, structural part of the coat glycoprotein, (ii) immunization by engineered fused epitopes of gp120 and encephalogenic peptide, a part of myelin basic protein, and (iii) combined vaccination by DNA and corresponding gp120 fragments incorporated into liposomes were investigated. In the first two cases monoclonal antibodies and their recombinant fragments with amidolytic and gp120-specific proteolytic activities were characterized. In the last case, catalytic antibodies with virus neutralizing activity proved in cell line models were harvested.

  12. Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

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    Wibmer, Constantinos Kurt; Gorman, Jason; Ozorowski, Gabriel; Bhiman, Jinal N.; Sheward, Daniel J.; Elliott, Debra H.; Rouelle, Julie; Smira, Ashley; Joyce, M. Gordon; Ndabambi, Nonkululeko; Druz, Aliaksandr; Asokan, Mangai; Burton, Dennis R.; Connors, Mark; Abdool Karim, Salim S.; Mascola, John R.; Robinson, James E.; Ward, Andrew B.; Williamson, Carolyn; Kwong, Peter D.; Morris, Lynn; Moore, Penny L.; Desrosiers, Ronald C.

    2017-01-11

    A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing

  13. Structural Basis for Species Selectivity in the HIV-1 gp120-CD4 Interaction: Restoring Affinity to gp120 in Murine CD4 Mimetic Peptides

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    Kristin Kassler

    2011-01-01

    Full Text Available The first step of HIV-1 infection involves interaction between the viral glycoprotein gp120 and the human cellular receptor CD4. Inhibition of the gp120-CD4 interaction represents an attractive strategy to block HIV-1 infection. In an attempt to explore the known lack of affinity of murine CD4 to gp120, we have investigated peptides presenting the putative gp120-binding site of murine CD4 (mCD4. Molecular modeling indicates that mCD4 protein cannot bind gp120 due to steric clashes, while the larger conformational flexibility of mCD4 peptides allows an interaction. This finding is confirmed by experimental binding assays, which also evidenced specificity of the peptide-gp120 interaction. Molecular dynamics simulations indicate that the mCD4-peptide stably interacts with gp120 via an intermolecular β-sheet, while an important salt-bridge formed by a C-terminal lysine is lost. Fixation of the C-terminus by introducing a disulfide bridge between the N- and C-termini of the peptide significantly enhanced the affinity to gp120.

  14. Anti-HIV double variable domain immunoglobulins binding both gp41 and gp120 for targeted delivery of immunoconjugates.

    Directory of Open Access Journals (Sweden)

    Ryan B Craig

    Full Text Available BACKGROUND: Anti-HIV immunoconjugates targeted to the HIV envelope protein may be used to eradicate the latent reservoir of HIV infection using activate-and-purge protocols. Previous studies have identified the two target epitopes most effective for the delivery of cytotoxic immunoconjugates the CD4-binding site of gp120, and the hairpin loop of gp41. Here we construct and test tetravalent double variable domain immunoglobulin molecules (DVD-Igs that bind to both epitopes. METHODS: Synthetic genes that encode DVD-Igs utilizing V-domains derived from human anti-gp120 and anti-gp41 Abs were designed and expressed in 293F cells. A series of constructs tested different inter-V-linker domains and orientations of the two V domains. Antibodies were tested for binding to recombinant Ag and native Env expressed on infected cells, for neutralization of infectious HIV, and for their ability to deliver cytotoxic immunoconjugates to infected cells. FINDINGS: The outer V-domain was the major determinant of binding and functional activity of the DVD-Ig. Function of the inner V-domain and bifunctional binding required at least 15 AA in the inter-V-domain linker. A molecular model showing the spatial orientation of the two epitopes is consistent with this observation. Linkers that incorporated helical domains (A[EAAAK](nA resulted in more effective DVD-Igs than those based solely on flexible domains ([GGGGS](n. In general, the DVD-Igs outperformed the less effective parental antibody and equaled the activity of the more effective. The ability of the DVD-Igs to deliver cytotoxic immunoconjugates in the absence of soluble CD4 was improved over that of either parent. CONCLUSIONS: DVD-Igs can be designed that bind to both gp120 and gp41 on the HIV envelope. DVD-Igs are effective in delivering cytotoxic immunoconjugates. The optimal design of these DVD-Igs, in which both domains are fully functional, has not yet been achieved.

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

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    Xiao Dan

    2010-11-01

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

  16. HIV-1 gp41 Fusion Intermediate: A Target for HIV Therapeutics

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    Chungen Pan

    2010-02-01

    Full Text Available Human immunodeficiency virus (HIV-1 infection is initiated by the binding of gp120 envelope glyco-protein to its cell receptor (CD4 and a coreceptor (CXCR4 or CCR5, followed by a series of conformational changes in the gp41 transmembrane subunit. These changes include insertion of fusion peptide into the target cell membrane and association of C-heptad repeat (CHR peptide with the N-heptad repeat (NHR trimer, a pre-hairpin fusion intermediate. A stable six-helix bundle core is then formed, bringing the viral envelope and target cell membrane into close proximity for fusion. Peptides derived from the CHR region, such as T20 and C34, inhibit HIV-1 fusion by interacting with the gp41 fusion intermediate. A number of anti-HIV-1 peptides and small molecule compounds targeting the gp41 NHR-trimer have been identified. By combining HIV fusion/entry inhibitors targeting different sites in the gp41 fusion intermediate, a potent synergistic effect takes place, resulting in a potential new therapeutic strategy for the HIV infection/AIDS. Here, we present an overview of the current development of anti-HIV drugs, particularly those targeting the gp41 fusion intermediate.

  17. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

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    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark [NIH

    2015-10-15

    The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

  18. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    International Nuclear Information System (INIS)

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph

    2005-01-01

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate

  19. Binding kinetics of aptamers to gp120 derived from HIV-1 subtype C

    CSIR Research Space (South Africa)

    Millroy, L

    2011-02-01

    Full Text Available aptamers with specific and strong affinity to the HIV-1 envelope glycoprotein gp120 and act as novel HIV-1 entry inhibitor drugs or as targeted drug delivery systems to HIV-1 infected cells. Prior to any downstream applications, novel gp120 aptamers need...

  20. Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

    International Nuclear Information System (INIS)

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Depetris, Rafael S.; Thomas, Antonia M.; Klasse, Per Johan; Moore, John P.

    2011-01-01

    Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.

  1. Induction of a protein-targeted catalytic response in autoimmune prone mice: antibody-mediated cleavage of HIV-1 glycoprotein GP120.

    Science.gov (United States)

    Ponomarenko, Natalia A; Vorobiev, Ivan I; Alexandrova, Elena S; Reshetnyak, Andrew V; Telegin, Georgy B; Khaidukov, Sergey V; Avalle, Bérangère; Karavanov, Alexander; Morse, Herbert C; Thomas, Daniel; Friboulet, Alain; Gabibov, Alexander G

    2006-01-10

    We have induced a polyclonal IgG that degrades the HIV-1 surface antigen, glycoprotein gp120, by taking advantage of the susceptibility of SJL mice to a peptide-induced autoimmune disorder, experimental autoimmune encephalomyelitis (EAE). Specific pathogen-free SJL mice were immunized with structural fragments of gp120, fused in-frame with encephalitogenic peptide MBP(85-101). It has resulted in a pronounced disease-associated immune response against antigens. A dramatic increase of gp120 degradation level by purified polyclonal IgG from immunized versus nonimmunized mice has been demonstrated by a newly developed fluorescence-based assay. This activity was inhibited by anti-mouse immunoglobulin antibodies as well as by Ser- and His-reactive covalent inhibitors. A dominant proteolysis site in recombinant gp120 incubated with purified polyclonal IgG from immunized mice was shown by SDS-PAGE. The SELDI-based mass spectrometry revealed that these antibodies exhibited significant specificity toward the Pro484-Leu485 peptide bond. The sequence surrounding this site is present in nearly half of the HIV-I variants. This novel strategy can be generalized for creating a catalytic vaccine against viral pathogens.

  2. The Effects of IGF-1 on Trk Expressing DRG Neurons with HIV-gp120- Induced Neurotoxicity.

    Science.gov (United States)

    Li, Hao; Liu, Zhen; Chi, Heng; Bi, Yanwen; Song, Lijun; Liu, Huaxiang

    2016-01-01

    HIV envelope glycoprotein gp120 is the main protein that causes HIVassociated sensory neuropathy. However, the underlying mechanisms of gp120-induced neurotoxicity are still unclear. There are lack effective treatments for relieving HIV-related neuropathic symptoms caused by gp120-induced neurotoxicity. In the present study, tyrosine kinase receptor (Trk)A, TrkB, and TrkC expression in primary cultured dorsal root ganglion (DRG) neurons with gp120-induced neurotoxicity was investigated. The effects of IGF-1 on distinct Trk-positive DRG neurons with gp120-induced neurotoxicity were also determined. The results showed that gp120 not only dose-dependently induced DRG neuronal apoptosis and inhibited neuronal survival and neurite outgrowth, but also decreased distinct Trk expression levels. IGF-1 rescued DRG neurons from apoptosis and improved neuronal survival of gp120 neurotoxic DRG neurons in vitro. IGF-1 also improved TrkA and TrkB, but not TrkC, expression in gp120 neurotoxic conditions. The effects of IGF-1 could be blocked by preincubation with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results suggested that gp120 may have a wide range of neurotoxicity on different subpopulations of DRG neurons, while IGF-1 might only relieve some subpopulations of DRG neurons with gp120-induced neurotoxicity. These data provide novel information of mechanisms of gp120 neurotoxicity on primary sensory neurons and the potential therapeutic effects of IGF-1 on gp120-induced neurotoxicity.

  3. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    Energy Technology Data Exchange (ETDEWEB)

    Costantini, Lindsey M. [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Irvin, Susan C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Kennedy, Steven C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Guo, Feng [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Goldstein, Harris; Herold, Betsy C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Snapp, Erik L., E-mail: erik-lee.snapp@einstein.yu.edu [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  4. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    International Nuclear Information System (INIS)

    Costantini, Lindsey M.; Irvin, Susan C.; Kennedy, Steven C.; Guo, Feng; Goldstein, Harris; Herold, Betsy C.; Snapp, Erik L.

    2015-01-01

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells

  5. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Weizao, E-mail: chenw3@mail.nih.gov [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Feng, Yang [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Wang, Yanping [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); The Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Zhu, Zhongyu; Dimitrov, Dimiter S. [Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  6. HIV-gp120 and physical dependence to buprenorphine.

    Science.gov (United States)

    Palma, J; Abood, M E; Benamar, K

    2015-05-01

    Opioids are among the most effective and commonly used analgesics in clinical practice for severe pain. However, the use of opioid medications is clinically limited by several adverse properties including dependence. While opioid dependence is a complex health condition, the treatment of HIV-infected individuals with opioid dependence presents additional challenges. The goal of this study was to examine the physical dependence to buprenorphine in the context of HIV. Young adult male rats (Sprague-Dawley) were pretreated with HIV-1 envelope glycoprotein 120 (gp120) injected into the periaqueductal gray area (PAG) and we examined the impact on physical dependence to opioid. It was found that the physical dependence to methadone occurred earlier than that to buprenorphine, and that gp120 did not enhance or precipitate the buprenorphine withdrawal. The results suggest that buprenorphine could be the better therapeutic option to manage opioid dependence in HIV. Copyright © 2015. Published by Elsevier Ireland Ltd.

  7. Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

    International Nuclear Information System (INIS)

    Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A.

    2004-01-01

    Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function

  8. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

    International Nuclear Information System (INIS)

    Koch, Markus; Pancera, Marie; Kwong, Peter D.; Kolchinsky, Peter; Grundner, Christoph; Wang Liping; Hendrickson, Wayne A.; Sodroski, Joseph; Wyatt, Richard

    2003-01-01

    The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization

  9. Ninety-five- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor

    International Nuclear Information System (INIS)

    Nygren, A.; Bergman, T.; Matthews, T.; Joernvall, H.; Wigzell, H.

    1988-01-01

    Iodine-125-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extend with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25- kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing 125 I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule

  10. Comparative Glycoprofiling of HIV gp120 Immunogens by Capillary Electrophoresis and MALDI Mass Spectrometry

    Science.gov (United States)

    Guttman, Miklós; Váradi, Csaba; Lee, Kelly K.; Guttman, András

    2015-01-01

    The Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) is the primary antigenic feature on the surface of the virus and is of key importance in HIV vaccinology. Vaccine trials with the gp120 subunit of Env are ongoing with the recent RV144 trial showing moderate efficacy. gp120 is densely covered with N-linked glycans that are thought to help evade the host's humoral immune response. To assess how the global glycosylation patterns vary between gp120 constructs, the glycan profiles of several gp120s were examined by capillary electrophoresis with laser induced fluorescence detection and MALDI-MS. The glycosylation profiles were found to be similar for chronic vs. transmitter/founder isolates and only varied moderately between gp120s from different clades. This study revealed that the addition of specific tags, such as the gD tag used in the RV144 trial, had significant effects on the overall glycosylation patterns. Such effects are likely to influence the immunogenicity of various Env immunogens and should be considered for future vaccine strategies, emphasizing the importance of the glycosylation analysis approach described in this paper. PMID:25809283

  11. Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs.

    Science.gov (United States)

    Jiang, Xiaohong; Dalebout, Tim J; Bredenbeek, Peter J; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S; Lukashevich, Igor S

    2011-02-01

    Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

    Science.gov (United States)

    Jiang, Xiaohong; Dalebout, Tim J.; Bredenbeek, Peter J.; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S.; Lukashevich, Igor S.

    2010-01-01

    Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV GP1 and GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and –GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF and LASV GP proteins in infected cells. YF17D/LASV-GP1&GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1&GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. PMID:21145373

  13. HIV-1 gp120 and drugs of abuse: interactions in the central nervous system.

    Science.gov (United States)

    Silverstein, Peter S; Shah, Ankit; Weemhoff, James; Kumar, Santosh; Singh, D P; Kumar, Anil

    2012-07-01

    HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs.

  14. Specific assay measuring binding of /sup 125/I-Gp 120 from HIV to T4/sup +//CD4/sup +/ cells

    Energy Technology Data Exchange (ETDEWEB)

    Lundin, K.; Nygren, A.; Ramstedt, U.; Gidlund, M.; Wigzell, H.; Arthur, L.O.; Robey, W.G.; Morein, B.

    1987-02-26

    The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4/sup +/ (T4/sup +/) cells. /sup 125/I-Gp120 could be shown to selectively bind to CD4/sup +/ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4/sup +/ cells. Monoclonal antibodies to other cell surface components do not interfere with /sup 125/I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block /sup 125/I-GP120 binding, though with variable titers. The authors believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding. (Auth.). 24 refs.; 2 figs.; 8 tabs.

  15. HIV-1 gp120 neurotoxicity proximally and at a distance from the point of exposure: protection by rSV40 delivery of antioxidant enzymes.

    Science.gov (United States)

    Louboutin, Jean-Pierre; Agrawal, Lokesh; Reyes, Beverly A S; Van Bockstaele, Elisabeth J; Strayer, David S

    2009-06-01

    Toxicity of HIV-1 envelope glycoprotein (gp120) for substantia nigra (SN) neurons may contribute to the Parkinsonian manifestations often seen in HIV-1-associated dementia (HAD). We studied the neurotoxicity of gp120 for dopaminergic neurons and potential neuroprotection by antioxidant gene delivery. Rats were injected stereotaxically into their caudate-putamen (CP); CP and (substantia nigra) SN neuron loss was quantified. The area of neuron loss extended several millimeters from the injection site, approximately 35% of the CP area. SN neurons, outside of this area of direct neurotoxicity, were also severely affected. Dopaminergic SN neurons (expressing tyrosine hydroxylase, TH, in the SN and dopamine transporter, DAT, in the CP) were mostly affected: intra-CP gp120 caused approximately 50% DAT+ SN neuron loss. Prior intra-CP gene delivery of Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) protected SN neurons from intra-CP gp120. Thus, SN dopaminergic neurons are highly sensitive to HIV-1 gp120-induced neurotoxicity, and antioxidant gene delivery, even at a distance, is protective.

  16. Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.

    Science.gov (United States)

    Pallesen, Jesper; Murin, Charles D; de Val, Natalia; Cottrell, Christopher A; Hastie, Kathryn M; Turner, Hannah L; Fusco, Marnie L; Flyak, Andrew I; Zeitlin, Larry; Crowe, James E; Andersen, Kristian G; Saphire, Erica Ollmann; Ward, Andrew B

    2016-08-08

    The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.

  17. 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

    ), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define...... 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....

  18. Prediction of the Secondary Structure of HIV-1 gp120

    DEFF Research Database (Denmark)

    Hansen, Jan; Lund, Ole; Nielsen, Jens O.

    1996-01-01

    Fourier transform infrared spectroscopy. The predicted secondary structure of gp120 compared well with data from NMR analysis of synthetic peptides from the V3 loop and the C4 region. As a first step towards modeling the tertiary structure of gp120, the predicted secondary structure may guide the design......The secondary structure of HIV-1 gp120 was predicted using multiple alignment and a combination of two independent methods based on neural network and nearest-neighbor algorithms. The methods agreed on the secondary structure for 80% of the residues in BH10 gp120. Six helices were predicted in HIV...

  19. N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction

    Directory of Open Access Journals (Sweden)

    Costa Nicola

    2007-12-01

    Full Text Available Abstract Background HIV envelope gp 120 glycoprotein is released during active HIV infection of brain macrophages thereby generating inflammation and oxidative stress which contribute to the development of the AIDS-Dementia Complex (ADC. Gp120 has also been found capable to generate excitotoxic effect on brain tissue via enhancement of glutamatergic neurotransmission, leading to neuronal and astroglial damage, though the mechanism is still to be better understood. Here we investigated on the effect of N-acetylcysteine (NAC, on gp120-induced damage in human cultured astroglial cells and the possible contribution of gp120-related reacting oxygen species (ROS in the imbalanced activity of glutamine synthase (GS, the enzyme that metabolizes glutamate into glutamine within astroglial cells playing a neuroprotective role in brain disorders. Results Incubation of Lipari human cultured astroglial cells with gp 120 (0.1–10 nM produced a significant reduction of astroglial cell viability and apoptosis as evaluated by TUNEL reaction and flow cytometric analysis (FACS. This effect was accompanied by lipid peroxidation as detected by means of malondialdehyde assay (MDA. In addition, gp 120 reduced both glutamine concentration in astroglial cell supernatants and GS expression as detected by immunocytochemistry and western blotting analysis. Pre-treatment of cells with NAC (0.5–5 mM, dose-dependently antagonised astroglial apoptotic cell death induced by gp 120, an effect accompanied by significant attenuation of MDA accumulation. Furthermore, both effects were closely associated with a significant recovery of glutamine levels in cell supernatants and by GS expression, thus suggesting that overproduction of free radicals might contribute in gp 120-related dysfunction of GS in astroglial cells. Conclusion In conclusion, the present experiments demonstrate that gp 120 is toxic to astroglial cells, an effect accompanied by lipid peroxidation and by altered

  20. Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

    International Nuclear Information System (INIS)

    Roche, Julien; Louis, John M.; Aniana, Annie; Ghirlando, Rodolfo; Bax, Ad

    2015-01-01

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41’s ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion

  1. Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

    Energy Technology Data Exchange (ETDEWEB)

    Roche, Julien; Louis, John M.; Aniana, Annie [National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Chemical Physics (United States); Ghirlando, Rodolfo [National Institutes of Health, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Bax, Ad, E-mail: bax@nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Chemical Physics (United States)

    2015-04-15

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41’s ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

  2. Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

    Directory of Open Access Journals (Sweden)

    Barna Dey

    2009-05-01

    Full Text Available The human immunodeficiency virus type 1 (HIV-1 exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

  3. Gp120 stability on HIV-1 virions and Gag-Env pseudovirions is enhanced by an uncleaved Gag core

    International Nuclear Information System (INIS)

    Hammonds, Jason; Chen Xuemin; Ding Lingmei; Fouts, Timothy; De Vico, Anthony; Megede, Jan zur; Barnett, Susan; Spearman, Paul

    2003-01-01

    Human immunodeficiency virus type-1 (HIV-1) particles incorporate a trimeric envelope complex (Env) made of gp120 (SU) and gp41 (TM) heterodimers. It has been previously established that soluble CD4 (sCD4) interaction leads to shedding of gp120 from viral particles, and that gp120 may also be easily lost from virions during incubation or particle purification procedures. In the design of HIV particle or pseudovirion-based HIV vaccines, it may be important to develop strategies to maximize the gp120 content of particles. We analyzed the gp120 retention of HIV-1 laboratory-adapted isolates and primary isolates following incubation with sCD4 and variations in temperature. NL4-3 shed gp120 readily in a temperature- and sCD4-dependent manner. Surprisingly, inactivation of the viral protease led to markedly reduced shedding of gp120. Gp120 shedding was shown to vary markedly between HIV-1 strains, and was not strictly determined by whether the isolate was adapted to growth on immortalized T cell lines or was a primary isolate. Pseudovirions produced by expression of codon-optimized gag and env genes also demonstrated enhanced gp120 retention when an immature core structure was maintained. Pseudovirions of optimal stability were produced through a combination of an immature Gag protein core and a primary isolate Env. These results support the feasibility of utilizing pseudovirion particles as immunogens for the induction of humoral responses directed against native envelope structures

  4. Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120

    International Nuclear Information System (INIS)

    Nara, P.L.; Robey, W.G.; Gonda, M.A.; Carter, S.G.; Fischinger, P.J.

    1987-01-01

    The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies has no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as well as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represent a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans

  5. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    International Nuclear Information System (INIS)

    Hollier, Mark J.; Dimmock, Nigel J.

    2005-01-01

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, β-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell

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

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    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

  7. Conformational alterations in the CD4 binding cavity of HIV-1 gp120 influencing gp120-CD4 interactions and fusogenicity of HIV-1 envelopes derived from brain and other tissues

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    Ramsland Paul A

    2011-06-01

    Full Text Available Abstract Background CD4-binding site (CD4bs alterations in gp120 contribute to HIV-1 envelope (Env mediated fusogenicity and the ability of gp120 to utilize low levels of cell-surface CD4. In a recent study, we constructed three-dimensional models of gp120 to illustrate CD4bs conformations associated with enhanced fusogenicity and enhanced CD4-usage of a modestly-sized panel of blood-derived HIV-1 Envs (n = 16. These conformations were characterized by a wider aperture of the CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop. Here, we sought to provide further validation of the utility of these models for understanding mechanisms that influence Env function, by characterizing the structure-function relationships of a larger panel of Envs derived from brain and other tissues (n = 81. Findings Three-dimensional models of gp120 were generated by our recently validated homology modelling protocol. Analysis of predicted CD4bs structures showed correlations between the aperture width of the CD4bs cavity and ability of the Envs to mediate cell-cell fusion, scavenge low-levels of cell-surface CD4, bind directly to soluble CD4, and bind to the Env mAb IgG1b12 whose epitope overlaps the gp120 CD4bs. These structural alterations in the CD4bs cavity were associated with repositioning of the V5 loop. Conclusions Using a large, independent panel of Envs, we can confirm the utility of three-dimensional gp120 structural models for illustrating CD4bs alterations that can affect Env function. Furthermore, we now provide new evidence that these CD4bs alterations augment the ability of gp120 to interact with CD4 by increasing the exposure of the CD4bs.

  8. Human CNS cultures exposed to HIV-1 gp120 reproduce dendritic injuries of HIV-1-associated dementia

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    Hammond Robert R

    2004-05-01

    Full Text Available Abstract HIV-1-associated dementia remains a common subacute to chronic central nervous system degeneration in adult and pediatric HIV-1 infected populations. A number of viral and host factors have been implicated including the HIV-1 120 kDa envelope glycoprotein (gp120. In human post-mortem studies using confocal scanning laser microscopy for microtubule-associated protein 2 and synaptophysin, neuronal dendritic pathology correlated with dementia. In the present study, primary human CNS cultures exposed to HIV-1 gp120 at 4 weeks in vitro suffered gliosis and dendritic damage analogous to that described in association with HIV-1-associated dementia.

  9. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    International Nuclear Information System (INIS)

    Grose, C.; Jackson, W.; Traugh, J.A.

    1989-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ- 32 P]ATP. The same glycoprotein was phosphorylated when [ 32 P]GTP was substituted for [ 32 P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

  10. HIV-1 specific IgA detected in vaginal secretions of HIV uninfected women participating in a microbicide trial in Southern Africa are primarily directed toward gp120 and gp140 specificities.

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    Kelly E Seaton

    Full Text Available Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035. We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.

  11. Insight derived from molecular dynamics simulations into molecular motions, thermodynamics and kinetics of HIV-1 gp120.

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    Peng Sang

    Full Text Available Although the crystal structures of the HIV-1 gp120 core bound and pre-bound by CD4 are known, the details of dynamics involved in conformational equilibrium and transition in relation to gp120 function have remained elusive. The homology models of gp120 comprising the N- and C-termini and loops V3 and V4 in the CD4-bound and CD4-unbound states were built and subjected to molecular dynamics (MD simulations to investigate the differences in dynamic properties and molecular motions between them. The results indicate that the CD4-bound gp120 adopted a more compact and stable conformation than the unbound form during simulations. For both the unbound and bound gp120, the large concerted motions derived from essential dynamics (ED analyses can influence the size/shape of the ligand-binding channel/cavity of gp120 and, therefore, were related to its functional properties. The differences in motion direction between certain structural components of these two forms of gp120 were related to the conformational interconversion between them. The free energy calculations based on the metadynamics simulations reveal a more rugged and complex free energy landscape (FEL for the unbound than for the bound gp120, implying that gp120 has a richer conformational diversity in the unbound form. The estimated free energy difference of ∼-6.0 kJ/mol between the global minimum free energy states of the unbound and bound gp120 indicates that gp120 can transform spontaneously from the unbound to bound states, revealing that the bound state represents a high-probability "ground state" for gp120 and explaining why the unbound state resists crystallization. Our results provide insight into the dynamics-and-function relationship of gp120, and facilitate understandings of the thermodynamics, kinetics and conformational control mechanism of HIV-1 gp120.

  12. Curcumin protects microglia and primary rat cortical neurons against HIV-1 gp120-mediated inflammation and apoptosis.

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    Luyan Guo

    Full Text Available Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9 and primary rat cortical neurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS, tumor necrosis factor-α (TNF-α and monocyte chemoattractant protein-1 (MCP-1. HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K(+ current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K(+ channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the delayed rectification and transient outward K(+ channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects cortical neurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K(+ current.

  13. Computational study of HIV gp120 as a target for polyanionic entry inhibitors: Exploiting the V3 loop region.

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    Louis R Hollingsworth

    Full Text Available Multiple approaches are being utilized to develop therapeutics to treat HIV infection. One approach is designed to inhibit entry of HIV into host cells, with a target being the viral envelope glycoprotein, gp120. Polyanionic compounds have been shown to be effective in inhibiting HIV entry, with a mechanism involving electrostatic interactions with the V3 loop of gp120 being proposed. In this study, we applied computational methods to elucidate molecular interactions between the repeat unit of the precisely alternating polyanion, Poly(4,4'-stilbenedicarboxylate-alt-maleic acid (DCSti-alt-MA and the V3 loop of gp120 from strains of HIV against which these polyanions were previously tested (IIIb, BaL, 92UG037, JR-CSF as well as two strains for which gp120 crystal structures are available (YU2, 2B4C. Homology modeling was used to create models of the gp120 proteins. Using monomers of the gp120 protein, we applied extensive molecular dynamics simulations to obtain dominant morphologies that represent a variety of open-closed states of the V3 loop to examine the interaction of 112 ligands of the repeating units of DCSti-alt-MA docked to the V3 loop and surrounding residues. Using the distance between the V1/V2 and V3 loops of gp120 as a metric, we revealed through MD simulations that gp120 from the lab-adapted strains (BaL and IIIb, which are more susceptible to inhibition by DCSti-alt-MA, clearly transitioned to the closed state in one replicate of each simulation set, whereas none of the replicates from the Tier II strains (92UG037 and JR-CSF did so. Docking repeat unit microspecies to the gp120 protein before and after MD simulation enabled identification of residues that were key for binding. Notably, only a few residues were found to be important for docking both before and after MD simulation as a result of the conformational heterogeneity provided by the simulations. Consideration of the residues that were consistently involved in interactions

  14. Molecular determinants of the V3 loop of human immunodeficiency virus type 1 glycoprotein gp120 responsible for controlling cell tropism.

    Science.gov (United States)

    Chavda, S C; Griffin, P; Han-Liu, Z; Keys, B; Vekony, M A; Cann, A J

    1994-11-01

    We and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gp120. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal beta-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gp120 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.

  15. Mutations in gp41 are correlated with coreceptor tropism but do not improve prediction methods substantially.

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    Thielen, Alexander; Lengauer, Thomas; Swenson, Luke C; Dong, Winnie W Y; McGovern, Rachel A; Lewis, Marilyn; James, Ian; Heera, Jayvant; Valdez, Hernan; Harrigan, P Richard

    2011-01-01

    The main determinants of HIV-1 coreceptor usage are located in the V3-loop of gp120, although mutations in V2 and gp41 are also known. Incorporation of V2 is known to improve prediction algorithms; however, this has not been confirmed for gp41 mutations. Samples with V3 and gp41 genotypes and Trofile assay (Monogram Biosciences, South San Francisco, CA, USA) results were taken from the HOMER cohort (n=444) and from patients screened for the MOTIVATE studies (n=1,916; 859 with maraviroc outcome data). Correlations of mutations with tropism were assessed using Fisher's exact test and prediction models trained using support vector machines. Models were validated by cross-validation, by testing models from one dataset on the other, and by analysing virological outcome. Several mutations within gp41 were highly significant for CXCR4 usage; most strikingly an insertion occurring in 7.7% of HOMER-R5 and 46.3% of HOMER-X4 samples (MOTIVATE 5.7% and 25.2%, respectively). Models trained on gp41 sequence alone achieved relatively high areas under the receiver-operating characteristic curve (AUCs; HOMER 0.713 and MOTIVATE 0.736) that were almost as good as V3 models (0.773 and 0.884, respectively). However, combining the two regions improved predictions only marginally (0.813 and 0.902, respectively). Similar results were found when models were trained on HOMER and validated on MOTIVATE or vice versa. The difference in median log viral load decrease at week 24 between patients with R5 and X4 virus was 1.65 (HOMER 2.45 and MOTIVATE 0.79) for V3 models, 1.59 for gp41-models (2.42 and 0.83, respectively) and 1.58 for the combined predictor (2.44 and 0.86, respectively). Several mutations within gp41 showed strong correlation with tropism in two independent datasets. However, incorporating gp41 mutations into prediction models is not mandatory because they do not improve substantially on models trained on V3 sequences alone.

  16. Glial TNFα in the spinal cord regulates neuropathic pain induced by HIV gp120 application in rats

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    Ouyang Handong

    2011-05-01

    Full Text Available Abstract Background HIV-associated sensory neuropathy (HIV-SN is one of the most common forms of peripheral neuropathy, affecting about 30% of people with acquired immune deficiency syndrome (AIDS. The symptoms of HIV-SN are dominated by neuropathic pain. Glia activation in the spinal cord has become an attractive target for attenuating chronic pain. This study will investigate the role of spinal TNFα released from glia in HIV-related neuropathic pain. Results Peripheral gp120 application into the rat sciatic nerve induced mechanical allodynia for more than 7 weeks, and upregulated the expression of spinal TNFα in the mRNA and the protein levels at 2 weeks after gp120 application. Spinal TNFα was colocalized with GFAP (a marker of astrocytes and Iba1 (a marker of microglia in immunostaining, suggesting that glia produce TNFα in the spinal cord in this model. Peripheral gp120 application also increased TNFα in the L4/5 DRG. Furthermore, intrathecal administration of TNFα siRNA or soluble TNF receptor reduced gp120 application-induced mechanical allodynia. Conclusions Our results indicate that TNFα in the spinal cord and the DRG are involved in neuropathic pain, following the peripheral HIV gp120 application, and that blockade of the glial product TNFα reverses neuropathic pain induced by HIV gp120 application.

  17. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice

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    Kesby, James P.; Hubbard, David T.; Markou, Athina; Semenova, Svetlana

    2012-01-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  18. Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120

    International Nuclear Information System (INIS)

    Wang Zhuying; Pekarskaya, Olga; Bencheikh, Meryem; Chao Wei; Gelbard, Harris A.; Ghorpade, Anuja; Rothstein, Jeffrey D.; Volsky, David J.

    2003-01-01

    L-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V max for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40-70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-α (TNF-α) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-α production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease

  19. The Protective Effects of IGF-1 on Different Subpopulations of DRG Neurons with Neurotoxicity Induced by gp120 and Dideoxycytidine In Vitro.

    Science.gov (United States)

    Lu, Lin; Dong, Haixia; Liu, Guixiang; Yuan, Bin; Li, Yizhao; Liu, Huaxiang

    2014-11-01

    Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC (50 μmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC (50 μmol/L) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (>25 μm), whereas ddC mainly affected small diameter DRG neurons (≤25 μm). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies.

  20. Cognitive deficits associated with combined HIV gp120 expression and chronic methamphetamine exposure in mice

    Science.gov (United States)

    Kesby, James P.; Markou, Athina; Semenova, Svetlana

    2014-01-01

    Methamphetamine abuse is common among individuals infected by human immunodeficiency virus (HIV). Neurocognitive outcomes tend to be worse in methamphetamine users with HIV. However, it is unclear whether discrete cognitive domains are susceptible to impairment after combined HIV infection and methamphetamine abuse. The expression of HIV/gp120 protein induces neuropathology in mice similar to HIV-induced pathology in humans. We investigated the separate and combined effects of methamphetamine exposure and gp120 expression on cognitive function in transgenic (gp120-tg) and control mice. The mice underwent an escalating methamphetamine binge regimen and were tested in novel object/location recognition, object-in-place recognition, and Barnes maze tests. gp120 expression disrupted performance in the object-in-place test (i.e., similar time spent with all objects, regardless of location), indicating deficits in associative recognition memory. gp120 expression also altered reversal learning in the Barnes maze, suggesting impairments in executive function. Methamphetamine exposure impaired spatial strategy in the Barnes maze, indicating deficits in spatial learning. Methamphetamine-exposed gp120-tg mice had the lowest spatial strategy scores in the final acquisition trials in the Barnes maze, suggesting greater deficits in spatial learning than all of the other groups. Although HIV infection involves interactions between multiple proteins and processes, in addition to gp120, our findings in gp120-tg mice suggest that humans with the dual insult of HIV infection and methamphetamine abuse may exhibit a broader spectrum of cognitive deficits than those with either factor alone. Depending on the cognitive domain, the combination of both insults may exacerbate deficits in cognitive performance compared with each individual insult. PMID:25476577

  1. Curcumin improves synaptic plasticity impairment induced by HIV-1gp120 V3 loop

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    Ling-ling Shen

    2015-01-01

    Full Text Available Curcumin has been shown to significantly improve spatial memory impairment induced by HIV-1 gp120 V3 in rats, but the electrophysiological mechanism remains unknown. Using extracellular microelectrode recording techniques, this study confirmed that the gp120 V3 loop could suppress long-term potentiation in the rat hippocampal CA1 region and synaptic plasticity, and that curcumin could antagonize these inhibitory effects. Using a Fura-2/AM calcium ion probe, we found that curcumin resisted the effects of the gp120 V3 loop on hippocampal synaptosomes and decreased Ca 2+ concentration in synaptosomes. This effect of curcumin was identical to nimodipine, suggesting that curcumin improved the inhibitory effects of gp120 on synaptic plasticity, ameliorated damage caused to the central nervous system, and might be a potential neuroprotective drug.

  2. Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

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    Lijun Chao

    Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

  3. Conserved region at the COOH terminus of human immunodeficiency virus gp120 envelope protein contains an immunodominant epitope

    International Nuclear Information System (INIS)

    Palker, T.J.; Matthews, T.J.; Clark, M.E.

    1987-01-01

    A highly immunogenic epitope from a conserved COOH-terminal region of the human immunodeficiency virus (HIV) gp120 envelope protein has been identified with antisera from HIV-seropositive subjects and a synthetic peptide (SP-22) containing 15 amino acids from this region (Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg). Peptide SP-22 absorbed up to 100% of anti-gp120 antibody reactivity from select HIV + patient sera in immunoblot assays and up to 79% of serum anti-gp120 antibody reactivity in competition RIA. In RIA, 45% of HIV-seropositive subjects had antibodies that bound to peptide SP-22. Human anti-SP-22 antibodies that bound to and were eluted from an SP-22 affinity column reacted with gp120 in RIA and immunoblot assays but did not neutralize HIV or inhibit HIV-induced syncytium formation in vitro, even though these antibodies comprised 70% of all anti-gp120 antibodies in the test serum. In contrast, the remaining 30% of SP-22 nonreactive anti-gp120 antibodies did not react with gp120 in immunoblot assays but did react in RIA and neutralized HIV in vitro. Thus, ≅ 50% of HIV-seropositive patients make high titers of nonneutralizing antibodies to an immunodominant antigen on gp120 defined by SP-22. Moreover, the COOH terminus of gp120 contains the major antigen or antigens identified by human anti-gp120 antibodies in immunoblot assays

  4. 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

  5. Cloning, expression and preliminary crystallographic analysis of the equine infectious anaemia virus (EIAV) gp45 ectodomain

    International Nuclear Information System (INIS)

    Sun, Pei-Long; Lv, Shu-Xia; Zhou, Jian-Hua; Liu, Xin-Qi

    2011-01-01

    The equine infectious anaemia virus gp45 ectodomain was cloned, expressed and crystallized. Preliminary crystallographic analysis showed that the protein belonged to space group P6 3 and contained one molecule per asymmetric unit. Like human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV) belongs to the lentivirus genus. The first successful lentiviral vaccine was developed for EIAV. Thus, EIAV may serve as a valuable model for HIV vaccine research. EIAV glycoprotein 45 (gp45) plays a similar role to gp41 in HIV by mediating virus–host membrane fusion. The gp45 ectodomain was constructed according to the structure of HIV gp41, with removal of the disulfide-bond loop region. The protein was expressed in Escherichia coli and crystallized following purification. However, most of the crystals grew as aggregates and could not be used for data collection. By extensively screening hundreds of crystals, a 2.7 Å resolution data set was collected from a single crystal. The crystal belonged to space group P6 3 , with unit-cell parameters a = b = 46.84, c = 101.61 Å, α = β = 90, γ = 120°. Molecular replacement was performed using the coordinates of various lengths of HIV gp41 as search models. A long bent helix was identified and a well defined electron-density map around the long helix was obtained. This primary model provided the starting point for further refinement

  6. Structural improvement of unliganded simian immunodeficiency virus gp120 core by normal-mode-based X-ray crystallographic refinement

    International Nuclear Information System (INIS)

    Chen, Xiaorui; Lu, Mingyang; Poon, Billy K.; Wang, Qinghua; Ma, Jianpeng

    2009-01-01

    The structural model of the unliganded and fully glycosylated simian immunodeficiency virus gp120 core determined to 4.0 Å resolution was substantially improved using a recently developed normal-mode-based anisotropic B-factor refinement method. The envelope protein gp120/gp41 of simian and human immunodeficiency viruses plays a critical role in viral entry into host cells. However, the extraordinarily high structural flexibility and heavy glycosylation of the protein have presented enormous difficulties in the pursuit of high-resolution structural investigation of some of its conformational states. An unliganded and fully glycosylated gp120 core structure was recently determined to 4.0 Å resolution. The rather low data-to-parameter ratio limited refinement efforts in the original structure determination. In this work, refinement of this gp120 core structure was carried out using a normal-mode-based refinement method that has been shown in previous studies to be effective in improving models of a supramolecular complex at 3.42 Å resolution and of a membrane protein at 3.2 Å resolution. By using only the first four nonzero lowest-frequency normal modes to construct the anisotropic thermal parameters, combined with manual adjustments and standard positional refinement using REFMAC5, the structural model of the gp120 core was significantly improved in many aspects, including substantial decreases in R factors, better fitting of several flexible regions in electron-density maps, the addition of five new sugar rings at four glycan chains and an excellent correlation of the B-factor distribution with known structural flexibility. These results further underscore the effectiveness of this normal-mode-based method in improving models of protein and nonprotein components in low-resolution X-ray structures

  7. Validating glycoprotein non-metastatic melanoma B (gpNMB, osteoactivin), a new biomarker of Gaucher disease.

    Science.gov (United States)

    Murugesan, Vagishwari; Liu, Jun; Yang, Ruhua; Lin, Haiquin; Lischuk, Andrew; Pastores, Gregory; Zhang, Xiaokui; Chuang, Wei-Lien; Mistry, Pramod K

    2018-02-01

    In the spleens of Gaucher disease mice and patients, there is a striking elevation of expression of glycoprotein non-Metastatic Melanoma B (gpNMB). We conducted a study in a large cohort of patients with Gaucher disease to assess the utility of serum levels of soluble fragment of gpNMB as a biomarker of disease activity. There was >15-fold elevation of gpNMB in sera of untreated patients with Gaucher disease. gpNMB levels correlated with overall disease severity as well as the severity of individual organ compartments: liver, spleen, bone and hematological disease. Imiglucerase enzyme replacement therapy resulted in significant reduction of gpNMB. Serum levels of gpNMB were highly correlated with accumulation of bioactive lipid substrate of Gaucher disease, glucosylsphingosine as well as established biomarkers, chitotriosidase and chemokine, CCL18. Our results suggest utility of gpNMB as a biomarker of Gaucher disease to monitor individual patients and cohorts of patients for disease progression or response to therapy. Investigation of gpNMB in Gaucher disease pathophysiology is likely to illuminate our understanding disease mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Antioxidant enzyme gene delivery to protect from HIV-1 gp120-induced neuronal apoptosis.

    Science.gov (United States)

    Agrawal, L; Louboutin, J-P; Reyes, B A S; Van Bockstaele, E J; Strayer, D S

    2006-12-01

    Human immunodeficiency virus-1 (HIV-1) infection in the central nervous system (CNS) may lead to neuronal loss and progressively deteriorating CNS function: HIV-1 gene products, especially gp120, induce free radical-mediated apoptosis. Reactive oxygen species (ROS), are among the potential mediators of these effects. Neurons readily form ROS after gp120 exposure, and so might be protected from ROS-mediated injury by antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD1) and/or glutathione peroxidase (GPx1). Both enzymes detoxify oxygen free radicals. As they are highly efficient gene delivery vehicles for neurons, recombinant SV40-derived vectors were used for these studies. Cultured mature neurons derived from NT2 cells and primary fetal neurons were transduced with rSV40 vectors carrying human SOD1 and/or GPx1 cDNAs, then exposed to gp120. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Transduction efficiency of both neuron populations was >95%, as assayed by immunostaining. Transgene expression was also ascertained by Western blotting and direct assays of enzyme activity. Gp120 induced apoptosis in a high percentage of unprotected NT2-N. Transduction with SV(SOD1) and SV(GPx1) before gp120 challenge reduced neuronal apoptosis by >90%. Even greater protection was seen in cells treated with both vectors in sequence. Given singly or in combination, they protect neuronal cells from HIV-1-gp120 induced apoptosis. We tested whether rSV40 s can deliver antioxidant enzymes to the CNS in vivo: intracerebral injection of SV(SOD1) or SV(GPx1) into the caudate putamen of rat brain yielded excellent transgene expression in neurons. In vivo transduction using SV(SOD1) also protected neurons from subsequent gp120-induced apoptosis after injection of both into the caudate putamen of rat brain. Thus, SOD1 and GPx1 can be delivered by SV40 vectors in vitro or in vivo. This approach may merit consideration for

  9. Surfactant protein D binds to human immunodeficiency virus (HIV) envelope protein gp120 and inhibits HIV replication

    DEFF Research Database (Denmark)

    Meschi, Joseph; Crouch, Erika C; Skolnik, Paul

    2005-01-01

    The envelope protein (gp120) of human immunodeficiency virus (HIV) contains highly conserved mannosylated oligosaccharides. These glycoconjugates contribute to resistance to antibody neutralization, and binding to cell surface lectins on macrophages and dendritic cells. Mannose-binding lectin (MBL......) binds to gp120 and plays a role in defence against the virus. In this study it is demonstrated that surfactant protein D (SP-D) binds to gp120 and inhibits HIV infectivity at significantly lower concentrations than MBL. The binding of SP-D was mediated by its calcium-dependent carbohydrate......-binding activity and was dependent on glycosylation of gp120. Native dodecameric SP-D bound to HIV gp120 more strongly than native trimeric SP-D. Since one common polymorphic form of SP-D is predominantly expressed as trimers and associated with lower blood levels, these individuals may have less effective innate...

  10. Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

    Science.gov (United States)

    Liao, Hua-Xin; Tomaras, Georgia D.; Bonsignori, Mattia; Tsao, Chun-Yen; Hwang, Kwan-Ki; Chen, Haiyan; Lloyd, Krissey E.; Bowman, Cindy; Sutherland, Laura; Jeffries, Thomas L.; Kozink, Daniel M.; Stewart, Shelley; Anasti, Kara; Jaeger, Frederick H.; Parks, Robert; Yates, Nicole L.; Overman, R. Glenn; Sinangil, Faruk; Berman, Phillip W.; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Karasavva, Nicos; Rerks-Ngarm, Supachai; Kim, Jerome H.; Michael, Nelson L.; Zolla-Pazner, Susan; Santra, Sampa; Letvin, Norman L.; Harrison, Stephen C.

    2013-01-01

    An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity. PMID:23175357

  11. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Directory of Open Access Journals (Sweden)

    Beuy Joob

    2014-12-01

    Full Text Available The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present. Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus. The in-depth analysis of the viral protein to find the binding site, active pocket is needed. Here, the authors analyzed the envelope glycoprotein GP2 from Ebola virus. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus was done. According to this assessment, 7 active pockets with varied druggability could be identified.

  12. Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

    International Nuclear Information System (INIS)

    Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

    2016-01-01

    Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4. (paper)

  13. Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

    Science.gov (United States)

    Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

    2016-10-01

    Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4.

  14. Marburg Virus Glycoprotein GP2: pH-Dependent Stability of the Ectodomain α-Helical Bundle†

    Science.gov (United States)

    Harrison, Joseph S.; Koellhoffer, Jayne F.; Chandran, Kartik; Lai, Jonathan R.

    2012-01-01

    Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV is required for fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔGunf H2O, of 33.4 ± 2.5 kcal/mol and melting temperature, Tm, of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH with higher stability under lower pH conditions (Tm values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. Based on these results, we hypothesize that pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism to control conformational preferences such that the six-helix bundle ‘post-fusion’ state is preferred under conditions of appropriately matured endosomes. PMID:22369502

  15. CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

    International Nuclear Information System (INIS)

    Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael; Ellett, Anne; Farrugia, William; Wesselingh, Steven L.; Cunningham, Anthony L.; Ramsland, Paul A.; Gorry, Paul R.

    2011-01-01

    CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

  16. Indirect detection of an epitope-specific response to HIV-1 gp120 immunization in human subjects.

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

    Full Text Available A specific response of human serum neutralizing antibodies (nAb to a conformational epitope as a result of vaccination of human subjects with the surface envelope glycoprotein (gp120 of HIV-1 has not previously been documented. Here, we used computational analysis to assess the epitope-specific responses of human subjects, which were immunized with recombinant gp120 immunogens in the VAX003 and VAX004 clinical trials. Our computational methodology--a variation of sieve analysis--compares the occurrence of specific nAb targeted conformational 3D epitopes on viruses from infected individuals who received vaccination to the occurrence of matched epitopes in the viruses infecting placebo subjects. We specifically studied seven crystallographically defined nAb targeted conformational epitopes in the V3 loop, an immunogenic region of gp120. Of the six epitopes present in the immunogens and targeted by known monoclonal neutralizing antibodies, only the one targeted by the anti-V3 nAb 2219 exhibited a significant reduction in occurrence in vaccinated subjects compared to the placebo group. This difference occurred only in the VAX003 Thailand cohort. No difference was seen between vaccinated and placebo groups for the occurrence of an epitope that was not present in the immunogen. Thus, it can be theorized that a specific 2219-like human neutralizing antibody immune response to AIDSVAX immunization occurred in the VAX003 cohort, and that this response protected subjects from a narrow subset of HIV-1 viruses circulating in Thailand in the 1990s and bearing the conformational epitope targeted by the neutralizing antibody 2219.

  17. Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

    Directory of Open Access Journals (Sweden)

    Wang Bin

    2007-12-01

    Full Text Available Abstract Background CCR5-restricted (R5 human immunodeficiency virus type 1 (HIV-1 variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA and AIDS (A R5 Envs, respectively. Results Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362, a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. Conclusion Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.

  18. Human immunodeficiency virus envelope protein Gp120 induces proliferation but not apoptosis in osteoblasts at physiologic concentrations.

    Directory of Open Access Journals (Sweden)

    Nathan W Cummins

    Full Text Available Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05, which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism.

  19. Systematic Protein-Protein Docking and Molecular Dynamics Studies of HIV-1 gp120 and CD4: Insights for New Drug Development

    Directory of Open Access Journals (Sweden)

    M. Rizman-Idid

    2011-12-01

    Full Text Available Background and the purpose of the study: The interactions between HIV-1 gp120 and mutated CD4 proteins were investigated in order to identify a lead structure for therapy based on competitive blocking of the HIV binding receptor for human T-cells. Crystal structures of HIV gp120-CD4 complexes reveal a close interaction of the virus receptor with CD4 Phe43, which is embedded in a pocket of the virus protein.Methods: This study applies computer simulations to determine the best binding of amino acid 43 CD4 mutants to HIV gp120. Besides natural CD4, three mutants carrying alternate aromatic residues His, Trp and Tyr at position 43 were investigated. Several docking programs were applied on isolated proteins based on selected crystal structures of gp120-CD4 complexes, as well as a 5 ns molecular dynamics study on the protein complexes. The initial structures were minimized in Gromacs to avoid crystal packing effects, and then subjected to docking experiments using AutoDock4, FireDock, ClusPro and ZDock. In molecular dynamics, the Gibbs free binding energy was calculated for the gp120-CD4 complexes. The docking outputs were analyzed on energy within the respective docking software.Results and conclusion: Visualization and hydrogen bonding analysis were performed using the Swiss-PdbViewer. Strong binding to HIV gp120 can be achieved with an extended aromatic group (Trp. However, the sterical demand of the interaction affects the binding kinetics. In conclusion, a ligand for an efficient blocking of HIV gp120 should involve an extended but conformational flexible aromatic group, i.e. a biphenyl. A docking study on biphenylalanine-43 confirms this expectation

  20. Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4

    DEFF Research Database (Denmark)

    Gram, G J; Hemming, A; Bolmstedt, A

    1994-01-01

    Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation...... in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor...... affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by Con...

  1. Investigation of the function of the putative self-association site of Epstein-Barr virus (EBV) glycoprotein 42 (gp42)

    International Nuclear Information System (INIS)

    Rowe, Cynthia L.; Matsuura, Hisae; Jardetzky, Theodore S.; Longnecker, Richard

    2011-01-01

    The Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a type II membrane protein essential for entry into B cells but inhibits entry into epithelial cells. X-ray crystallography suggests that gp42 may form dimers when bound to human leukocyte antigen (HLA) class II receptor (Mullen et al., 2002) or multimerize when not bound to HLA class II (Kirschner et al., 2009). We investigated this self-association of gp42 using several different approaches. We generated soluble mutants of gp42 containing mutations within the self-association site and found that these mutants have a defect in fusion. The gp42 mutants bound to gH/gL and HLA class II, but were unable to bind wild-type gp42 or a cleavage mutant of gp42. Using purified gp42, gH/gL, and HLA, we found these proteins associate 1:1:1 by gel filtration suggesting that gp42 dimerization or multimerization does not occur or is a transient event undetectable by our methods.

  2. Human antibody response to a strain-specific HIV-1 gp120 epitope associated with cell fusion inhibition

    NARCIS (Netherlands)

    Goudsmit, J.; Boucher, C. A.; Meloen, R. H.; Epstein, L. G.; Smit, L.; van der Hoek, L.; Bakker, M.

    1988-01-01

    PEPSCAN analysis, performed using 536 overlapping nonapeptides derived from the HTLV-III B nucleotide sequence of the region encoding the external envelope protein of 120 kDa (gp120), identified in the V3 region of gp120 a major binding site for antibodies of HIV-1-infected humans. The minimal amino

  3. HIV gp120 binds to mannose receptor on vaginal epithelial cells and induces production of matrix metalloproteinases.

    Directory of Open Access Journals (Sweden)

    Sashaina E Fanibunda

    Full Text Available BACKGROUND: During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs downstream of HIV gp120 binding to hMR. PRINCIPAL FINDINGS: Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody. CONCLUSION: hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the

  4. Mimicking protein-protein interactions through peptide-peptide interactions: HIV-1 gp120 and CXCR4

    Directory of Open Access Journals (Sweden)

    Andrea eGross

    2013-09-01

    Full Text Available We have recently designed a soluble synthetic peptide that functionally mimics the HIV-1 coreceptor CXCR4, which is a chemokine receptor that belongs to the family of seven-transmembrane GPCRs. This CXCR4 mimetic peptide, termed CX4-M1, presents the three extracellular loops (ECLs of the receptor. In binding assays involving recombinant proteins, as well as in cellular infection assays, CX4-M1 was found to selectively recognize gp120 from HIV-1 strains that use CXCR4 for cell entry (X4 tropic HIV-1. Furthermore, anti-HIV-1 antibodies modulate this interaction in a molecular mechanism related to that of their impact on the gp120-CXCR4 interaction. We could now show that the selectivity of CX4-M1 pertains not only to gp120 from X4 tropic HIV-1, but also to synthetic peptides presenting the V3 loops of these gp120 proteins. The V3 loop is thought to be an essential part of the coreceptor binding site of gp120 that contacts the second ECL of the coreceptor. We were able to experimentally confirm this notion in binding assays using substitution analogs of CX4-M1 and the V3 loop peptides, respectively, as well as in cellular infection assays. These results indicate that interactions of the HIV-1 Env with coreceptors can be mimicked by synthetic peptides, which may be useful to explore these interactions at the molecular level in more detail.

  5. A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics.

    Science.gov (United States)

    Saaby, Lasse; Brodin, Birger

    2017-09-01

    Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate kinetic parameters, such as K m and V max , for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC 50 ) and the disassociation constant for an inhibitor/P-gp complex (K i ) have been determined to estimate P-gp inhibition. This review addresses in vitro methods commonly used to study P-gp transport kinetics and aims at providing a critical evaluation of the application of steady-state Michaelis-Menten analysis of kinetic parameters for substrate/P-gp interactions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  6. Antiviral Activity of HIV gp120 Targeting Bispecific T Cell Engager (BiTE®) Antibody Constructs.

    Science.gov (United States)

    Brozy, Johannes; Schlaepfer, Erika; Mueller, Christina K S; Rochat, Mary-Aude; Rampini, Silvana K; Myburgh, Renier; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A; Muenz, Markus; Speck, Roberto F

    2018-05-02

    Today's gold standard in HIV therapy is the combined antiretroviral therapy (cART). It requires strict adherence by patients and life-long medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency, but cannot cure patients. The bispecific T cell engaging (BiTE®) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. We here generated BiTE® antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1+2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ϵ scFv. These engineered human BiTE® antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in PBMCs as well as in macrophages co-cultured with autologous CD8+ T-cells, the most potent being the human CD4(1+2) BiTE® antibody construct and the CD4(1+2)L17b BiTE® antibody construct. The CD4(1+2) h BiTE® antibody construct promoted HIV infection of human CD4-/CD8+ T cells. In contrast, the neutralizing B12 and the VRC01 BiTE® antibody constructs as well as the CD4(1+2)L17b BiTE® antibody construct did not. Thus, BiTE® antibody constructs targeting HIV gp120 are very promising for constraining HIV and warrant further development as novel antiviral therapy with curative potential. Importance HIV is a chronic infection well controlled with the current cART. However, we lack cure of HIV, and the HIV pandemic goes on. Here we showed in vitro and ex vivo t hat a bispecific T-cell engaging (BiTE®) antibody construct targeting HIV gp120 resulted in substantially reduced HIV replication. In addition, these BiTE® antibody constructs display efficient killing of gp120 expressing cells and inhibited replication in ex vivo

  7. Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

    Science.gov (United States)

    deCamp, Allan C; Rolland, Morgane; Edlefsen, Paul T; Sanders-Buell, Eric; Hall, Breana; Magaret, Craig A; Fiore-Gartland, Andrew J; Juraska, Michal; Carpp, Lindsay N; Karuna, Shelly T; Bose, Meera; LePore, Steven; Miller, Shana; O'Sullivan, Annemarie; Poltavee, Kultida; Bai, Hongjun; Dommaraju, Kalpana; Zhao, Hong; Wong, Kim; Chen, Lennie; Ahmed, Hasan; Goodman, Derrick; Tay, Matthew Z; Gottardo, Raphael; Koup, Richard A; Bailer, Robert; Mascola, John R; Graham, Barney S; Roederer, Mario; O'Connell, Robert J; Michael, Nelson L; Robb, Merlin L; Adams, Elizabeth; D'Souza, Patricia; Kublin, James; Corey, Lawrence; Geraghty, Daniel E; Frahm, Nicole; Tomaras, Georgia D; McElrath, M Juliana; Frenkel, Lisa; Styrchak, Sheila; Tovanabutra, Sodsai; Sobieszczyk, Magdalena E; Hammer, Scott M; Kim, Jerome H; Mullins, James I; Gilbert, Peter B

    2017-01-01

    Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

  8. The Ixodes scapularis Salivary Protein, Salp15, Prevents the Association of HIV-1 gp120 and CD4

    OpenAIRE

    Juncadella, Ignacio J.; Garg, Renu; Bates, Tonya C.; Olivera, Elias R.; Anguita, Juan

    2007-01-01

    Ixodes scapularis salivary protein, Salp15, inhibits CD4+ T cell activation by binding to the most-extracellular domains of the CD4 molecule, potentially overlapping with the gp120-binding region. We now show that Salp15 inhibits the interaction of gp120 and CD4. Furthermore, Salp15 prevents syncytia formation between HL2/3 (a stable HeLa cell line expressing the envelope protein) and CD4-expressing cells. Salp15 prevented gp120-CD4 interaction at least partially through its direct interactio...

  9. HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Pasquinelli Gianandrea

    2011-05-01

    Full Text Available Abstract Background HIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs. Results MSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPARγ activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context. Conclusions The HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients.

  10. Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4

    DEFF Research Database (Denmark)

    Gram, G J; Hemming, A; Bolmstedt, A

    1994-01-01

    affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by Con...... in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor...

  11. HIV-1 gp120 Upregulates Brain-Derived Neurotrophic Factor (BDNF) Expression in BV2 Cells via the Wnt/β-Catenin Signaling Pathway.

    Science.gov (United States)

    Wang, Yongdi; Liao, Jinxu; Tang, Shao-Jun; Shu, Jianhong; Zhang, Wenping

    2017-06-01

    HIV-1 gp120 plays a critical role in the pathogenesis of HIV-associated pain, but the underlying molecular mechanisms are incompletely understood. This study aims to determine the effect and possible mechanism of HIV-1 gp120 on BDNF expression in BV2 cells (a murine-derived microglial cell line). We observed that gp120 (10 ng/ml) activated BV2 cells in cultures and upregulated proBDNF/mBDNF. Furthermore, gp120-treated BV2 also accumulated Wnt3a and β-catenin, suggesting the activation of the Wnt/β-catenin pathway. We demonstrated that activation of the pathway by Wnt3a upregulated BDNF expression. In contrast, inhibition of the Wnt/β-catenin pathway by either DKK1 or IWR-1 attenuated BDNF upregulation induced by gp120 or Wnt3a. These findings collectively suggest that gp120 stimulates BDNF expression in BV2 cells via the Wnt/β-catenin signaling pathway.

  12. A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics

    DEFF Research Database (Denmark)

    Saaby, Lasse; Brodin, Birger

    2017-01-01

    Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties...... and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may...... mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate...

  13. iP-gp , a novel cell line with tight barrier function and expression of human P-glycoprotein (ABCB1) for drug screening

    DEFF Research Database (Denmark)

    Brodin, Birger; Ozgür, Burak; Saaby, Lasse

    that new API's are evaluated with respect to P-gp interactions.  Aim : The aim of the present work was to validate the suitability of the newly developed iP-gp cell line for investigating P-gp interactions with human P-gp. Methods: IPEC-J2 MDR1 (iP-gp) cells were cultured on permeable supports for 17......Background : The efflux transporter P-glycoprotein (P-gp, product of the MDR1/ABCB1 gene) hinders uptake of drug compounds to the brain, limits intestinal uptake, is a cause of resistance to chemoterapeutics and a potential "site" for drug-drug interaction. Regulatory agencies therefore recommend.......04 +/- 0.01 µM in transport experiments including digoxin and rhodamine 123, respectively. Summary/Conclusion : The iP-gp cell line may become a useful screening tool for interactions between drug compounds and human P-gp....

  14. Limonene reduces hyperalgesia induced by gp120 and cytokines by modulation of IL-1 β and protein expression in spinal cord of mice.

    Science.gov (United States)

    Piccinelli, Ana Claudia; Morato, Priscila Neder; Dos Santos Barbosa, Marcelo; Croda, Julio; Sampson, Jared; Kong, Xiangpeng; Konkiewitz, Elisabete Castelon; Ziff, Edward B; Amaya-Farfan, Jaime; Kassuya, Cândida Aparecida Leite

    2017-04-01

    We have investigated the antihyperalgesic effects of limonene in mice that received intrathecal injection of gp120. Male Swiss mice received gp120, IL-1β or TNF-α intrathecally or sterile saline as a control. A mechanical sensitivity test was performed at 2 and 3h after the injection. Spinal cord and blood samples were isolated for protein quantification. Intrathecal administration of gp120 increased mechanical sensitivity measured with an electronic Von Frey apparatus, at 2 and 3h after the injections. Limonene administered orally prior to gp120 administration significantly decreased this mechanical sensitivity at 3h after the gp120 injection. In addition, intrathecal injection of gp120 increased IL-1β and IL-10 in serum, and limonene prevented the ability of gp120 to increase these cytokines. Limonene also inhibited TNF-α and IL-1β-induced mechanical hyperalgesia. Western blot assay demonstrated limonene was capable of increasing SOD expression in the cytoplasm of cells from spinal cord at 4h after intrathecal IL-1β injection. These results demonstrate that gp120 causes mechanical hyperalgesia and a peripheral increase in IL-1β and IL-10, and that prior administration of limonene inhibits these changes. Also limonene modulates the activation of SOD expression in the spinal cord after spinal IL-1β application. The ability of limonene to inhibit the mechanical hyperalgesia induced by gp120, TNF-α and IL-1β emphasizes the anti-inflammatory action of limonene, specifically its ability to inhibit cytokine production and its consequences. Copyright © 2016. Published by Elsevier Inc.

  15. Functional, non-clonal IgMa-restricted B cell receptor interactions with the HIV-1 envelope gp41 membrane proximal external region.

    Directory of Open Access Journals (Sweden)

    Laurent Verkoczy

    Full Text Available The membrane proximal external region (MPER of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR. To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (approximately 7% fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgM(hi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (approximately 15% of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igh(a (BALB/c and Igh(b (C57BL/6 congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igh(a locus. Mapping of MPER gp41 interactions with IgM(a identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc C(H regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgM(a determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses.

  16. Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization.

    Directory of Open Access Journals (Sweden)

    Arthur S Kim

    2014-07-01

    Full Text Available Human antibody 10E8 targets the conserved membrane proximal external region (MPER of envelope glycoprotein (Env subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design.

  17. Conglutinin binds the HIV-1 envelope glycoprotein gp 160 and inhibits its interaction with cell membrane CD4

    DEFF Research Database (Denmark)

    Andersen, Ove; Sørensen, A M; Svehag, S E

    1991-01-01

    The highly glycosylated envelope glycoprotein (gp 160) of human immunodeficiency virus (HIV) interacts with the CD4 molecule present on the membrane of CD4+ cells and is involved in the pathobiology of HIV infection. Lectins bind glycoproteins through non-covalent interactions with specific hexose...... residues. The mammalian C-type lectin bovine conglutinin was examined for its ability to interact with recombinant gp160 (rgp160) produced in vaccinia virus-infected BHK21 cells. Specific binding of conglutinin to rgp160 was demonstrated by ELISA. The interaction of bovine conglutinin with rgp160...... of the binding of rgp160 to the CD4 receptor on CEM 13 cells, as demonstrated by FACS analyses. These results indicate that conglutinin may inhibit the infection with HIV-1 through its interaction with the viral envelope glycoprotein....

  18. Correlation between carbohydrate structures on the envelope glycoprotein gp120 of HIV-1 and HIV-2 and syncytium inhibition with lectins

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C M; Nielsen, C

    1989-01-01

    The binding of 13 different lectins to gp120 partially purified from two HIV-1 isolates and one HIV-2 isolate was studied by in situ staining on electrophoretically separated and electroblotted HIV antigens. The lectins concanavalin A, wheat germ agglutinin, Lens culinaris agglutinin, Vicia faba...

  19. 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

  20. The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

    Science.gov (United States)

    Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

    2008-01-01

    Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

  1. In silico analysis of HIV-1 Env-gp120 reveals structural bases for viral adaptation in growth-restrictive cells

    Directory of Open Access Journals (Sweden)

    Masaru eYokoyama

    2016-02-01

    Full Text Available Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1 envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness.

  2. Molecular docking guided structure based design of symmetrical N,N'-disubstituted urea/thiourea as HIV-1 gp120-CD4 binding inhibitors.

    Science.gov (United States)

    Sivan, Sree Kanth; Vangala, Radhika; Manga, Vijjulatha

    2013-08-01

    Induced fit molecular docking studies were performed on BMS-806 derivatives reported as small molecule inhibitors of HIV-1 gp120-CD4 binding. Comprehensive study of protein-ligand interactions guided in identification and design of novel symmetrical N,N'-disubstituted urea and thiourea as HIV-1 gp120-CD4 binding inhibitors. These molecules were synthesized in aqueous medium using microwave irradiation. Synthesized molecules were screened for their inhibitory ability by HIV-1 gp120-CD4 capture enzyme-linked immunosorbent assay (ELISA). Designed compounds were found to inhibit HIV-1 gp120-CD4 binding in micromolar (0.013-0.247 μM) concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. The bacteria binding glycoprotein salivary agglutinin (SAG/gp340) activates complement via the lectin pathway

    NARCIS (Netherlands)

    Leito, Jelani T. D.; Ligtenberg, Antoon J. M.; van Houdt, Michel; van den Berg, Timo K.; Wouters, Diana

    2011-01-01

    Salivary agglutinin (SAG), also known as gp-340 and Deleted in Malignant Brain Tumours 1, is a glycoprotein that is present in tears, lung fluid and mucosal surfaces along the gastrointestinal tract. It is encoded by the Deleted in Malignant Brain Tumours 1 gene, a member of the Scavenger Receptor

  4. DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

    Science.gov (United States)

    Ribeck, Noah; Saleh, Omar A

    2013-01-01

    The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

  5. The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies.

    Science.gov (United States)

    Brinkmann, Constantin; Nehlmeier, Inga; Walendy-Gnirß, Kerstin; Nehls, Julia; González Hernández, Mariana; Hoffmann, Markus; Qiu, Xiangguo; Takada, Ayato; Schindler, Michael; Pöhlmann, Stefan

    2016-12-15

    The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae, facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism. Filoviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The

  6. Binding of HIV-1 gp120 to DC-SIGN promotes ASK-1-dependent activation-induced apoptosis of human dendritic cells.

    Directory of Open Access Journals (Sweden)

    Yongxiong Chen

    2013-01-01

    Full Text Available During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC, including DC-SIGN⁺ cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN⁺ blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV⁺ serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN⁺ DC that were isolated directly from HIV-1⁺ individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential

  7. DMPD: Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12960231 Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited sign...82. Epub 2003 Jul 22. (.png) (.svg) (.html) (.csml) Show Macrophage activation through CCR5- and CXCR4-media...on through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. Authors Lee C, Liu QH, Tomkowicz B, Yi

  8. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

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    Cátia Teixeira

    Full Text Available HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules.

  9. Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

    NARCIS (Netherlands)

    Sanders, Rogier W.; Vesanen, Mika; Schuelke, Norbert; Master, Aditi; Schiffner, Linnea; Kalyanaraman, Roopa; Paluch, Maciej; Berkhout, Ben; Maddon, Paul J.; Olson, William C.; Lu, Min; Moore, John P.

    2002-01-01

    The envelope glycoprotein (Env) complex of human immunodeficiency virus type I has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41

  10. Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

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    Garry Robert F

    2008-02-01

    Full Text Available Abstract Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G, were established for Thogoto virus (THOV GP and Autographa california multiple NPV (AcMNPV GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes.

  11. Development of Classification Models for Identifying “True” P-glycoprotein (P-gp Inhibitors Through Inhibition, ATPase Activation and Monolayer Efflux Assays

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    Anna Maria Bianucci

    2012-06-01

    Full Text Available P-glycoprotein (P-gp is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as “true” P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

  12. The major surface glycoprotein (gp63) from Leishmania major and Leishmania donovani cleaves CD4 molecules on human T cells

    DEFF Research Database (Denmark)

    Hey, A S; Theander, T G; Hviid, L

    1994-01-01

    The effect of Leishmania major and L. donovani surface protease gp63 on surface markers on human T cells was studied using fluorescence-activated flow cytometry. Purified gp63 (63,000 m.w. glycoprotein) at concentrations above 10 micrograms/ml completely inhibited binding of six different anti-CD4......-expression of CD4, reaching 50% of the initial level after 72 h of incubation in medium. Preincubation of cells with live promastigotes showed an inhibitory effect on CD4 comparable to that seen with purified gp63. The binding of Abs directed against other surface markers present on human T-cells--CD2, CD3, CD5......, CD8, CD11A, CD25, CD45RO, CD45RA, CD58, TCR-alpha, TCR-gamma, and HLA DQ--was not inhibited by gp63. These data suggest that gp63, both in its purified form and in the form anchored to the parasite membrane, cleaves CD4 on human T cells. The cleavage of CD4 by the protease might play a role...

  13. Positive evolution of the glycoprotein (GP) gene is related to transmission of the Ebola virus.

    Science.gov (United States)

    Jing, Y X; Wang, L N; Wu, X M; Song, C X

    2016-03-28

    Ebola hemorrhagic fever is a fatal disease caused by the negative-strand RNA of the Ebola virus. A high-intensity outbreak of this fever was reported in West Africa last year; however, there is currently no definitive treatment strategy available for this disease. In this study, we analyzed the molecular evolutionary history and attempted to determine the positive selection sites in the Ebola genes using multiple-genomic sequences of the various Ebola virus subtypes, in order to gain greater clarity into the evolution of the virus and its various subtypes. Only the glycoprotein (GP) gene was positively selected among the 8 Ebola genes, with the other genes remaining in the purification stage. The positive selection sites in the GP gene were identified by a random-site model; these sites were found to be located in the mucin-like region, which is associated with transmembrane protein binding. Additionally, different branches of the phylogenetic tree displayed different positive sites, which in turn was responsible for differences in the cell adhesion ability of the virus. In conclusion, the pattern of positive sites in the GP gene is associated with the epidemiology and prevalence of Ebola in different areas.

  14. Binding of recombinant HIV coat protein gp120 to human monocytes

    International Nuclear Information System (INIS)

    Finbloom, D.S.; Hoover, D.L.; Meltzer, M.S.

    1991-01-01

    Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication

  15. Characterization of HIV-1 gp120 antibody specificities induced in anogenital secretions of RV144 vaccine recipients after late boost immunizations.

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    Siriwat Akapirat

    Full Text Available Sexual transmission is the principal driver of the human immunodeficiency virus (HIV pandemic. Understanding HIV vaccine-induced immune responses at mucosal surfaces can generate hypotheses regarding mechanisms of protection, and may influence vaccine development. The RV144 (ClinicalTrials.gov NCT00223080 efficacy trial showed protection against HIV infections but mucosal samples were not collected, therefore, the contribution of mucosal antibodies to preventing HIV-1 acquisition is unknown. Here, we report the generation, magnitude and persistence of antibody responses to recombinant gp120 envelope and antigens including variable one and two loop scaffold antigens (gp70V1V2 previously shown to correlate with risk in RV144. We evaluated antibody responses to gp120 A244gD and gp70V1V2 92TH023 (both CRF01_AE and Case A2 (subtype B in cervico-vaginal mucus (CVM, seminal plasma (SP and rectal secretions (RS from HIV-uninfected RV144 vaccine recipients, who were randomized to receive two late boosts of ALVAC-HIV/AIDSVAX®B/E, AIDSVAX®B/E, or ALVAC-HIV alone at 0 and 6 months. Late vaccine boosting increased IgG geometric mean titers (GMT to gp120 A244gD in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM (28 and 17 fold, respectively, followed by SP and RS. IgG to gp70V1V2 92TH023 increased in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM (11-17 fold and SP (2 fold two weeks post first boost. IgG to Case A2 was only detected in AIDSVAX®B/E and ALVAC-HIV/AIDSVAX®B/E CVM. Mucosal IgG to gp120 A244gD (CVM, SP, RS, gp70V1V2 92TH023 (CVM, SP, and Case A2 (CVM correlated with plasma IgG levels (p<0.001. Although the magnitude of IgG responses declined after boosting, anti-gp120 A244gD IgG responses in CVM persisted for 12 months post final vaccination. Further studies in localization, persistence and magnitude of envelope specific antibodies (IgG and dimeric IgA in anogenital secretions will help determine their role in preventing mucosal HIV acquisition.

  16. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail

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    Takeo eKuwata

    2013-05-01

    Full Text Available The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1 infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of nonhuman primates with simian immunodeficiency virus (SIV is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7900-fold, 1000-fold, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody.

  17. Comprehensive functional analysis of N-linked glycans on Ebola virus GP1.

    Science.gov (United States)

    Lennemann, Nicholas J; Rhein, Bethany A; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

    2014-01-28

    Ebola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca(2+)-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency. Filovirus outbreaks occur sporadically throughout central Africa, causing high fatality rates among the general public and health care workers. These unpredictable hemorrhagic fever outbreaks are caused by multiple species of Ebola viruses, as well as Marburg virus. While filovirus

  18. P2Y12 receptor upregulation in satellite glial cells is involved in neuropathic pain induced by HIV glycoprotein 120 and 2',3'-dideoxycytidine.

    Science.gov (United States)

    Yi, Zhihua; Xie, Lihui; Zhou, Congfa; Yuan, Huilong; Ouyang, Shuai; Fang, Zhi; Zhao, Shanhong; Jia, Tianyu; Zou, Lifang; Wang, Shouyu; Xue, Yun; Wu, Bing; Gao, Yun; Li, Guilin; Liu, Shuangmei; Xu, Hong; Xu, Changshui; Zhang, Chunping; Liang, Shangdong

    2018-03-01

    The direct neurotoxicity of HIV and neurotoxicity of combination antiretroviral therapy medications both contribute to the development of neuropathic pain. Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in mechanical and thermal hyperalgesia. The P2Y 12 receptor expressed in SGCs of the DRG is involved in pain transmission. In this study, we explored the role of the P2Y 12 receptor in neuropathic pain induced by HIV envelope glycoprotein 120 (gp120) combined with ddC (2',3'-dideoxycytidine). A rat model of gp120+ddC-induced neuropathic pain was used. Peripheral nerve exposure to HIV-gp120+ddC increased mechanical and thermal hyperalgesia in gp120+ddC-treated model rats. The gp120+ddC treatment increased expression of P2Y 12 receptor mRNA and protein in DRG SGCs. In primary cultured DRG SGCs treated with gp120+ddC, the levels of [Ca 2+ ] i activated by the P2Y 12 receptor agonist 2-(Methylthio) adenosine 5'-diphosphate trisodium salt (2-MeSADP) were significantly increased. P2Y 12 receptor shRNA treatment inhibited 2-MeSADP-induced [Ca 2+ ] i in primary cultured DRG SGCs treated with gp120+ddC. Intrathecal treatment with a shRNA against P2Y 12 receptor in DRG SGCs reduced the release of pro-inflammatory cytokines, decreased phosphorylation of p38 MAPK in the DRG of gp120+ddC-treated rats. Thus, downregulating the P2Y 12 receptor relieved mechanical and thermal hyperalgesia in gp120+ddC-treated rats.

  19. Prime-Boost Vaccination Using Chemokine-Fused gp120 DNA and HIV Envelope Peptides Activates Both Immediate and Long-Term Memory Cellular Responses in Rhesus Macaques

    Directory of Open Access Journals (Sweden)

    Hong Qin

    2010-01-01

    Full Text Available HIV vaccine candidates with improved immunogenicity and induction of mucosal T-cell immunity are needed. A prime-boost strategy using a novel HIV glycoprotein 120 DNA vaccine was employed to immunize rhesus macaques. The DNA vaccine encoded a chimeric gp120 protein in fusion with monocyte chemoattractant protein-3, which was hypothesized to improve the ability of antigen-presenting cells to capture viral antigen through chemokine receptor-mediated endocytosis. DNA vaccination induced virus-reactive T cells in peripheral blood, detectable by T cell proliferation, INFγ ELISPOT and sustained IL-6 production, without humoral responses. With a peptide-cocktail vaccine containing a set of conserved polypeptides of HIV-1 envelope protein, given by nasogastric administration, primed T-cell immunity was significantly boosted. Surprisingly, long-term and peptide-specific mucosal memory T-cell immunity was detected in both vaccinated macaques after one year. Therefore, data from this investigation offer proof-of-principle for potential effectiveness of the prime-boost strategy with a chemokine-fused gp120 DNA and warrant further testing in the nonhuman primate models for developing as a potential HIV vaccine candidate in humans.

  20. 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.

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

    International Nuclear Information System (INIS)

    Rusche, J.R.; Lynn, D.L.; Robert-Guroff, M.

    1987-01-01

    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

  2. Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein.

    Science.gov (United States)

    Murphy, R Elliot; Samal, Alexandra B; Vlach, Jiri; Saad, Jamil S

    2017-11-07

    The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Accurate and efficient gp120 V3 loop structure based models for the determination of HIV-1 co-receptor usage

    Directory of Open Access Journals (Sweden)

    Vaisman Iosif I

    2010-10-01

    Full Text Available Abstract Background HIV-1 targets human cells expressing both the CD4 receptor, which binds the viral envelope glycoprotein gp120, as well as either the CCR5 (R5 or CXCR4 (X4 co-receptors, which interact primarily with the third hypervariable loop (V3 loop of gp120. Determination of HIV-1 affinity for either the R5 or X4 co-receptor on host cells facilitates the inclusion of co-receptor antagonists as a part of patient treatment strategies. A dataset of 1193 distinct gp120 V3 loop peptide sequences (989 R5-utilizing, 204 X4-capable is utilized to train predictive classifiers based on implementations of random forest, support vector machine, boosted decision tree, and neural network machine learning algorithms. An in silico mutagenesis procedure employing multibody statistical potentials, computational geometry, and threading of variant V3 sequences onto an experimental structure, is used to generate a feature vector representation for each variant whose components measure environmental perturbations at corresponding structural positions. Results Classifier performance is evaluated based on stratified 10-fold cross-validation, stratified dataset splits (2/3 training, 1/3 validation, and leave-one-out cross-validation. Best reported values of sensitivity (85%, specificity (100%, and precision (98% for predicting X4-capable HIV-1 virus, overall accuracy (97%, Matthew's correlation coefficient (89%, balanced error rate (0.08, and ROC area (0.97 all reach critical thresholds, suggesting that the models outperform six other state-of-the-art methods and come closer to competing with phenotype assays. Conclusions The trained classifiers provide instantaneous and reliable predictions regarding HIV-1 co-receptor usage, requiring only translated V3 loop genotypes as input. Furthermore, the novelty of these computational mutagenesis based predictor attributes distinguishes the models as orthogonal and complementary to previous methods that utilize sequence

  4. Structure and dynamics of the gp120 V3 loop that confers noncompetitive resistance in R5 HIV-1(JR-FL to maraviroc.

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    Yuzhe Yuan

    Full Text Available Maraviroc, an (HIV-1 entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1 from using CCR5 as a coreceptor for entry into CD4(+ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1(V3-M5 derived from HIV-1(JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1(JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i changes in V3 configuration on the gp120 outer domain, (ii reduction of an anti-parallel β-sheet in the V3 stem region, (iii reduction in fluctuations of the V3 tip and stem regions, and (iv a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

  5. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    International Nuclear Information System (INIS)

    Mefford, Megan E.; Kunstman, Kevin; Wolinsky, Steven M.; Gabuzda, Dana

    2015-01-01

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions

  6. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    Energy Technology Data Exchange (ETDEWEB)

    Mefford, Megan E., E-mail: megan_mefford@hms.harvard.edu [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Kunstman, Kevin, E-mail: kunstman@northwestern.edu [Northwestern University Medical School, Chicago, IL (United States); Wolinsky, Steven M., E-mail: s-wolinsky@northwestern.edu [Northwestern University Medical School, Chicago, IL (United States); Gabuzda, Dana, E-mail: dana_gabuzda@dfci.harvard.edu [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Department of Neurology (Microbiology and Immunobiology), Harvard Medical School, Boston, MA (United States)

    2015-07-15

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.

  7. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  8. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

    Science.gov (United States)

    Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

    2010-01-01

    Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

  9. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

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    Joan Joseph

    2010-01-01

    Full Text Available Mycobacterium bovis Bacillus Calmette-Guérin (BCG as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261 and Mycobacteria spp. α-antigen promoter (in plasmid pJH222. Among 14 rBCG:HIV-1gp120 (pMV261 colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222 colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.

  10. HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells

    International Nuclear Information System (INIS)

    Cicala, Claudia; Arthos, James; Censoplano, Nina; Cruz, Catherine; Chung, Eva; Martinelli, Elena; Lempicki, Richard A.; Natarajan, Ven; VanRyk, Donald; Daucher, Marybeth; Fauci, Anthony S.

    2006-01-01

    The replication of human immunodeficiency virus (HIV) in CD4+ T-cells is strongly dependent upon the state of activation of infected cells. Infection of sub-optimally activated cells is believed to play a critical role in both the transmission of virus and the persistence of CD4+ T-cell reservoirs. There is accumulating evidence that HIV can modulate signal-transduction pathways in a manner that may facilitate replication in such cells. We previously demonstrated that HIV gp120 induces virus replication in resting CD4+ T cells isolated from HIV-infected individuals. Here, we show that in resting CD4+ T-cells, gp120 activates NFATs and induces their translocation into the nucleus. The HIV LTR encodes NFAT recognition sites, and NFATs may play a critical role in promoting viral replication in sub-optimally activated cells. These observations provide insight into a potential mechanism by which HIV is able to establish infection in resting cells, which may have implications for both transmission of HIV and the persistence of viral reservoirs

  11. Gp96 Peptide Antagonist gp96-II Confers Therapeutic Effects in Murine Intestinal Inflammation

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    Claudia A. Nold-Petry

    2017-12-01

    Full Text Available BackgroundThe expression of heat shock protein gp96 is strongly correlated with the degree of tissue inflammation in ulcerative colitis and Crohn’s disease, thereby leading us to the hypothesis that inhibition of expression via gp96-II peptide prevents intestinal inflammation.MethodsWe employed daily injections of gp96-II peptide in two murine models of intestinal inflammation, the first resulting from five daily injections of IL-12/IL-18, the second via a single intrarectal application of TNBS (2,4,6-trinitrobenzenesulfonic acid. We also assessed the effectiveness of gp96-II peptide in murine and human primary cell culture.ResultsIn the IL-12/IL-18 model, all gp96-II peptide-treated animals survived until day 5, whereas 80% of placebo-injected animals died. gp96-II peptide reduced IL-12/IL-18-induced plasma IFNγ by 89%, IL-1β by 63%, IL-6 by 43% and tumor necrosis factor (TNF by 70% compared to controls. The clinical assessment Disease Activity Index of intestinal inflammation severity was found to be significantly lower in the gp96-II-treated animals when compared to vehicle-injected mice. gp96-II peptide treatment in the TNBS model limited weight loss to 5% on day 7 compared with prednisolone treatment, whereas placebo-treated animals suffered a 20% weight loss. Histological disease severity was reduced equally by prednisolone (by 40% and gp96-II peptide (35%. Mice treated with either gp96-II peptide or prednisolone exhibited improved endoscopic scores compared with vehicle-treated control mice: vascularity, fibrin, granularity, and translucency scores were reduced by up to 49% by prednisolone and by up to 30% by gp96-II peptide. In vitro, gp96-II peptide reduced TLR2-, TLR4- and IL-12/IL-18-induced cytokine expression in murine splenocytes, with declines in constitutive IL-6 (54%, lipopolysaccharide-induced TNF (48%, IL-6 (81% and in Staphylococcus epidermidis-induced TNF (67% and IL-6 (81%, as well as IL-12/IL-18-induced IFNγ (75%. gp

  12. [Expression and significance of P-gp/mdr1 mRNA, MRP and LRP in non-Hodgkin's lymphoma].

    Science.gov (United States)

    Li, Le; Su, Li-ping; Ma, Li; Zhao, Jin; Zhu, Lei; Zhou, Yong-an

    2009-03-01

    To explore the expression and clinical significance of P-glycoprotein (P-gp)/mdr1mRNA, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in newly diagnosed non-Hodgkin's lymphoma. mdr1 mRNA of in 41 patients with non-Hodgkin's lymphoma was assayed by semi-quantitative RT-PCR. The expressions of P-gp, MRP and LRP proteins in lymph node viable blasts were identified by flow cytometry. The results were compared with those obtained from control cases, and the correlation of the changes with clinical outcomes was analyzed. (1) Among the 41 cases, the positive expression of P-gp protein was detected in 8 cases, MRP in 7 cases, LRP in 15 cases, and mdr 1 mRNA in 11 cases. (2) The P-gp and LRP levels in NHL were significantly higher than those in control group, but MRP wasn't. The P-gp over-expression was significantly associated with mdr1mRNA (r = 0.396, P = 0.01). No correlation was showed among the expressions of P-gp, MRP and LRP. (3) Patients with P-gp expression had a poorer outcome of chemotherapy than those with P-gp-negative (P = 0.005). P-gp expression was significantly associated with higher clinical stage (P = 0.046) and elevated serum lactate dehydrogenase level (P = 0.032), but not associated with malignant degree (P = 0.298). MRP had no impact on the outcome of chemotherapy (P = 0.212), and wasn't significantly associated with higher clinical stage (P = 0.369), elevated LDH (P = 0.762) and higher malignant degree (P = 0.451). Patients with LRP expression had a poorer outcome of chemotherapy than those LRP-negative (P = 0.012). LRP expression was significantly associated with higher clinical stage (P = 0.0019), elevated LDH (P = 0.02) and higher malignant degree (P = 0.01). The data of this study indicate that P-gp and LRP expressions but not MRP expression are important in the mechanism of drug resistance associated with a poor clinical outcome in previously untreated NHL.

  13. Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xue, E-mail: lixue@imm.ac.cn; Hu, Jinping, E-mail: hujp@imm.ac.cn; Wang, Baolian, E-mail: wangbaolian@imm.ac.cn; Sheng, Li, E-mail: shengli@imm.ac.cn; Liu, Zhihao, E-mail: liuzhihao@imm.ac.cn; Yang, Shuang, E-mail: yangsh@imm.ac.cn; Li, Yan, E-mail: yanli@imm.ac.cn

    2014-03-01

    Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb–drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F{sub 1} [20(S)-GF{sub 1}] exhibited significant inhibition (> 50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC{sub 50} = 9.42 μM), followed by 18β-GA (IC{sub 50} = 21.78 μM), 20(S)-GF{sub 1} (IC{sub 50} = 76.08 μM) and DAG (IC{sub 50} = 77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with K{sub m} and V{sub max} values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF{sub 1} exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure–inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC{sub 0−t} and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF{sub 1} and Rh{sub 1} at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb–drug interactions in humans. - Highlights: • Emodin, 18

  14. Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp

    International Nuclear Information System (INIS)

    Li, Xue; Hu, Jinping; Wang, Baolian; Sheng, Li; Liu, Zhihao; Yang, Shuang; Li, Yan

    2014-01-01

    Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb–drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F 1 [20(S)-GF 1 ] exhibited significant inhibition (> 50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC 50 = 9.42 μM), followed by 18β-GA (IC 50 = 21.78 μM), 20(S)-GF 1 (IC 50 = 76.08 μM) and DAG (IC 50 = 77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with K m and V max values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF 1 exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure–inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC 0−t and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF 1 and Rh 1 at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb–drug interactions in humans. - Highlights: • Emodin, 18β-GA, DAG, and 20(S)-GF 1 significantly inhibited P-gp in vitro

  15. Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines.

    Directory of Open Access Journals (Sweden)

    Ling Ye

    Full Text Available Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14 in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.

  16. Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines

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    Valeria Feinshtein

    2013-09-01

    Full Text Available Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD, a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp and Breast Cancer Resistance Protein (BCRP expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp for comparison. Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24–72 h exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3 and rhodamine123(rh123. Cyclosporine A (CsA served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3 and rh123 was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.

  17. Mucoadhesive properties and interaction with P-glycoprotein (P-gp) of thiolated-chitosans and -glycol chitosans and corresponding parent polymers: a comparative study.

    Science.gov (United States)

    Trapani, Adriana; Palazzo, Claudio; Contino, Marialessandra; Perrone, Maria Grazia; Cioffi, Nicola; Ditaranto, Nicoletta; Colabufo, Nicola Antonio; Conese, Massimo; Trapani, Giuseppe; Puglisi, Giovanni

    2014-03-10

    The aim of the present work was to compare the mucoadhesive and efflux pump P-glycoprotein (P-gp) interacting properties of chitosan (CS)- and glycolchitosan (GCS)-based thiomers and corresponding unmodified parent polymers. For this purpose, the glycol chitosan-N-acetyl-cysteine (GCS-NAC) and glycol chitosan-glutathione (GCS-GSH) thiomers were prepared under simple and mild conditions. Their mucoadhesive characteristics were studied by turbidimetric and zeta potential measurements. The P-gp interacting properties were evaluated measuring the effects of thiolated- and unmodified-polymers on the bidirectional transport (BA/AB) of rhodamine-123 across Caco-2 cells as well as in the calcein-AM and ATPase activity assays. Although all the thiomers and unmodified polymers showed optimal-excellent mucoadhesive properties, the best mucoadhesive performances have been obtained by CS and CS-based thiomers. Moreover, it was found that the pretreatment of Caco-2 cell monolayer with GCS-NAC or GCS restores Rho-123 cell entrance by inhibiting P-gp activity. Hence, GCS-NAC and GCS may constitute new biomaterials useful for improving the bioavailability of P-gp substrates.

  18. Psychosocial factors in GP work: the effects of taking a GP position or leaving GP work.

    Science.gov (United States)

    Heponiemi, Tarja; Kouvonen, Anne; Aalto, Anna-Mari; Elovainio, Marko

    2013-06-01

    We examined the effects of leaving public sector general practitioner (GP) work and of taking a GP position on changes in work-related psychosocial factors, such as time pressure, patient-related stress, distress and work interference with family. In addition, we examined whether changes in time pressure and patient-related stress mediated the association of employment change with changes of distress and work interference with family. Participants were 1705 Finnish physicians (60% women) who responded to surveys in 2006 and 2010. Analyses of covariance were conducted to examine the effect of employment change to outcome changes adjusted for gender, age and response format. Mediational effects were tested following the procedures outlined by Baron and Kenny. Employment change was significantly associated with all the outcomes. Leaving public sector GP work was associated with substantially decreased time pressure, patient-related stress, distress and work interference with family. In contrast, taking a position as a public sector GP was associated with an increase in these factors. Mediation tests suggested that the associations of employment change with distress change and work interference with family change were partially explained by the changes in time pressure and patient-related stress. Our results showed that leaving public sector GP work is associated with favourable outcomes, whereas taking a GP position in the public sector is associated with adverse effects. Primary health-care organizations should pay more attention to the working conditions of their GPs, in particular, to time pressure and patient-related stress.

  19. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

    International Nuclear Information System (INIS)

    Usami, Katsuaki; Matsuno, Keita; Igarashi, Manabu; Denda-Nagai, Kaori; Takada, Ayato; Irimura, Tatsuro

    2011-01-01

    Highlights: → Ebola virus infection is mediated by binding to and fusion with the target cells. → Structural feature of the viral glycoprotein determines the infectivity. → Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. → GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. → There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

  20. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

    Energy Technology Data Exchange (ETDEWEB)

    Usami, Katsuaki [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Matsuno, Keita; Igarashi, Manabu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Denda-Nagai, Kaori [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan); Takada, Ayato [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020 (Japan); Irimura, Tatsuro, E-mail: irimura@mol.f.u-tokyo.ac.jp [Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033 (Japan)

    2011-04-01

    Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses, i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.

  1. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    International Nuclear Information System (INIS)

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-01-01

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  2. Antibodies with High Avidity to the gp120 Envelope Protein in Protection from Simian Immunodeficiency Virus SIVmac251 Acquisition in an Immunization Regimen That Mimics the RV-144 Thai Trial

    Science.gov (United States)

    Pegu, Poonam; Vaccari, Monica; Gordon, Shari; Keele, Brandon F.; Doster, Melvin; Guan, Yongjun; Ferrari, Guido; Pal, Ranajit; Ferrari, Maria Grazia; Whitney, Stephen; Hudacik, Lauren; Billings, Erik; Rao, Mangala; Montefiori, David; Tomaras, Georgia; Alam, S. Munir; Fenizia, Claudio; Lifson, Jeffrey D.; Stablein, Donald; Tartaglia, Jim; Michael, Nelson; Kim, Jerome; Venzon, David

    2013-01-01

    The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8+ T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIVmac251 that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4+ and CD8+ T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIVmac251 acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIVmac251-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIVmac251 infectivity in cells that express high levels of α4β7 integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines. PMID:23175374

  3. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    International Nuclear Information System (INIS)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-01-01

    Research highlights: → One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. → N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. → These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  4. The putative P-gp inhibitor telmisartan does not affect the transcellular permeability and cellular uptake of the calcium channel antagonist verapamil in the P-glycoprotein expressing cell line MDCK II MDR1

    DEFF Research Database (Denmark)

    Saaby, Lasse; Tfelt-Hansen, Peer; Brodin, Birger

    2015-01-01

    monolayers with a permeability of 5.7 × 10−5 cm sec−1 compared to an apical to basolateral permeability of 1.3 × 10−5 cm sec-1. The efflux could be inhibited with the P-gp inhibitor zosuquidar. Zosuquidar (0.4 μmol/L) reduced the efflux ratio (PB-A/PA-B) for verapamil 4.6–1.6. The presence of telmisartan......Verapamil is used in high doses for the treatment of cluster headache. Verapamil has been described as a P-glycoprotein (P-gp, ABCB1) substrate. We wished to evaluate in vitro whether co administration of a P-gp inhibitor with verapamil could be a feasible strategy for increasing CNS uptake...... of verapamil. Fluxes of radiolabelled verapamil across MDCK II MDR1 monolayers were measured in the absence and presence of the putative P-gp inhibitor telmisartan (a clinically approved drug compound). Verapamil displayed a vectorial basolateral-to-apical transepithelial efflux across the MDCK II MDR1...

  5. Ion Mobility Mass Spectrometry for Extracting Spectra of N-Glycans Directly from Incubation Mixtures Following Glycan Release: Application to Glycans from Engineered Glycoforms of Intact, Folded HIV gp120

    Science.gov (United States)

    Harvey, David J.; Sobott, Frank; Crispin, Max; Wrobel, Antoni; Bonomelli, Camille; Vasiljevic, Snezana; Scanlan, Christopher N.; Scarff, Charlotte A.; Thalassinos, Konstantinos; Scrivens, James H.

    2011-03-01

    The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/ m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.

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

    Science.gov (United States)

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

    2016-06-01

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

  7. Identification of N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamides as a new class of HIV-1 entry inhibitors that prevent gp120 binding to CD4

    International Nuclear Information System (INIS)

    Zhao Qian; Ma Liying; Jiang Shibo; Lu Hong; Liu Shuwen; He Yuxian; Strick, Nathan; Neamati, Nouri; Debnath, Asim Kumar

    2005-01-01

    We have identified two N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide analogs as a novel class of human immunodeficiency virus type 1 (HIV-1) entry inhibitors that block the gp120-CD4 interaction, using database screening techniques. The lead compounds, NBD-556 and NBD-557, are small molecule organic compounds with drug-like properties. These compounds showed potent cell fusion and virus-cell fusion inhibitory activity at low micromolar levels. A systematic study showed that these compounds target viral entry by inhibiting the binding of HIV-1 envelope glycoprotein gp120 to the cellular receptor CD4 but did not inhibit reverse transcriptase, integrase, or protease, indicating that they do not target the later stages of the HIV-1 life cycle to inhibit HIV-1 infection. These compounds were equally potent inhibitors of both X4 and R5 viruses tested in CXCR4 and CCR5 expressing cell lines, respectively, indicating that their anti-HIV-1 activity is not dependent on the coreceptor tropism of the virus. A surface plasmon resonance study, which measures binding affinity, clearly demonstrated that these compounds bind to unliganded HIV-1 gp120 but not to the cellular receptor CD4. NBD-556 and NBD-557 were active against HIV-1 laboratory-adapted strains including an AZT-resistant strain and HIV-1 primary isolates, indicating that these compounds can potentially be further modified to become potent HIV-1 entry inhibitors

  8. Antibodies with high avidity to the gp120 envelope protein in protection from simian immunodeficiency virus SIV(mac251) acquisition in an immunization regimen that mimics the RV-144 Thai trial.

    Science.gov (United States)

    Pegu, Poonam; Vaccari, Monica; Gordon, Shari; Keele, Brandon F; Doster, Melvin; Guan, Yongjun; Ferrari, Guido; Pal, Ranajit; Ferrari, Maria Grazia; Whitney, Stephen; Hudacik, Lauren; Billings, Erik; Rao, Mangala; Montefiori, David; Tomaras, Georgia; Alam, S Munir; Fenizia, Claudio; Lifson, Jeffrey D; Stablein, Donald; Tartaglia, Jim; Michael, Nelson; Kim, Jerome; Venzon, David; Franchini, Genoveffa

    2013-02-01

    The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8(+) T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIV(mac251) that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4(+) and CD8(+) T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIV(mac251) acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIV(mac251)-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIV(mac251) infectivity in cells that express high levels of α(4)β(7) integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines.

  9. Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

    Science.gov (United States)

    Cai, Lifeng; Gochin, Miriam; Liu, Keliang

    2011-12-01

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

  10. 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.

  11. Structure-guided Design and Immunological Characterization of Immunogens Presenting the HIV-1 gp120 V3 Loop on a CTB Scaffold

    Energy Technology Data Exchange (ETDEWEB)

    M Totrov; X Jiang; X Kong; S Cohen; C Krachmarov; A Salomon; C Williams; M Seaman; R Abagyan; et al.

    2011-12-31

    V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.

  12. Microsoft Dynamics GP 2013 implementation

    CERN Document Server

    Yudin, Victoria

    2013-01-01

    A step-by-step guide for planning and carrying out your Microsoft Dynamics GP 2013 implementation. Detailed descriptions and illustrations of setup screens and practical examples and advice are included for the Dynamics GP system and core modules.If you are a new or existing Microsoft Dynamics GP consultant or an end user who wants to implement, install, and set up core modules of Dynamics GP 2013, then this book is for you. A basic understanding of business management systems and either Dynamics GP or a similar application is recommended.

  13. Humoral immune response to hypervariable region 1 of the putative envelope glycoprotein (gp70) of hepatitis C virus.

    OpenAIRE

    Kato, N; Sekiya, H; Ootsuyama, Y; Nakazawa, T; Hijikata, M; Ohkoshi, S; Shimotohno, K

    1993-01-01

    We recently found that alterations of amino acids in hypervariable region 1 (HVR1) of the putative envelope glycoprotein (gp70) of hepatitis C virus (HCV) occurred sequentially in the chronic phase of hepatitis at intervals of several months. This finding suggests that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection involving escape from the immunosurveillance system. To explore this possibility, we examined the humoral immune response to HVR1 with our assa...

  14. Levels of HIV1 gp120 3D B-cell epitopes mutability and variability: searching for possible vaccine epitopes.

    Science.gov (United States)

    Khrustalev, Vladislav Victorovich

    2010-01-01

    We used a DiscoTope 1.2 (http://www.cbs.dtu.dk/services/DiscoTope/), Epitopia (http://epitopia.tau.ac.il/) and EPCES (http://www.t38.physik.tu-muenchen.de/programs.htm) algorithms to map discontinuous B-cell epitopes in HIV1 gp120. The most mutable nucleotides in HIV genes are guanine (because of G to A hypermutagenesis) and cytosine (because of C to U and C to A mutations). The higher is the level of guanine and cytosine usage in third (neutral) codon positions and the lower is their level in first and second codon positions of the coding region, the more stable should be an epitope encoded by this region. We compared guanine and cytosine usage in regions coding for five predicted 3D B-cell epitopes of gp120. To make this comparison we used GenBank resource: 385 sequences of env gene obtained from ten HIV1-infected individuals were studied (http://www.barkovsky.hotmail.ru/Data/Seqgp120.htm). The most protected from nonsynonymous nucleotide mutations of guanine and cytosine 3D B-cell epitope is situated in the first conserved region of gp120 (it is mapped from 66th to 86th amino acid residue). We applied a test of variability to confirm this finding. Indeed, the less mutable predicted B-cell epitope is the less variable one. MEGA4 (standard PAM matrix) was used for the alignments and "VVK Consensus" algorithm (http://www.barkovsky.hotmail.ru) was used for the calculations.

  15. Effects of Agitation, Aeration and Temperature on Production of a Novel Glycoprotein GP-1 by Streptomyces kanasenisi ZX01 and Scale-Up Based on Volumetric Oxygen Transfer Coefficient

    Directory of Open Access Journals (Sweden)

    Yong Zhou

    2018-01-01

    Full Text Available The effects of temperature, agitation and aeration on glycoprotein GP-1 production by Streptomyces kanasenisi ZX01 in bench-scale fermentors were systematically investigated. The maximum final GP-1 production was achieved at an agitation speed of 200 rpm, aeration rate of 2.0 vvm and temperature of 30 °C. By using a dynamic gassing out method, the effects of agitation and aeration on volumetric oxygen transfer coefficient (kLa were also studied. The values of volumetric oxygen transfer coefficient in the logarithmic phase increased with increase of agitation speed (from 14.53 to 32.82 h−1 and aeration rate (from 13.21 to 22.43 h−1. In addition, a successful scale-up from bench-scale to pilot-scale was performed based on volumetric oxygen transfer coefficient, resulting in final GP-1 production of 3.92, 4.03, 3.82 and 4.20 mg/L in 5 L, 15 L, 70 L and 500 L fermentors, respectively. These results indicated that constant volumetric oxygen transfer coefficient was appropriate for the scale-up of batch fermentation of glycoprotein GP-1 by Streptomyces kanasenisi ZX01, and this scale-up strategy successfully achieved 100-fold scale-up from bench-scale to pilot-scale fermentor.

  16. Serotonin transporter protein (SERT) and P-glycoprotein (P-gp) binding activity of montanine and coccinine from three species of Haemanthus L. (Amaryllidaceae)

    DEFF Research Database (Denmark)

    Stafford, Gary Ivan; Birer, C.; Brodin, Birger

    2013-01-01

    The alkaloid rich extracts from an acid/base extraction of bulb material of Haemanthus coccineus L., H. montanus Baker and H. sanguineus Jacq. revealed that two montanine type Amaryllidaceae alkaloids, montanine (1) and coccinine (2) were the major alkaloid constituents. Together these two...... to the relative proportions of coccinine and montanine in the extracts and thus are likely to be due to more potent unidentified minor constituents. Both alkaloids exhibited low binding affinity to P-glycoprotein (P-gp) as demonstrated by low inhibition of calcein-AM efflux in the MDCK-MDR1 cell line....... This indicates that P-gp efflux will not be limiting for blood-brain-barrier passage of the alkaloids....

  17. Characterization of the immuno dominant regions within gp41 of env gene of HIV in Pakistan

    International Nuclear Information System (INIS)

    Nisar, L.; Qadir, M.I.; Nisa, T.; Malik, S.A.; Tabassum, N.

    2011-01-01

    Objective of the present study was to characterize the immuno dominant sequences of gp41 in HIV strain in Pakistani population so that vaccine may be prepared based upon these regions. A total of 25 specimens were collected from HIV patients of different areas of Pakistan. The viral RNA was isolated using QIAamp MinElute Spin Kit manufactured by Quiagen, California, USA. RT-PCR was done for amplification of the required region and confirmed by gel electrophoresis. The nucleotides of the required regions were sequenced using Big Dye terminator. Less than 20% of the specimens had mutations in immuno dominant regions, so it may be concluded that immuno dominant regions of gp41 in HIV strain in Pakistani population are conservatives and vaccine based upon these regions may prove active immunization against the disease. (author)

  18. Characterization of the immuno dominant regions within gp41 of env gene of HIV in Pakistan

    Energy Technology Data Exchange (ETDEWEB)

    Nisar, L; Qadir, M I; Nisa, T; Malik, S A; Tabassum, N

    2011-08-15

    Objective of the present study was to characterize the immuno dominant sequences of gp41 in HIV strain in Pakistani population so that vaccine may be prepared based upon these regions. A total of 25 specimens were collected from HIV patients of different areas of Pakistan. The viral RNA was isolated using QIAamp MinElute Spin Kit manufactured by Quiagen, California, USA. RT-PCR was done for amplification of the required region and confirmed by gel electrophoresis. The nucleotides of the required regions were sequenced using Big Dye terminator. Less than 20% of the specimens had mutations in immuno dominant regions, so it may be concluded that immuno dominant regions of gp41 in HIV strain in Pakistani population are conservatives and vaccine based upon these regions may prove active immunization against the disease. (author)

  19. Spinal CPEB-mtROS-CBP signaling pathway contributes to perineural HIV gp120 with ddC-related neuropathic pain in rats.

    Science.gov (United States)

    Iida, Takafumi; Yi, Hyun; Liu, Shue; Huang, Wan; Kanda, Hirotsugu; Lubarsky, David A; Hao, Shuanglin

    2016-07-01

    Human immunodeficiency virus (HIV) patients treated with nucleoside reverse transcriptase inhibitors (NRTIs), have been known to develop neuropathic pain. While there has been a major shift away from some neurotoxic NRTIs in current antiretroviral therapy, a large number of HIV patients alive today have previously received them, and many have developed painful peripheral neuropathy. The exact mechanisms by which HIV with NRTIs contribute to the development of neuropathic pain are not known. Previous studies suggest that cytoplasmic polyadenylation element-binding protein (CPEB), reactive oxygen species (ROS), and cAMP-response element-binding protein (CREB)-binding protein (CBP), are involved in the neuroimmunological diseases including inflammatory/neuropathic pain. In this study, we investigated the role of CPEB, mitochondrial ROS (mtROS), or CBP in neuropathic pain induced by HIV envelope protein gp120 combined with antiretroviral drug. The application of recombinant gp120 into the sciatic nerve plus systemic ddC (one of NRTIs) induced mechanical allodynia. Knockdown of CPEB or CBP using intrathecal antisense oligodeoxynucleotide (AS-ODN) reduced mechanical allodynia. Intrathecal mitochondrial superoxide scavenger mito-tempol (Mito-T) increased mechanical withdrawal threshold. Knockdown of CPEB using intrathecal AS-ODN, reduced the up-regulated mitochondrial superoxide in the spinal dorsal horn in rats with gp120 combined with ddC. Intrathecal Mito-T lowered the increased expression of CBP in the spinal dorsal horn. Immunostaining studies showed that neuronal CPEB positive cells were co-localized with MitoSox positive profiles, and that MitoSox positive profiles were co-localized with neuronal CBP. Our studies suggest that neuronal CPEB-mtROS-CBP pathway in the spinal dorsal horn, plays an important role in the gp120/ddC-induced neuropathic pain in rats. Copyright © 2016. Published by Elsevier Inc.

  20. Evaluation of "credit card" libraries for inhibition of HIV-1 gp41 fusogenic core formation.

    Science.gov (United States)

    Xu, Yang; Lu, Hong; Kennedy, Jack P; Yan, Xuxia; McAllister, Laura A; Yamamoto, Noboru; Moss, Jason A; Boldt, Grant E; Jiang, Shibo; Janda, Kim D

    2006-01-01

    Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.

  1. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    Energy Technology Data Exchange (ETDEWEB)

    Du, Yijun [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan (China); Pattnaik, Asit K. [School of Veterinary Medicine and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900 (United States); Song, Cheng [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Yoo, Dongwan, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Li, Gang, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing (China)

    2012-03-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  2. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    International Nuclear Information System (INIS)

    Du, Yijun; Pattnaik, Asit K.; Song, Cheng; Yoo, Dongwan; Li, Gang

    2012-01-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 (ω − 2, where ω is the GPI moiety at E160), P159 (ω − 1), and M162 (ω + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide–anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  3. Natural mannosylation of HIV-1 gp120 imposes no immunoregulatory effects in primary human plasmacytoid dendritic cells

    NARCIS (Netherlands)

    Sondergaard, J.N.; Vinner, L.; Brix, S.

    2014-01-01

    Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity, and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs, but no receptors for high-mannose have so far

  4. Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate

    DEFF Research Database (Denmark)

    Visciano, Maria Luisa; Tagliamonte, Maria; Stewart-Jones, Guillaume

    2013-01-01

    Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutr...

  5. Differential effect on TCR:CD3 stimulation of a 90-kD glycoprotein (gp90/Mac-2BP), a member of the scavenger receptor cysteine-rich domain protein family

    DEFF Research Database (Denmark)

    Silvestri, B; Calderazzo, F; Coppola, V

    1998-01-01

    We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non-specific ......We studied the effects of a 90-kD glycoprotein (gp90/Mac-2BP) belonging to the scavenger receptor family, present in normal serum and at increased levels in inflammatory disease and cancer patients, on some T cell function parameters. Whereas the lymphocyte proliferative response to non......-specific mitogens such as phytohaemagglutinin (PHA) and concanavalin A (Con A), but not pokeweed mitogen (PWM), was strongly reduced, probably due to the lectin-binding properties of gp90/Mac-2BP, the response to T cell receptor (TCR) agonists such as superantigens and allogeneic cells was potentiated. When...... lymphocytes were stimulated with different anti-TCR:CD3 MoAbs, both in soluble and solid-phase form, gp90/Mac-2BP was able to down-regulate the proliferative response to anti-CD3 MoAb, whereas the response to anti-TCR alphabeta MoAb was enhanced. A similar differential effect was observed when a MoAb against...

  6. A mechanistic understanding of allosteric immune escape pathways in the HIV-1 envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Anurag Sethi

    Full Text Available The HIV-1 envelope (Env spike, which consists of a compact, heterodimeric trimer of the glycoproteins gp120 and gp41, is the target of neutralizing antibodies. However, the high mutation rate of HIV-1 and plasticity of Env facilitates viral evasion from neutralizing antibodies through various mechanisms. Mutations that are distant from the antibody binding site can lead to escape, probably by changing the conformation or dynamics of Env; however, these changes are difficult to identify and define mechanistically. Here we describe a network analysis-based approach to identify potential allosteric immune evasion mechanisms using three known HIV-1 Env gp120 protein structures from two different clades, B and C. First, correlation and principal component analyses of molecular dynamics (MD simulations identified a high degree of long-distance coupled motions that exist between functionally distant regions within the intrinsic dynamics of the gp120 core, supporting the presence of long-distance communication in the protein. Then, by integrating MD simulations with network theory, we identified the optimal and suboptimal communication pathways and modules within the gp120 core. The results unveil both strain-dependent and -independent characteristics of the communication pathways in gp120. We show that within the context of three structurally homologous gp120 cores, the optimal pathway for communication is sequence sensitive, i.e. a suboptimal pathway in one strain becomes the optimal pathway in another strain. Yet the identification of conserved elements within these communication pathways, termed inter-modular hotspots, could present a new opportunity for immunogen design, as this could be an additional mechanism that HIV-1 uses to shield vulnerable antibody targets in Env that induce neutralizing antibody breadth.

  7. A gp41-based heteroduplex mobility assay provides rapid and accurate assessment of intrasubtype epidemiological linkage in HIV type 1 heterosexual transmission Pairs.

    Science.gov (United States)

    Manigart, Olivier; Boeras, Debrah I; Karita, Etienne; Hawkins, Paulina A; Vwalika, Cheswa; Makombe, Nathan; Mulenga, Joseph; Derdeyn, Cynthia A; Allen, Susan; Hunter, Eric

    2012-12-01

    A critical step in HIV-1 transmission studies is the rapid and accurate identification of epidemiologically linked transmission pairs. To date, this has been accomplished by comparison of polymerase chain reaction (PCR)-amplified nucleotide sequences from potential transmission pairs, which can be cost-prohibitive for use in resource-limited settings. Here we describe a rapid, cost-effective approach to determine transmission linkage based on the heteroduplex mobility assay (HMA), and validate this approach by comparison to nucleotide sequencing. A total of 102 HIV-1-infected Zambian and Rwandan couples, with known linkage, were analyzed by gp41-HMA. A 400-base pair fragment within the envelope gp41 region of the HIV proviral genome was PCR amplified and HMA was applied to both partners' amplicons separately (autologous) and as a mixture (heterologous). If the diversity between gp41 sequences was low (<5%), a homoduplex was observed upon gel electrophoresis and the transmission was characterized as having occurred between partners (linked). If a new heteroduplex formed, within the heterologous migration, the transmission was determined to be unlinked. Initial blind validation of gp-41 HMA demonstrated 90% concordance between HMA and sequencing with 100% concordance in the case of linked transmissions. Following validation, 25 newly infected partners in Kigali and 12 in Lusaka were evaluated prospectively using both HMA and nucleotide sequences. Concordant results were obtained in all but one case (97.3%). The gp41-HMA technique is a reliable and feasible tool to detect linked transmissions in the field. All identified unlinked results should be confirmed by sequence analyses.

  8. Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.

    Science.gov (United States)

    Bornholdt, Zachary A; Ndungo, Esther; Fusco, Marnie L; Bale, Shridhar; Flyak, Andrew I; Crowe, James E; Chandran, Kartik; Saphire, Erica Ollmann

    2016-02-23

    The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics. Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs. Copyright © 2016 Bornholdt et al.

  9. Effect of partial and complete variable loop deletions of the human immunodeficiency virus type 1 envelope glycoprotein on the breadth of gp160-specific immune responses

    International Nuclear Information System (INIS)

    Gzyl, Jaroslaw; Bolesta, Elizabeth; Wierzbicki, Andrew; Kmieciak, Dariusz; Naito, Toshio; Honda, Mitsuo; Komuro, Katsutoshi; Kaneko, Yutaro; Kozbor, Danuta

    2004-01-01

    Induction of cross-reactive cellular and humoral responses to the HIV-1 envelope (env) glycoprotein was examined after DNA immunization of BALB/c mice with gp140 89.6 -derived constructs exhibiting partial or complete deletions of the V1, V2, and V3 domains. It was demonstrated that specific modification of the V3 loop (mV3) in combination with the V2-modified (mV2) or V1/V2-deleted (ΔV1/V2) region elicited increased levels of cross-reactive CD8 + T cell responses. Mice immunized with the mV2/mV3 or ΔV1/V2/mV3 gp140 89.6 plasmid DNA were greater than 50-fold more resistant to challenge with recombinant vaccinia virus (rVV) expressing heterologous env gene products than animals immunized with the wild-type (WT) counterpart. Sera from mV2/mV3- and ΔV1/V2/mV3-immunized mice exhibited the highest cross-neutralizing activity and displayed intermediate antibody avidity values which were further enhanced by challenge with rVV expressing the homologous gp160 glycoprotein. In contrast, complete deletion of the variable regions had little or no effect on the cross-reactive antibody responses. The results of these experiments indicate that the breadth of antibody responses to the HIV-1 env glycoprotein may not be increased by removal of the variable domains. Instead, partial deletions within these regions may redirect specific responses toward conserved epitopes and facilitate approaches for boosting cross-reactive cellular and antibody responses to the env glycoprotein

  10. Up-regulation of the Neuronal Nicotinic Receptor α7 by HIV Glycoprotein 120

    Science.gov (United States)

    Ballester, Leomar Y.; Capó-Vélez, Coral M.; García-Beltrán, Wilfredo F.; Ramos, Félix M.; Vázquez-Rosa, Edwin; Ríos, Raymond; Mercado, José R.; Meléndez, Roberto I.; Lasalde-Dominicci, José A.

    2012-01-01

    Approximately 30–50% of the >30 million HIV-infected subjects develop neurological complications ranging from mild symptoms to dementia. HIV does not infect neurons, and the molecular mechanisms behind HIV-associated neurocognitive decline are not understood. There are several hypotheses to explain the development of dementia in HIV+ individuals, including neuroinflammation mediated by infected microglia and neuronal toxicity by HIV proteins. A key protein associated with the neurological complications of HIV, gp120, forms part of the viral envelope and can be found in the CSF of infected individuals. HIV-1-gp120 interacts with several receptors including CD4, CCR5, CXCR4, and nicotinic acetylcholine receptors (nAChRs). However, the role of nAChRs in HIV-associated neurocognitive disorder has not been investigated. We studied the effects of gp120IIIB on the expression and function of the nicotinic receptor α7 (α7-nAChR). Our results show that gp120, through activation of the CXCR4 chemokine receptor, induces a functional up-regulation of α7-nAChRs. Because α7-nAChRs have a high permeability to Ca2+, we performed TUNEL staining to investigate the effects of receptor up-regulation on cell viability. Our data revealed an increase in cell death, which was blocked by the selective antagonist α-bungarotoxin. The in vitro data are supported by RT-PCR and Western blot analysis, confirming a remarkable up-regulation of the α7-nAChR in gp120-transgenic mice brains. Specifically, α7-nAChR up-regulation is observed in mouse striatum, a region severely affected in HIV+ patients. In summary, CXCR4 activation induces up-regulation of α7-nAChR, causing cell death, suggesting that α7-nAChR is a previously unrecognized contributor to the neurotoxicity associated with HIV infection. PMID:22084248

  11. Delivery of siRNA silencing P-gp in peptide-functionalized nanoparticles causes efflux modulation at the blood-brain barrier

    DEFF Research Database (Denmark)

    Gomes, Maria João; Kennedy, Patrick J; Martins, Susana

    2017-01-01

    AIM: Explore the use of transferrin-receptor peptide-functionalized nanoparticles (NPs) targeting blood-brain barrier (BBB) as siRNA carriers to silence P-glycoprotein (P-gp). MATERIALS & METHODS: Permeability experiments were assessed through a developed BBB cell-based model; P-gp mRNA expression...

  12. Mechanism of feline immunodeficiency virus envelope glycoprotein-mediated fusion

    International Nuclear Information System (INIS)

    Garg, Himanshu; Fuller, Frederick J.; Tompkins, Wayne A.F.

    2004-01-01

    Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU 1 failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using site-directed mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation

  13. Esters of the Marine-Derived Triterpene Sipholenol A Reverse P-GP-Mediated Drug Resistance

    Directory of Open Access Journals (Sweden)

    Yongchao Zhang

    2015-04-01

    Full Text Available Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1. Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers.

  14. Evaluation of “Credit Card” Libraries for Inhibition of HIV-1 gp41 Fusogenic Core Formation

    Science.gov (United States)

    Xu, Yang; Lu, Hong; Kennedy, Jack P.; Yan, Xuxia; McAllister, Laura; Yamamoto, Noboru; Moss, Jason A.; Boldt, Grant E.; Jiang, Shibo; Janda, Kim D.

    2008-01-01

    Protein-protein interactions are of critical importance in biological systems and small molecule modulators of such protein recognition and intervention processes are of particular interests. To investigate this area of research, we have synthesized small molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motifs, appended with a variety of chemical functions, which we have termed as “credit-card” structures. From two of our “credit-card” libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors. PMID:16827565

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

    International Nuclear Information System (INIS)

    Halaban, R.; Moellmann, G.

    1990-01-01

    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 lt /B 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 lt mutation renders the protein susceptible to rapid proteolytic degradation

  16. Structural and functional characterization of EIAV gp45 fusion peptide proximal region and asparagine-rich layer

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Liangwei; Du, Jiansen [State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Wang, Xuefeng; Zhou, Jianhua; Wang, Xiaojun [State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001 (China); Liu, Xinqi, E-mail: liu2008@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2016-04-15

    Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesis of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine. - Highlights: • The crystal structure of EIAV gp45 was determined. • The fusion peptide proximal region adopts a novel conformation different to HIV-1. • The asparagine-rich layer includes an extensive hydrogen-bond network. • These regions of EIAV are highly tolerant to mutations. • The results provide insight into the mechanism of gp41/gp45-mediated membrane fusion.

  17. Association between patients' recommendation of their GP and their evaluation of the GP.

    Science.gov (United States)

    Vedsted, Peter; Heje, Hanne N

    2008-01-01

    Patient priorities and patient evaluations indicate that accessibility should receive more attention to increase quality in general practice. The definition of family medicine emphasizes the patient-centred approach, communication skills, continuity, and clinical skills. We aimed to explore the associations between the 23 items in the Europep questionnaire measuring patient evaluation of general practice and the patients' recommendation of their general practitioner (GP) to friends and to study the relationship of these items with the core competences of family medicine. Cross-sectional study where patients aged 18 years and over attending the practice were included. Patients completed the Danish version of the 23 item Europep questionnaire and an additional item about the degree to which they could recommend their GP to friends. Danish general practice (the DanPEP study). A total of 50 191 patients and 690 GPs were included in the analyses. For each item, associations were calculated between a positive answer and the degree to which the patient could recommend the GP. Analyses were made at patient and GP levels. We found 12 items that covered the 10 most strongly associated items from both analyses: four of six items from the "doctor-patient relationship", two of five items from "medical care", and all items from "information and support" and "organization of services". No items from "accessibility" were among the 12 items. Recommending the GP to others was most strongly associated with the "emphatic", "patient-oriented", "informative and coordinating", and "competent/skilled" GP and to a lesser degree with accessibility to general practice.

  18. Microsoft Dynamics GP 2013 financial management

    CERN Document Server

    Grieve, Ian

    2013-01-01

    A standard tutorial-based approach covering Microsoft Dynamics GP 2013 and its six financial modules. The book is intended to allow users to improve their system use and workflow by introducing new modules to assist in financial management.This book is for you if you're a Dynamics GP partner, or Dynamics GP user, primarily focused on delivering application optimizations. This book assumes that you have a working knowledge of Microsoft Dynamics GP and have an understanding of the requirements of financial management.

  19. Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 onto plates coated with anti-CD4bs antibodies b12, b6 or the CD4 receptor mimetic, CD4-IgG2...

  20. On πgp-continuous functions in topological spaces

    International Nuclear Information System (INIS)

    Park, Jin Han; Park, Jin Keun

    2004-01-01

    The concept of πgp-closed sets was introduced by Park [On πgp-closed sets in topological spaces, Indian J. Pure Appl. Math., in press]. The aim of this paper is to consider and characterize πgp-irresolute and πgp-continuous functions via the concept of πgp-closed sets and to relate these concepts to the classes of πGPO-compact spaces and πGP-connected spaces

  1. Pseudotyping Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs are functionally analogous to AcMNPV GP64

    NARCIS (Netherlands)

    Lung, O.; Westenberg, M.; Vlak, J.M.; Zuidema, D.; Blissard, G.W.

    2002-01-01

    GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral

  2. Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120.

    Science.gov (United States)

    Curreli, Francesca; Belov, Dmitry S; Kwon, Young Do; Ramesh, Ranjith; Furimsky, Anna M; O'Loughlin, Kathleen; Byrge, Patricia C; Iyer, Lalitha V; Mirsalis, Jon C; Kurkin, Alexander V; Altieri, Andrea; Debnath, Asim K

    2018-05-12

    We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  3. Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II recepter HLA-DR1

    Energy Technology Data Exchange (ETDEWEB)

    Mullen, M.; Haan, K.M.; Longnecker, R.; Jardetzky, T.

    2010-03-08

    Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.

  4. Release of Glycoprotein (GP1 from the Tegumental Surface of Taenia solium by Phospholipase C from Clostridium perfringens Suggests a Novel Protein-Anchor to Membranes

    Directory of Open Access Journals (Sweden)

    Abraham Landa

    2010-01-01

    Full Text Available In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC. Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43 kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, αmethyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD, suggesting a novel anchor to the membrane for the glycoprotein GP1.

  5. Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Zachary A. Bornholdt

    2016-02-01

    Full Text Available The filovirus surface glycoprotein (GP mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.

  6. Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

    DEFF Research Database (Denmark)

    Schønning, Kristian; Bolmstedt, A; Novotny, J

    1998-01-01

    An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected...... immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation....

  7. P-gp and MRP1 Expression in Parathyroid Tumors Related to Histology, Weight and Tc-99m-Sestamibi Imaging Results

    NARCIS (Netherlands)

    Jorna, F. H.; Hollema, H.; Hendrikse, H. N.; Bart, J.; Brouwers, A. H.; Plukker, J. T. M.

    Objective: P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) are membrane efflux pumps that may have a role in the kinetics of Tc-99m-sestamibi (MIBI) in parathyroid tumors. P-gp and MRP1 expression in parathyroid tumors was studied and related to histology, weight and pre- and

  8. Estimating the probability of polyreactive antibodies 4E10 and 2F5 disabling a gp41 trimer after T cell-HIV adhesion.

    Directory of Open Access Journals (Sweden)

    Bin Hu

    2014-01-01

    Full Text Available A few broadly neutralizing antibodies, isolated from HIV-1 infected individuals, recognize epitopes in the membrane proximal external region (MPER of gp41 that are transiently exposed during viral entry. The best characterized, 4E10 and 2F5, are polyreactive, binding to the viral membrane and their epitopes in the MPER. We present a model to calculate, for any antibody concentration, the probability that during the pre-hairpin intermediate, the transient period when the epitopes are first exposed, a bound antibody will disable a trivalent gp41 before fusion is complete. When 4E10 or 2F5 bind to the MPER, a conformational change is induced that results in a stably bound complex. The model predicts that for these antibodies to be effective at neutralization, the time to disable an epitope must be shorter than the time the antibody remains bound in this conformation, about five minutes or less for 4E10 and 2F5. We investigate the role of avidity in neutralization and show that 2F5 IgG, but not 4E10, is much more effective at neutralization than its Fab fragment. We attribute this to 2F5 interacting more stably than 4E10 with the viral membrane. We use the model to elucidate the parameters that determine the ability of these antibodies to disable epitopes and propose an extension of the model to analyze neutralization data. The extended model predicts the dependencies of IC50 for neutralization on the rate constants that characterize antibody binding, the rate of fusion of gp41, and the number of gp41 bridging the virus and target cell at the start of the pre-hairpin intermediate. Analysis of neutralization experiments indicate that only a small number of gp41 bridges must be disabled to prevent fusion. However, the model cannot determine the exact number from neutralization experiments alone.

  9. Optional part-time and longer GP training modules in GP practices associated with more trainees becoming GPs - a cohort study in Switzerland.

    Science.gov (United States)

    Studerus, Lara; Ahrens, Regina; Häuptle, Christian; Goeldlin, Adrian; Streit, Sven

    2018-01-05

    Switzerland, like many other countries, has a shortage of General Practitioners (GPs). Optional GP training modules in GP practices were offered during the at least 5-year GP training program to increase student and trainee interest in becoming a GP. The training modules had not yet been evaluated. We determined how many Swiss GP trainees became practicing GPs after they completed optional training modules, and if longer modules were associated with higher rates of GP specialization. In this population-based cohort study, we included GP trainees who chose an optional GP training module in GP practice, provided by the Foundation to Promote Training in General Practice (WHM) between 2006 and 2015. GP trainees were invited to complete an online survey to assess the primary outcome (becoming a practicing GP by 2016). Data on non-responders was collected via an internet search. We calculated univariate time-to-event curves to become a practicing GP, stratified by trainee's gender, length, part-time training, and number of years after graduation until training modules were completed. We used a multivariate model to adjust for characteristics of participants, training, and satisfaction with training modules. We assessed primary outcome for 351 (92.1%) of 381 former GP trainees who participated in a WHM program between 2006 and 2015. Of these 218 (57%) were practicing GPs by 2016. When focusing on the trainees who had completed training between 2006 and 2010, the rate of practicing GPs was even 73%. Longer (p = 0.018) and part-time training modules (p = 0.003) were associated with higher rates of being a practicing GP. Most (81%) practicing GPs thought their optional GP training module was (very) important in their choice of specialty. GP trainees who spent more time training in a GP practice, or who trained part-time were more likely to become practicing GPs. Most (80%) rated their training module as (very) important in their choice of career, highlighting that

  10. GP registrar well-being: a cross-sectional survey

    OpenAIRE

    Schattner, Peter; Mazalin, Dennis; Pier, Ciaran; Wainer, Jo; Ling, Mee Yoke

    2010-01-01

    Abstract Objectives To investigate the major stressors affecting GP registrars, how those at risk can be best identified and the most useful methods of managing or reducing their stress. Design, setting and participants Cross-sectional postal questionnaire of all GP registrars in one large regional training provider's catchment area. Main outcome measures The Depression, Anxiety and Stress Scale (DASS), a specifically developed Registrar Stressor Scale consisting of five subscales of potentia...

  11. Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

    Science.gov (United States)

    d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

    2015-04-16

    ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. stableGP

    Data.gov (United States)

    National Aeronautics and Space Administration — The code in the stableGP package implements Gaussian process calculations using efficient and numerically stable algorithms. Description of the algorithms is in the...

  13. A Directed Molecular Evolution Approach to Improved Immunogenicity of the HIV-1 Envelope Glycoprotein

    Science.gov (United States)

    Du, Sean X.; Xu, Li; Zhang, Wenge; Tang, Susan; Boenig, Rebecca I.; Chen, Helen; Mariano, Ellaine B.; Zwick, Michael B.; Parren, Paul W. H. I.; Burton, Dennis R.; Wrin, Terri; Petropoulos, Christos J.; Ballantyne, John A.; Chambers, Michael; Whalen, Robert G.

    2011-01-01

    A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences. PMID:21738594

  14. Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate.

    Science.gov (United States)

    Visciano, Maria Luisa; Tagliamonte, Maria; Stewart-Jones, Guillaume; Heyndrickx, Leo; Vanham, Guido; Jansson, Marianne; Fomsgaard, Anders; Grevstad, Berit; Ramaswamy, Meghna; Buonaguro, Franco M; Tornesello, Maria Lina; Biswas, Priscilla; Scarlatti, Gabriella; Buonaguro, Luigi

    2013-07-08

    Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

  15. Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

    Directory of Open Access Journals (Sweden)

    Shoufeng Ren

    2018-01-01

    Full Text Available Ebola virus (EBOV causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP is the major protective antigen of EBOV, and can generate virus-like particles (VLPs by co-expression with matrix protein (VP40. In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV replicon vector DREP to express EBOV GP and matrix viral protein (VP40. EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40. Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

  16. Soluble rhesus lymphocryptovirus gp350 protects against infection and reduces viral loads in animals that become infected with virus after challenge.

    Directory of Open Access Journals (Sweden)

    Junji Sashihara

    2011-10-01

    Full Text Available Epstein-Barr virus (EBV is a human lymphocryptovirus that is associated with several malignancies. Elevated EBV DNA in the blood is observed in transplant recipients prior to, and at the time of post-transplant lymphoproliferative disease; thus, a vaccine that either prevents EBV infection or lowers the viral load might reduce certain EBV malignancies. Two major approaches have been suggested for an EBV vaccine- immunization with either EBV glycoprotein 350 (gp350 or EBV latency proteins (e.g. EBV nuclear antigens [EBNAs]. No comparative trials, however, have been performed. Rhesus lymphocryptovirus (LCV encodes a homolog for each gene in EBV and infection of monkeys reproduces the clinical, immunologic, and virologic features of both acute and latent EBV infection. We vaccinated rhesus monkeys at 0, 4 and 12 weeks with (a soluble rhesus LCV gp350, (b virus-like replicon particles (VRPs expressing rhesus LCV gp350, (c VRPs expressing rhesus LCV gp350, EBNA-3A, and EBNA-3B, or (d PBS. Animals vaccinated with soluble gp350 produced higher levels of antibody to the glycoprotein than those vaccinated with VRPs expressing gp350. Animals vaccinated with VRPs expressing EBNA-3A and EBNA-3B developed LCV-specific CD4 and CD8 T cell immunity to these proteins, while VRPs expressing gp350 did not induce detectable T cell immunity to gp350. After challenge with rhesus LCV, animals vaccinated with soluble rhesus LCV gp350 had the best level of protection against infection based on seroconversion, viral DNA, and viral RNA in the blood after challenge. Surprisingly, animals vaccinated with gp350 that became infected had the lowest LCV DNA loads in the blood at 23 months after challenge. These studies indicate that gp350 is critical for both protection against infection with rhesus LCV and for reducing the viral load in animals that become infected after challenge. Our results suggest that additional trials with soluble EBV gp350 alone, or in combination with

  17. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    Science.gov (United States)

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

  18. Educational CPD: how UK GP trainers develop themselves as teachers.

    Science.gov (United States)

    Waters, Mark; Wall, David

    2007-09-01

    There is little in the literature giving the perspective of UK General Practice (GP) trainers on their development as teachers. What motivates GP trainers develop themselves as teachers? What obstacles to their professional development do GP trainers perceive? A questionnaire to all GP trainers in the West Midlands Deanery in 2004. 360/444 (81%) questionnaires were returned. 56.6% of GP trainers had another educational role in addition to training GP Registrars in the practice. 15.8% of trainers possessed an educational qualification. 13 had completed a Certificate in Medical Education and 28 were engaged in study towards that qualification. Trainers wanted more time to spend on their development as teachers than they presently have, and would then be interested in a wider variety of learning methods. However, 56.6% of trainers would still not choose to undertake a university-accredited course. Female GP trainers perceived more difficulty in obtaining protected time for their development as teachers (Educational CPD) (p = 0.021), were significantly less sure of their partners' support for this development (p = 0.033), and were more likely to agree with trainers undertaking a Certificate in Medical Education (p = 0.003). Having an additional educational role did not affect trainers' ability to take protected time, but significantly increased the amount of time aspired to (p = 0.005). Nothing made more difference to trainers' perception of their ability to undertake Educational CPD than did the perceived attitude of their partners. Educational CPD was very important to GP trainers, but getting protected time was difficult. Consideration of the needs and opinions of partners was a very strong barrier to trainers taking sufficient protected time. Given more available time, GP trainers would be more likely to consider gaining academic qualifications in education. However, this was not be something that all trainers wanted.

  19. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H.; Qin, Yali; Cho, Michael W., E-mail: mcho@iastate.edu

    2016-03-15

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; {sup 671}NWFDITNWLWYIK{sup 683}) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. - Highlights: • Four gp41 MPER-based immunogens that resemble fusion intermediates were generated. • C-terminal region of MPER that contains 4E10/10E8 epitopes was highly immunogenic. • Altering 6HB structure can influence immunogenic properties of the MPER. • Induced antibodies targeted multiple residues critical for 4E10/10E8 binding. • Development of immunogens based on fusion intermediates is a promising strategy.

  20. Determination of the equilibrium micelle-inserting position of the fusion peptide of gp41 of human immunodeficiency virus type 1 at amino acid resolution by exchange broadening of amide proton resonances

    International Nuclear Information System (INIS)

    Chang, D.-K.; Cheng, S.-F.

    1998-01-01

    The exchange broadening of backbone amide proton resonances of a 23-mer fusion peptide of the transmembrane subunit of HIV-1 envelope glycoprotein gp41, gp41-FP, was investigated at pH 5 and 7 at room temperature in perdeuterated sodium dodecyl sulfate (SDS) micellar solution. Comparison of resonance peaks for these pHs revealed an insignificant change in exchange rate between pH 5 and 7 for amide protons of residues 4 through 14, while the exchange rate increase at neutral pH was more prominent for amide protons of the remaining residues, with peaks from some protons becoming undetectable. The relative insensitivity to pH of the exchange for the amide protons of residues 4 through 14 is attributable to the drastic reduction in [OH-] in the micellar interior, leading to a decreased exchange rate. The A15-G16 segment represents a transition between these two regimes. The data are thus consistent with the notion that the peptide inserts into the hydrophobic core of a membrane-like structure and the A15-G16 dipeptide is located at the micellar-aqueous boundary

  1. Polymorphisms in the HIV-1 gp41 env gene, natural resistance to enfuvirtide (T-20) and pol resistance among pregnant Brazilian women.

    Science.gov (United States)

    Reis, Mônica Nogueira da Guarda; de Alcântara, Keila Correa; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

    2014-01-01

    The selective pressure of antiretroviral drugs (ARVs) targeting HIV-1 pol can promote drug resistance mutations in other genomic regions, such as env. Drug resistance among women should be monitored to avoid horizontal and mother-to-child transmission. To describe natural resistance to T-20 (enfuvirtide), gp41 env polymorphisms, mutations in pol and HIV-1 subtypes, 124 pregnant women were recruited. For 98 patients, the gp41 env, protease (PR) and reverse transcriptase (RT) fragments were sequenced. The patients were ARV naïve (n = 30), taking mother-to-child transmission prophylaxis (n = 50), or being treated with highly active ARV therapy/HAART (n = 18). The Stanford and IAS/USA databases and other sources were used to analyze PR/RT, gp41 env resistance mutations. The HIV-1 genetic diversity was analyzed by REGA/phylogenetic analyses. The patients' median age was 25 years (range, 16-42), 18.4% had AIDS. The frequency of natural resistance to T-20 (N42D, L44M, and R46M-low-impact mutations) was 6.1% (6/98); 20.4% (20/98) had compensatory mutations in HR2. The prevalence of transmitted drug resistance in the pol was 13.3% (4/30), and the prevalence of secondary drug resistance was 33.3% (6/18). Two patients were infected with multidrug resistant/MDR viruses. The analysis of HIV-1 subtypes (PR/RT/gp41) revealed that 61.2% (60/98) were subtype B, 12.2% (12/98) were subtype C, 4.1% (4/98) were subtype F1, and 22.4% (22/98) were possible recombinants (BF1 = 20.4%; BC = 2%). Natural resistance to T-20 was not associated with pol resistance or previous ARV use. The high rate of secondary resistance, including MDR, indicates that the number of women that may need T-20 salvage therapy may be higher than anticipated. © 2013 Wiley Periodicals, Inc.

  2. The process behind the expression of mdr-1/P-gp and mrp/MRP in human leukemia/lymphoma.

    Science.gov (United States)

    Hirose, Masao

    2009-04-01

    There is a controversy over the link between phenotypes of multidrug resistance (MDR) and clinical outcome in leukemia/lymphoma patients. This may be because the process behind the induction and loss of expression of genotypes and phenotypes by which MDR develops and the role of MDR in fresh cells of human leukemia/lymphoma are not clearly defined. P-glycoprotein (P-gp) increased and decreased along with mdr-1 expression in three cell lines out of five vincristine (VCR)-resistant cell lines. MRP appeared with increased mrp expression in the other two cell lines. After the drug was removed from the culture system, mdr-1/P-gp changed in parallel with the level of VCR resistance, although mrp and MRP did not. It was concluded that P-gp is directly derived from mdr-1 and that mdr-1/P-gp supports the VCR-resistance but mrp/MRP is not directly linked to the VCR-resistance. These results should contribute to a better understanding of MDR phenomenon in cancer.

  3. Construction and Characterization of a Humanized Anti-Epstein-Barr Virus gp350 Antibody with Neutralizing Activity in Cell Culture

    Directory of Open Access Journals (Sweden)

    Jerome E. Tanner

    2018-04-01

    Full Text Available Acute Epstein-Barr virus (EBV infection in immunosuppressed transplant patients can give rise to a malignant B-cell proliferation known as post-transplant lymphoproliferative disease (PTLD. The EBV major virion surface glycoprotein (gp350 is a principal target of naturally occurring neutralizing antibodies and is viewed as the best target to prevent acute infection and PTLD in at-risk transplant recipients. We have constructed a humanized (hu version of the murine anti-gp350 neutralizing monoclonal antibody 72a1. The hu72a1 IgG1 antibody displayed no significant anti-mouse activity, recognized both gp350 and its splice variant gp220 as well as a gp350 peptide that was shown to constitute the principal EBV gp350 neutralizing epitope when tested in immunoassays. Hu72a1 antibody blocked in vitro EBV infection of B cells at a level which equaled that of a mouse-human chimeric 72a1 antibody construct. This work provides a further structural and immunological understanding of the 72a1 antibody interaction with EBV gp350, and constitutes a launch point for future anti-EBV therapeutic antibodies designed to block EBV infection and prevent PTLD while eliminating the deleterious antigenic murine features of the original 72a1 antibody.

  4. Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    S Gnanakaran

    Full Text Available A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an

  5. Increased Circulating Level of the Survival Factor GP88 (Progranulin in the Serum of Breast Cancer Patients When Compared to Healthy Subjects

    Directory of Open Access Journals (Sweden)

    Katherine Rak Tkaczuk

    2011-01-01

    Full Text Available Introduction GP88 (PC-Cell Derived Growth Factor, progranulin is a glycoprotein overexpressed in breast tumors and involved in their proliferation and survival. Since GP88 is secreted, an exploratory study was established to compare serum GP88 level between breast cancer patients (BC and healthy volunteers (HV. Methods An IRB approved prospective study enrolled 189 stage 1–4 BC patients and 18 HV. GP88 serum concentration was determined by immunoassay. Results Serum GP88 level was 28.7+ 5.8 ng/ml in HV and increased to 40.7+ 16.0 ng/ml ( P = 0.007 for stage 1-3 and 45.3 +23.3 ng/ml ( P = 0.0007 for stage 4 BC patients. There was no correlation between the GP88 level and BC characteristics such as age, race, tumor grade, ER, PR and HER-2 expression. Conclusion These data suggest that serial testing of serum GP88 levels may have value as a circulating biomarker for detection, monitoring and follow up of BC.

  6. High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

    Science.gov (United States)

    Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

    2016-02-01

    We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

  7. A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection.

    Science.gov (United States)

    Alsmadi, O; Herz, R; Murphy, E; Pinter, A; Tilley, S A

    1997-01-01

    Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection. PMID

  8. Membrane proximal external region of gp41 from HIV-1 strains HXB2 and JRFL has different sensitivity to alanine mutation

    Science.gov (United States)

    Yi, Hyun Ah; Diaz-Rohrer, Barbara; Saminathan, Priyanka; Jacobs, Amy

    2015-01-01

    The transmembrane subunit (gp41) of the HIV envelope protein complex (Env) mediates the viral fusion step of HIV entry. The membrane-proximal external region (MPER), one of the functional domains of gp41, has been the focus of a great deal of research because it is a target for neutralizing antibodies. In this study, we examined 23 amino acid residues in MPER (660-683) in both a CXCR4 co-receptor utilizing strain (HXB2) and a CCR5 utilizing strain (JRFL) by alanine scanning mutagenesis. Despite the high degree of gp41 sequence conservation, the effects of alanine mutation in the MPER were different between the two strains. Most mutations in HXB2 had fusogenicity and protein expression levels not less than 50% of wild type in the case of cell-cell fusion. However, about thirty percent of the mutants in HXB2 showed a severe defect in fusogenicity in viral entry. Mutations in the MPER of strain JRFL had more dramatic effects than HXB2 in cell-cell fusion and viral entry. The fact that there are large differences in the effects of mutation between two strains suggests the potential for MPER interaction with non-conserved sequences such as the fusion peptide and/or other NHR domains as well as potential long-range structural effects on the conformational changes that occur with the Env complex during membrane fusion. PMID:25649507

  9. A monocyte chemotaxis inhibiting factor in serum of HIV infected men shares epitopes with the HIV transmembrane protein gp41

    NARCIS (Netherlands)

    Tas, M.; Drexhage, H. A.; Goudsmit, J.

    1988-01-01

    This report describes that gp41, the transmembranous envelope protein of HIV, is able to inhibit monocyte chemotaxis (measured as FMLP-induced polarization). To study the presence of such immunosuppressive HIV env proteins in the circulation of HIV-infected men, fractions were prepared from serum

  10. GP registrar well-being: a cross-sectional survey.

    Science.gov (United States)

    Schattner, Peter; Mazalin, Dennis; Pier, Ciaran; Wainer, Jo; Ling, Mee Yoke

    2010-02-09

    To investigate the major stressors affecting GP registrars, how those at risk can be best identified and the most useful methods of managing or reducing their stress. Cross-sectional postal questionnaire of all GP registrars in one large regional training provider's catchment area. The Depression, Anxiety and Stress Scale (DASS), a specifically developed Registrar Stressor Scale consisting of five subscales of potential stressors, plus closed questions on how to identify and manage stress in GP registrars. Survey response rate of 51% (102/199). Rural difficulties followed by achieving a work/life balance were the principal stressors. Ten percent of registrars were mildly or moderately depressed or anxious (DASS) and 7% mild to moderately anxious (DASS). Registrars preferred informal means of identifying those under stress (a buddy system and talks with their supervisors); similarly, they preferred to manage stress by discussions with family and friends, debriefing with peers and colleagues, or undertaking sport and leisure activities. This study supports research which confirms that poor psychological well-being is an important issue for a significant minority of GP trainees. Regional training providers should ensure that they facilitate formal and informal strategies to identify those at risk and assist them to cope with their stress.

  11. CGP lil-gp 2.1;1.02 User's Manual

    Science.gov (United States)

    Janikow, Cezary Z.; DeWeese, Scott W.

    1997-01-01

    This document describes extensions provided to lil-gp facilitating dealing with constraints. This document deals specifically with lil-gp 1.02, and the resulting extension is referred to as CGP lil-gp 2.1; 1.02 (the first version is for the extension, the second for the utilized lil-gp version). Unless explicitly needed to avoid confusion, version numbers are omitted.

  12. 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

    - and O-linked carbohydrate epitopes (LeY, A1, and sialyl-Tn) were able to block infection by cell-free virus as well as inhibit syncytium formation. Inhibition of virus infectivity was independent of virus strain (HTLVIIIB or patient isolate SSI-002), the cell line used for virus propagation (H9 or MT4......), and the cell type used as the infection target (MT4, PMC, or selected T4 lymphocytes). Inhibition was observed when viruses were preincubated with MAbs but not when cells were preincubated with MAbs before inoculation, and the MAbs were shown to precipitate 125I-labeled gp120. The MAbs therefore define...

  13. The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors

    Directory of Open Access Journals (Sweden)

    Martín-García Julio

    2008-10-01

    Full Text Available Abstract Background HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2, we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env. Results Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283 has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env

  14. High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis.

    Science.gov (United States)

    Weiss, Eric R; Alter, Galit; Ogembo, Javier Gordon; Henderson, Jennifer L; Tabak, Barbara; Bakiş, Yasin; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Luzuriaga, Katherine

    2017-01-01

    The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a

  15. Interferon-β induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier.

    Science.gov (United States)

    Nazli, Aisha; Dizzell, Sara; Zahoor, Muhammad Atif; Ferreira, Victor H; Kafka, Jessica; Woods, Matthew William; Ouellet, Michel; Ashkar, Ali A; Tremblay, Michel J; Bowdish, Dawn Me; Kaushic, Charu

    2018-03-19

    More than 40% of HIV infections occur via female reproductive tract (FRT) through heterosexual transmission. Epithelial cells that line the female genital mucosa are the first line of defense against HIV-1 and other sexually transmitted pathogens. These sentient cells recognize and respond to external stimuli by induction of a range of carefully balanced innate immune responses. Previously, we have shown that in response to HIV-1 gp120, the genital epithelial cells (GECs) from upper reproductive tract induce an inflammatory response that may facilitate HIV-1 translocation and infection. In this study, we report that the endometrial and endocervical GECs simultaneously induce biologically active interferon-β (IFNβ) antiviral responses following exposure to HIV-1 that act to protect the epithelial tight junction barrier. The innate antiviral response was directly induced by HIV-1 envelope glycoprotein gp120 and addition of gp120 neutralizing antibody inhibited IFNβ production. Interferon-β was induced by gp120 in upper GECs through Toll-like receptor 2 signaling and required presence of heparan sulfate on epithelial cell surface. The induction of IFNβ was dependent upon activation of transcription factor IRF3 (interferon regulatory factor 3). The IFNβ was biologically active, had a protective effect on epithelial tight junction barrier and was able to inhibit HIV-1 infection in TZM-bl indicator cells and HIV-1 replication in T cells. This is the first report that recognition of HIV-1 by upper GECs leads to induction of innate antiviral pathways. This could explain the overall low infectivity of HIV-1 in the FRT and could be exploited for HIV-1 prophylaxis.Cellular and Molecular Immunology advance online publication, 19 March 2018; doi:10.1038/cmi.2017.168.

  16. Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract

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    Lixia Ji

    2017-01-01

    Full Text Available Objective. Lens osmotic expansion, provoked by overactivated aldose reductase (AR, is the most essential event of sugar cataract. Chloride channel 3 (Clcn3 is a volume-sensitive channel, mainly participating in the regulation of cell fundamental volume, and P-glycoprotein (P-gp acts as its modulator. We aim to study whether P-gp and Clcn3 are involved in lens osmotic expansion of galactosemic cataract. Methods and Results. In vitro, lens epithelial cells (LECs were primarily cultured in gradient galactose medium (10–60 mM, more and more vacuoles appeared in LEC cytoplasm, and mRNA and protein levels of AR, P-gp, and Clcn3 were synchronously upregulated along with the increase of galactose concentration. In vivo, we focused on the early stage of rat galactosemic cataract, amount of vacuoles arose from equatorial area and scattered to the whole anterior capsule of lenses from the 3rd day to the 9th day, and mRNA and protein levels of P-gp and Clcn3 reached the peak around the 9th or 12th day. Conclusion. Galactosemia caused the osmotic stress in lenses; it also markedly leads to the upregulations of AR, P-gp, and Clcn3 in LECs, together resulting in obvious osmotic expansion in vitro and in vivo.

  17. GP registrar well-being: a cross-sectional survey

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    Schattner Peter

    2010-02-01

    Full Text Available Abstract Objectives To investigate the major stressors affecting GP registrars, how those at risk can be best identified and the most useful methods of managing or reducing their stress. Design, setting and participants Cross-sectional postal questionnaire of all GP registrars in one large regional training provider's catchment area. Main outcome measures The Depression, Anxiety and Stress Scale (DASS, a specifically developed Registrar Stressor Scale consisting of five subscales of potential stressors, plus closed questions on how to identify and manage stress in GP registrars. Results Survey response rate of 51% (102/199. Rural difficulties followed by achieving a work/life balance were the principal stressors. Ten percent of registrars were mildly or moderately depressed or anxious (DASS and 7% mild to moderately anxious (DASS. Registrars preferred informal means of identifying those under stress (a buddy system and talks with their supervisors; similarly, they preferred to manage stress by discussions with family and friends, debriefing with peers and colleagues, or undertaking sport and leisure activities. Conclusions This study supports research which confirms that poor psychological well-being is an important issue for a significant minority of GP trainees. Regional training providers should ensure that they facilitate formal and informal strategies to identify those at risk and assist them to cope with their stress.

  18. Autographa californica multiple nucleopolyhedrovirus GP64 protein: Analysis of domain I and V amino acid interactions and membrane fusion activity

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Qianlong [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Blissard, Gary W. [Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, United State (United States); Liu, Tong-Xian [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Li, Zhaofei, E-mail: zhaofeili73@outlook.com [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China)

    2016-01-15

    The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.

  19. Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

    Directory of Open Access Journals (Sweden)

    Jacqueline Ameri

    2017-04-01

    Full Text Available Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2 as a specific cell surface marker for isolating pancreatic endoderm cells (PECs from differentiated hESCs and human fetal pancreas. Isolated GP2+ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2+ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

  20. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    Directory of Open Access Journals (Sweden)

    Elena Gustchina

    Full Text Available A series of mini-antibodies (monovalent and bivalent Fabs targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066 broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062 non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN363 or 3-H has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen

  1. Glycosaminoglycans are interactants of Langerin: comparison with gp120 highlights an unexpected calcium-independent binding mode.

    Science.gov (United States)

    Chabrol, Eric; Nurisso, Alessandra; Daina, Antoine; Vassal-Stermann, Emilie; Thepaut, Michel; Girard, Eric; Vivès, Romain R; Fieschi, Franck

    2012-01-01

    Langerin is a C-type lectin specifically expressed in Langerhans cells. As recently shown for HIV, Langerin is thought to capture pathogens and mediate their internalisation into Birbeck Granules for elimination. However, the precise functions of Langerin remain elusive, mostly because of the lack of information on its binding properties and physiological ligands. Based on recent reports that Langerin binds to sulfated sugars, we conducted here a comparative analysis of Langerin interaction with mannose-rich HIV glycoprotein gp120 and glycosaminoglycan (GAGs), a family of sulfated polysaccharides expressed at the surface of most mammalian cells. Our results first revealed that Langerin bound to these different glycans through very distinct mechanisms and led to the identification of a novel, GAG-specific binding mode within Langerin. In contrast to the canonical lectin domain, this new binding site showed no Ca(2+)-dependency, and could only be detected in entire, trimeric extracellular domains of Langerin. Interestingly binding to GAGs, did not simply rely on a net charge effect, but rather on more discrete saccharide features, such as 6-O-sulfation, or iduronic acid content. Using molecular modelling simulations, we proposed a model of Langerin/heparin complex, which located the GAG binding site at the interface of two of the three Carbohydrate-recognition domains of the protein, at the edge of the a-helix coiled-coil. To our knowledge, the binding properties that we have highlighted here for Langerin, have never been reported for C-type lectins before. These findings provide new insights towards the understanding of Langerin biological functions.

  2. Glycosaminoglycans are interactants of Langerin: comparison with gp120 highlights an unexpected calcium-independent binding mode.

    Directory of Open Access Journals (Sweden)

    Eric Chabrol

    Full Text Available Langerin is a C-type lectin specifically expressed in Langerhans cells. As recently shown for HIV, Langerin is thought to capture pathogens and mediate their internalisation into Birbeck Granules for elimination. However, the precise functions of Langerin remain elusive, mostly because of the lack of information on its binding properties and physiological ligands. Based on recent reports that Langerin binds to sulfated sugars, we conducted here a comparative analysis of Langerin interaction with mannose-rich HIV glycoprotein gp120 and glycosaminoglycan (GAGs, a family of sulfated polysaccharides expressed at the surface of most mammalian cells. Our results first revealed that Langerin bound to these different glycans through very distinct mechanisms and led to the identification of a novel, GAG-specific binding mode within Langerin. In contrast to the canonical lectin domain, this new binding site showed no Ca(2+-dependency, and could only be detected in entire, trimeric extracellular domains of Langerin. Interestingly binding to GAGs, did not simply rely on a net charge effect, but rather on more discrete saccharide features, such as 6-O-sulfation, or iduronic acid content. Using molecular modelling simulations, we proposed a model of Langerin/heparin complex, which located the GAG binding site at the interface of two of the three Carbohydrate-recognition domains of the protein, at the edge of the a-helix coiled-coil. To our knowledge, the binding properties that we have highlighted here for Langerin, have never been reported for C-type lectins before. These findings provide new insights towards the understanding of Langerin biological functions.

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

    Science.gov (United States)

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

    1999-01-01

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

  4. cAMP promotes the synthesis in early G1 of gp115, a yeast glycoprotein containing glycosyl-phosphatidylinositol.

    Science.gov (United States)

    Grandori, R; Popolo, L; Vai, M; Alberghina, L

    1990-08-25

    The glycoprotein gp115 (Mr = 115,000, pI 4.8-5) is localized in the plasma membrane of Saccharomyces cerevisiae cells and maximally expressed during G1 phase. To gain insight on the mechanism regulating its synthesis, we have examined various conditions of cell proliferation arrest. We used pulse-labeling experiments with [35S]methionine and two-dimensional gel electrophoresis analysis, which allow the detection of the well characterized 100-kDa precursor of gp115 (p100). In the cAMP-requiring mutant cyr1, p100 synthesis is active during exponential growth, shut off by cAMP removal, and induced when growth is restored by cAMP readdition. The inhibition of p100 synthesis also occurs in TS1 mutant cells (ras1ras2-ts1) shifted from 24 to 37 degrees C. During nitrogen starvation of rca1 cells, a mutant permeable to cAMP, p100 synthesis is also inhibited. cAMP complements the effect of ammonium deprivation, promoting p100 synthesis, even when added to cells which have already entered G0. Experiments with the bcy1 and cyr1bcy1 mutants have indicated the involvement of the cAMP-dependent protein kinases in the control of p100 synthesis. Moreover, the synthesis of p100 was unaffected in A364A cells, terminally arrested at START B by alpha-factor. These results indicate that the switch operating on p100 synthesis is localized in early G1 (START A) and is one of the multiple events controlled by the cAMP pathway.

  5. Natural mannosylation of HIV-1 gp120 imposes no immunoregulatory effects in primary human plasmacytoid dendritic cells

    DEFF Research Database (Denmark)

    Søndergaard, Jonas Nørskov; Vinner, Lasse; Pedersen, Susanne Brix

    2014-01-01

    Plasmacytoid dendritic cells (pDCs) play a vital role in activation of anti-HIV-1 immunity, and suppression of pDCs might mitigate immune responses against HIV-1. HIV-1 gp120 high-mannose has been attributed immunosuppressive roles in human myeloid DCs, but no receptors for high-mannose have so far...... or viable HIV-1 particles with various degrees of mannosylation were cultured with pDCs. Activation of pDCs was determined by assaying secretion of IFN-alpha, viability, and upregulation of several pDC-activation markers: CD40, CD86, HLA-DR, CCR7, and PD-L1. The level of activation negatively correlated...

  6. P-gp expression levels in the erythrocytes of brown trout: a new tool for aquatic sentinel biomarker development.

    Science.gov (United States)

    Valton, Emeline; Wawrzyniak, Ivan; Amblard, Christian; Combourieu, Bruno; Bayle, Marie-Laure; Desmolles, François; Kwiatkowski, Fabrice; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2017-09-01

    P-glycoprotein (P-gp) is a ubiquitous membrane detoxification pump involved in cellular defence against xenobiotics. Blood is a hub for the trade and transport of physiological molecules and xenobiotics. Our recent studies have highlighted the expression of a 140-kDa P-gp in brown trout erythrocytes in primary cell culture and its dose-dependent response to Benzo[a]pyrene pollutant. The purpose of this study was focused on using P-gp expression in brown trout erythrocytes as a biomarker for detecting the degree of river pollution. abcb1 gene and P-gp expression level were analysed by reverse transcriptase-PCR and Western blot, in the erythrocytes of brown trouts. The latter were collected in upstream and downstream of four rivers in which 17 polycyclic aromatic hydrocarbons and 348 varieties of pesticides micro-residues were analysed by liquid chromatography and mass spectrometry. The abcb1 gene and the 140-kDa P-gp were not expressed in trout erythrocytes from uncontaminated river. In contrast, they are clearly expressed in contaminated rivers, in correlation with the river pollution degree and the nature of the pollutants. This biological tool may offer considerable advantages since it provides an effective response to the increasing need for an early biomarker.

  7. Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS.

    Science.gov (United States)

    Xie, Weidong; Zhang, Yaou; Bu, Cunya; Sun, Shijing; Hu, Shaoliang; Cai, Guoping

    2011-01-01

    Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. An Electrically Tight In Vitro Blood-Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

    DEFF Research Database (Denmark)

    Helms, Hans Christian; Hersom, Maria; Kuhlmann, Louise Borella

    2014-01-01

    Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate......, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one...... isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport....

  9. HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer

    CSIR Research Space (South Africa)

    Rangel Lopes de Campos, W

    2014-10-01

    Full Text Available - surface receptors, the CXCR4 chemokine receptor and the TNF- R1 death receptor. The predominant apoptotic pathway varied from CXCR4-triggered, mitochondrion-initiated to CD95/Fas- ligand initiated, depending on the culture conditions as previously reported... virus type 1 gp120 to CXCR4 induces mitochondrial transmembrane depolarization and cytochrome c- mediated apoptosis independently of Fas signaling. J Virol 75: 7637–7650. 26. Bottarel F, Feito MJ, Bragardo M, Bonissoni S, Buonfiglio D, et al. (1999...

  10. Differences in serum IgA responses to HIV-1 gp41 in elite controllers compared to viral suppressors on highly active antiretroviral therapy.

    Directory of Open Access Journals (Sweden)

    Rafiq Nabi

    Full Text Available Mechanisms responsible for natural control of human immunodeficiency type 1 (HIV replication in elite controllers (EC remain incompletely defined. To determine if EC generate high quality HIV-specific IgA responses, we used Western blotting to compare the specificities and frequencies of IgA to HIV antigens in serum of gender-, age- and race-matched EC and aviremic controllers (HC and viremic noncontrollers (HN on highly active antiretroviral therapy (HAART. Concentrations and avidity of IgA to HIV antigens were measured using ELISA or multiplex assays. Measurements for IgG were performed in parallel. EC were found to have stronger p24- and V1V2-specific IgG responses than HN, but there were no IgG differences for EC and HC. In contrast, IgA in EC serum bound more frequently to gp160 and gag proteins than IgA in HC or HN. The avidity of anti-gp41 IgA was also greater in EC, and these subjects had stronger IgA responses to the gp41 heptad repeat region 1 (HR1, a reported target of anti-bacterial RNA polymerase antibodies that cross react with gp41. However, EC did not demonstrate greater IgA responses to E. coli RNA polymerase or to peptides containing the shared LRAI sequence, suggesting that most of their HR1-specific IgA antibodies were not induced by intestinal microbiota. In both EC and HAART recipients, the concentrations of HIV-specific IgG were greater than HIV-specific IgA, but their avidities were comparable, implying that they could compete for antigen. Exceptions were C1 peptides and V1V2 loops. IgG and IgA responses to these antigens were discordant, with IgG reacting to V1V2, and IgA reacting to C1, especially in EC. Interestingly, EC with IgG hypergammaglobulinemia had greater HIV-specific IgA and IgG responses than EC with normal total IgG levels. Heterogeneity in EC antibody responses may therefore be due to a more focused HIV-specific B cell response in some of these individuals. Overall, these data suggest that development of

  11. Determination of the minimal fusion peptide of bovine leukemia virus gp30

    International Nuclear Information System (INIS)

    Lorin, Aurelien; Lins, Laurence; Stroobant, Vincent; Brasseur, Robert; Charloteaux, Benoit

    2007-01-01

    In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide

  12. Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

    Science.gov (United States)

    Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

    2018-01-15

    Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

  13. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

    Directory of Open Access Journals (Sweden)

    Natalia Brzozowska

    2016-05-01

    Full Text Available Cannabidiol (CBD is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp and breast cancer resistance protein (Bcrp mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−, Bcrp knockout (Abcg2−∕−, combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕− and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

  14. A Phase I Trial of Epstein-Barr Virus Gp350 Vaccine for Children With Chronic Kidney Disease Awaiting Transplantation

    NARCIS (Netherlands)

    Rees, L.; Tizard, E.J.; Morgan, A.J.; Cubitt, W.D.; Finerty, S.; Oyewole-Eletu, T.A.; Owen, K.; Royed, C.; Stevens, S.J.C.; Shroff, R.C.; Tanday, M.K.; Wilson, A.; Middeldorp, J.M.; Amlot, P.L.; Steven, N.M.

    2009-01-01

    Background. Vaccination against Epstein-Barr virus (EBV), inducing an antibody response to the envelope glycoprotein gp350, might protect EBV-negative children with chronic kidney disease from lymphoproliferative disease after transplantation. Methods. A phase I trial recruited children with chronic

  15. Involvement of Leishmania donovani major surface glycoprotein ...

    Indian Academy of Sciences (India)

    The major surface glycoprotein gp63 of the kinetoplastid protozoal parasite Leishmania is implicated as a ligand mediating uptake of the parasite into, and survival within, the host macrophage. By expressing gp63 antisense RNA from an episomal vector in L. donovani promastigotes, gp63-deficient transfectants were ...

  16. Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment

    International Nuclear Information System (INIS)

    Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

    2008-01-01

    Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS

  17. Requirements within the Ebola Viral Glycoprotein for Tetherin Antagonism

    Directory of Open Access Journals (Sweden)

    Nathan H. Vande Burgt

    2015-10-01

    Full Text Available Tetherin is an interferon-induced, intrinsic cellular response factor that blocks release of numerous viruses, including Ebola virus, from infected cells. As with many viruses targeted by host factors, Ebola virus employs a tetherin antagonist, the viral glycoprotein (EboGP, to counteract restriction and promote virus release. Unlike other tetherin antagonists such as HIV-1 Vpu or KSHV K5, the features within EboGP needed to overcome tetherin are not well characterized. Here, we describe sequences within the EboGP ectodomain and membrane spanning domain (msd as necessary to relieve tetherin restriction of viral particle budding. Fusing the EboGP msd to a normally secreted form of the glycoprotein effectively promotes Ebola virus particle release. Cellular protein or lipid anchors could not substitute for the EboGP msd. The requirement for the EboGP msd was not specific for filovirus budding, as similar results were seen with HIV particles. Furthermore trafficking of chimeric proteins to budding sites did not correlate with an ability to counter tetherin. Additionally, we find that a glycoprotein construct, which mimics the cathepsin-activated species by proteolytic removal of the EboGP glycan cap and mucin domains, is unable to counteract tetherin. Combining these results suggests an important role for the EboGP glycan cap and msd in tetherin antagonism.

  18. Exploring resilience in rural GP registrars--implications for training.

    Science.gov (United States)

    Walters, Lucie; Laurence, Caroline O; Dollard, Joanne; Elliott, Taryn; Eley, Diann S

    2015-07-02

    Resilience can be defined as the ability to rebound from adversity and overcome difficult circumstances. General Practice (GP) registrars face many challenges in transitioning into general practice, and additional stressors and pressures apply for those choosing a career in rural practice. At this time of international rural generalist medical workforce shortages, it is important to focus on the needs of rural GP registrars and how to support them to become resilient health care providers. This study sought to explore GP registrars' perceptions of their resilience and strategies they used to maintain resilience in rural general practice. In this qualitative interpretive research, semi-structured interviews were recorded, transcribed and analysed using an inductive approach. Initial coding resulted in a coding framework which was refined using constant comparison and negative case analysis. Authors developed consensus around the final conceptual model. Eighteen GP registrars from: Australian College of Rural and Remote Medicine Independent Pathway, and three GP regional training programs with rural training posts. Six main themes emerged from the data. Firstly, rural GP registrars described four dichotomous tensions they faced: clinical caution versus clinical courage; flexibility versus persistence; reflective practice versus task-focused practice; and personal connections versus professional commitment. Further themes included: personal skills for balance which facilitated resilience including optimistic attitude, self-reflection and metacognition; and finally GP registrars recognised the role of their supervisors in supporting and stretching them to enhance their clinical resilience. Resilience is maintained as on a wobble board by balancing professional tensions within acceptable limits. These limits are unique to each individual, and may be expanded through personal growth and professional development as part of rural general practice training.

  19. A multi-system approach assessing the interaction of anticonvulsants with P-gp.

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    David Dickens

    Full Text Available 30% of epilepsy patients receiving antiepileptic drugs (AEDs are not fully controlled by therapy. The drug transporter hypothesis for refractory epilepsy proposes that P-gp is over expressed at the epileptic focus with a role of P-gp in extruding AEDs from the brain. However, there is controversy regarding whether all AEDs are substrates for this transporter. Our aim was to investigate transport of phenytoin, lamotrigine and carbamazepine by using seven in-vitro transport models. Uptake assays in CEM/VBL cell lines, oocytes expressing human P-gp and an immortalised human brain endothelial cell line (hCMEC/D3 were carried out. Concentration equilibrium transport assays were performed in Caco-2, MDCKII ±P-gp and LLC-PK1±P-gp in the absence or presence of tariquidar, an inhibitor of P-gp. Finally, primary porcine brain endothelial cells were used to determine the apparent permeability (Papp of the three AEDs in the absence or presence of P-gp inhibitors. We detected weak transport of phenytoin in two of the transport systems (MDCK and LLC-PK1 cells transfected with human P-gp but not in the remaining five. No P-gp interaction was observed for lamotrigine or carbamazepine in any of the seven validated in-vitro transport models. Neither lamotrigine nor carbamazepine was a substrate for P-gp in any of the model systems tested. Our data suggest that P-gp is unlikely to contribute to the pathogenesis of refractory epilepsy through transport of carbamazepine or lamotrigine.

  20. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

    Science.gov (United States)

    Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

    2010-11-18

    The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  1. Dual-phase 99mTc-MIBI imaging and the expressions of P-gp, GST-π, and MRP1 in hyperparathyroidism.

    Science.gov (United States)

    Xue, Jianjun; Liu, Yan; Yang, Danrong; Yu, Yan; Geng, Qianqian; Ji, Ting; Yang, Lulu; Wang, Qi; Wang, Yuanbo; Lu, Xueni; Yang, Aimin

    2017-10-01

    The aim of this study was to further elucidate the mechanisms of dual-phase technetium-99m methoxyisobutylisonitrile (Tc-MIBI) parathyroid imaging by exploring the association between early uptake results (EUR), delayed uptake results (DUR), and the retention index (RI) in dual-phase Tc-MIBI parathyroid imaging and P glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and glutathione S-transferase-π (GST-π) expression in hyperparathyroidism (HPT). Preoperative dual-phase (early and delayed) Tc-MIBI imaging was performed on 74 patients undergoing parathyroidectomy for HPT. EUR, DUR, and RI were calculated. P-gp, MRP1, and GST-π expressions were assessed using immunohistochemistry in resected tissue from HPT and control patients. The association between P-gp, MRP1, and GST-π expressions and EUR, DUR, and RI in HPT was evaluated. The positive rate of dual-phase T c-MIBI imaging was 91.89% (68/74) and the false-negative rate was 8.11% (6/74). P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients (47.37 and 81.5%, P<0.05); there was no difference in MRP1. EUR were associated with P-gp and GST-π expressions, and DUR were associated with MRP1 expression. There was a significant difference in MRP1 expression between RI greater than or equal to 0 and RI less than 0. There was no relationship between the sensitivity of dual-phase Tc-MIBI imaging and P-gp, MRP1, and GST-π expressions in resected parathyroid tissue. The six false-negative HPT cases consisted of three P-gp (-)/MRP1 (-) tissues, three P-gp (-)/GST-π (-) tissues, and four MRP1 (-)/GST-π (-) tissues. As P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients, Tc-MIBI may wash out faster from normal parathyroid tissue surrounding the lesion compared with the lesion itself, facilitating detection.

  2. Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

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    Mayumi Oakland

    2013-01-01

    Full Text Available Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.

  3. Screening and Identification of ssDNA Aptamer for Human GP73

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    Jingchun Du

    2015-01-01

    Full Text Available As one tumor marker of HCC, Golgi Protein 73 (GP73 is given more promise in the early diagnosis of HCC, and aptamers have been developed to compete with antibodies as biorecognition probes in different detection system. In this study, we utilized GP73 to screen specific ssDNA aptamers by SELEX technique. First, GP73 proteins were expressed and purified by prokaryotic expression system and Nickle ion affinity chromatography, respectively. At the same time, the immunogenicity of purified GP73 was confirmed by Western blotting. The enriched ssDNA library with high binding capacity for GP73 was obtained after ten rounds of SELEX. Then, thirty ssDNA aptamers were sequenced, in which two ssDNA aptamers with identical DNA sequence were confirmed, based on the alignment results, and designated as A10-2. Furthermore, the specific antibody could block the binding of A10-2 to GP73, and the specific binding of A10-2 to GP73 was also supported by the observation that several tumor cell lines exhibited variable expression level of GP73. Significantly, the identified aptamer A10-2 could distinguish normal and cancerous liver tissues. So, our results indicate that the aptamer A10-2 might be developed into one molecular probe to detect HCC from normal liver specimens.

  4. Application of interferon modulators to overcome partial resistance of human ovarian cancers to VSV-GP oncolytic viral therapy

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    Catherine Dold

    2016-01-01

    Full Text Available Previously, we described an oncolytic vesicular stomatitis virus variant pseudotyped with the nonneurotropic glycoprotein of the lymphocytic choriomeningitis virus, VSV-GP, which was highly effective in glioblastoma. Here, we tested its potency for the treatment of ovarian cancer, a leading cause of death from gynecological malignancies. Effective oncolytic activity of VSV-GP could be demonstrated in ovarian cancer cell lines and xenografts in mice; however, remission was temporary in most mice. Analysis of the innate immune response revealed that ovarian cancer cell lines were able to respond to and produce type I interferon, inducing an antiviral state upon virus infection. This is in stark contrast to published data for other cancer cell lines, which were mostly found to be interferon incompetent. We showed that in vitro this antiviral state could be reverted by combining VSV-GP with the JAK1/2-inhibitor ruxolitinib. In addition, for the first time, we report the in vivo enhancement of oncolytic virus treatment by ruxolitinib, both in subcutaneous as well as in orthotopic xenograft mouse models, without causing significant additional toxicity. In conclusion, VSV-GP has the potential to be a potent and safe oncolytic virus to treat ovarian cancer, especially when combined with an inhibitor of the interferon response.

  5. A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate- and Co-Receptor- Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation.

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    Bridgette Janine Connell

    Full Text Available Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1-7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS as well as to an antibody (mAb 17b directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1.

  6. Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells.

    Science.gov (United States)

    Jumnongprakhon, Pichaya; Sivasinprasasn, Sivanan; Govitrapong, Piyarat; Tocharus, Chainarong; Tocharus, Jiraporn

    2017-06-01

    Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood-brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV Infection

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    Yuan Liu

    2013-12-01

    Full Text Available Golgi protein 73 (GP73, which is up-regulated in hepatocellular carcinoma (HCC, has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.

  8. Primary structure of and immunoglobulin E response to the repeat subunit of gp15/400 from human lymphatic filarial parasites

    NARCIS (Netherlands)

    Paxton, W. A.; Yazdanbakhsh, M.; Kurniawan, A.; Partono, F.; Maizels, R. M.; Selkirk, M. E.

    1993-01-01

    We have isolated and sequenced clones encoding the repeated subunit of the surface-associated glycoprotein gp15/400 from the two nematode species predominantly responsible for lymphatic filariasis in humans: Brugia malayi and Wuchereria bancrofti. The amino acid sequence of the 15-kDa subunit,

  9. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

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    Charles Sabin

    Full Text Available The human monoclonal antibody (mAb HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1 of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  10. Selection for Dutch postgraduate GP training; time for improvement

    NARCIS (Netherlands)

    Vermeulen, M.I.; Kuyvenhoven, M.M.; Zuithoff, N.P.; Tromp, F.; Graaf, Y. van der; Pieters, R.H.

    2012-01-01

    Background: In the Netherlands we select candidates for the postgraduate GP training by assessing personal qualities in interviews. Because of differences in the ratio of number of candidates and number of vacancies between the eight departments of GP training we questioned whether the risk of being

  11. A single gp120 residue can affect HIV-1 tropism in macaques.

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    Gregory Q Del Prete

    2017-09-01

    Full Text Available Species-dependent variation in proteins that aid or limit virus replication determines the ability of lentiviruses to jump between host species. Identifying and overcoming these differences facilitates the development of animal models for HIV-1, including models based on chimeric SIVs that express HIV-1 envelope (Env glycoproteins, (SHIVs and simian-tropic HIV-1 (stHIV strains. Here, we demonstrate that the inherently poor ability of most HIV-1 Env proteins to use macaque CD4 as a receptor is improved during adaptation by virus passage in macaques. We identify a single amino acid, A281, in HIV-1 Env that consistently changes during adaptation in macaques and affects the ability of HIV-1 Env to use macaque CD4. Importantly, mutations at A281 do not markedly affect HIV-1 Env neutralization properties. Our findings should facilitate the design of HIV-1 Env proteins for use in non-human primate models and thus expedite the development of clinically relevant reagents for testing interventions against HIV-1.

  12. Characterization of Human Colorectal Cancer MDR1/P-gp Fab Antibody

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    Xuemei Zhang

    2013-01-01

    Full Text Available In this study, the peptide sized 21 kDa covering P-gp transmembrane region was first prepared for generating a novel mouse monoclonal antibody Fab fragment with biological activity against multiple drug resistance protein P-gp21 by phage display technology. Phage-displayed antibody library prepared from mice spleen tissues was selected against the recombinant protein P-gp21 with five rounds of panning. A number of clones expressing Fab bound to P-gp21, showing neutralized activity in vitro, were isolated and screened by enzyme-linked immunosorbent assay based on its recognition properties to P-gp21 and human colorectal cancer tissue homogenate, resulting in identification of an optimal recombinant Fab clone (Number 29. Further characterization by recloning number 29 into an expression vector showed significant induction of the Fab antibody in the clone number 29 by Isopropyl β-D-1-thiogalactopyranoside (IPTG. After purified by HiTrap Protein L, the specificity of the Fab antibody to P-gp21 was also confirmed. Not only was the targeted region of this monoclonal Fab antibody identified as a 16-peptide epitope (ALKDKKELEGSGKIAT comprising residues 883–898 within the transmembrane (TM domain of human P-gp, but also the binding ability with it was verified. The clinical implication of our results for development of personalized therapy of colorectal cancer will be further studied.

  13. Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

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    Annie Albin Lumen

    Full Text Available We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918 to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health, Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown. These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1 bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2 partition into the basolateral membrane and directly reduce membrane permeability; (3 aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while

  14. Study and preparation of 99Tcm-GP kit for lung ventilation imaging

    International Nuclear Information System (INIS)

    Zhu Lin; Meng Fanmin; Zhang Jihong; Hong Tao; Liu Yunzhong; Liu Xiujie

    1997-01-01

    The preparation of the lyophilizing reagent, D-glucose-l-phosphate (GP) kit and the method of using this kit to label 99 Tc m to form 99 Tc m -GP for lung ventilation imaging at room temperature in a simple, rapid procedure are described. The stability of the lyophilizing reagent kit under various stock conditions is examined. The results show that all of the 99 Tc m -GP yields by the lyophilizing reagent kit are above 95% at 4 degree C (cold), 20-25 degree C (room temperature) and 40 degree C (oven) for 180, 90 and 3 days, respectively. The clinical practice indicates that in comparison with 99 Tc m -DTPA, 99 Tc m -GP has remarkable difference (P 99 Tc m -GP is an ideal radioaerosol for SPECT studies of lung ventilation. It has high alveolar deposition rate but low adhesion in the major airways compared to those of 99 Tc m -DTPA. 99 Tc m -GP also features prolonged pulmonary clearance time

  15. Clustering of HIV-1 Subtypes Based on gp120 V3 Loop electrostatic properties

    International Nuclear Information System (INIS)

    López de Victoria, Aliana; Kieslich, Chris A; Rizos, Apostolos K; Krambovitis, Elias; Morikis, Dimitrios

    2012-01-01

    The V3 loop of the glycoprotein gp120 of HIV-1 plays an important role in viral entry into cells by utilizing as coreceptor CCR5 or CXCR4, and is implicated in the phenotypic tropisms of HIV viruses. It has been hypothesized that the interaction between the V3 loop and CCR5 or CXCR4 is mediated by electrostatics. We have performed hierarchical clustering analysis of the spatial distributions of electrostatic potentials and charges of V3 loop structures containing consensus sequences of HIV-1 subtypes. Although the majority of consensus sequences have a net charge of +3, the spatial distribution of their electrostatic potentials and charges may be a discriminating factor for binding and infectivity. This is demonstrated by the formation of several small subclusters, within major clusters, which indicates common origin but distinct spatial details of electrostatic properties. Some of this information may be present, in a coarse manner, in clustering of sequences, but the spatial details are largely lost. We show the effect of ionic strength on clustering of electrostatic potentials, information that is not present in clustering of charges or sequences. We also make correlations between clustering of electrostatic potentials and net charge, coreceptor selectivity, global prevalence, and geographic distribution. Finally, we interpret coreceptor selectivity based on the N 6 X 7 T 8 |S 8 X 9 sequence glycosylation motif, the specific positive charge location according to the 11/24/25 rule, and the overall charge and electrostatic potential distribution. We propose that in addition to the sequence and the net charge of the V3 loop of each subtype, the spatial distributions of electrostatic potentials and charges may also be important factors for receptor recognition and binding and subsequent viral entry into cells. This implies that the overall electrostatic potential is responsible for long-range recognition of the V3 loop with coreceptors CCR5/CXCR4, whereas the charge

  16. Clustering of HIV-1 Subtypes Based on gp120 V3 Loop electrostatic properties

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    López de Victoria Aliana

    2012-02-01

    Full Text Available Abstract Background The V3 loop of the glycoprotein gp120 of HIV-1 plays an important role in viral entry into cells by utilizing as coreceptor CCR5 or CXCR4, and is implicated in the phenotypic tropisms of HIV viruses. It has been hypothesized that the interaction between the V3 loop and CCR5 or CXCR4 is mediated by electrostatics. We have performed hierarchical clustering analysis of the spatial distributions of electrostatic potentials and charges of V3 loop structures containing consensus sequences of HIV-1 subtypes. Results Although the majority of consensus sequences have a net charge of +3, the spatial distribution of their electrostatic potentials and charges may be a discriminating factor for binding and infectivity. This is demonstrated by the formation of several small subclusters, within major clusters, which indicates common origin but distinct spatial details of electrostatic properties. Some of this information may be present, in a coarse manner, in clustering of sequences, but the spatial details are largely lost. We show the effect of ionic strength on clustering of electrostatic potentials, information that is not present in clustering of charges or sequences. We also make correlations between clustering of electrostatic potentials and net charge, coreceptor selectivity, global prevalence, and geographic distribution. Finally, we interpret coreceptor selectivity based on the N6X7T8|S8X9 sequence glycosylation motif, the specific positive charge location according to the 11/24/25 rule, and the overall charge and electrostatic potential distribution. Conclusions We propose that in addition to the sequence and the net charge of the V3 loop of each subtype, the spatial distributions of electrostatic potentials and charges may also be important factors for receptor recognition and binding and subsequent viral entry into cells. This implies that the overall electrostatic potential is responsible for long-range recognition of the V3

  17. Attitudes towards obesity treatment in GP training practices: a focus group study

    NARCIS (Netherlands)

    Jochemsen-van der Leeuw, H. G. A.; van Dijk, N.; Wieringa-de Waard, M.

    2011-01-01

    Both patients and government expect the GP to treat obesity. Previous studies reported a negative attitude of GPs towards this task. Little is known about the attitude of GP trainees. To assess the attitude and other factors that influence the willingness and ability of GP trainees to provide

  18. IL-10 mediated by herpes simplex virus vector reduces neuropathic pain induced by HIV gp120 combined with ddC in rats.

    Science.gov (United States)

    Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C; Candiotti, Keith A; Lubarsky, David A; Hao, Shuanglin

    2014-07-30

    HIV-associated sensory neuropathy affects over 50% of HIV patients and is a common peripheral nerve complication of HIV infection and highly active antiretroviral therapy (HAART). Evidence shows that painful HIV sensory neuropathy is influenced by neuroinflammatory events that include the proinflammatory molecules, MAP Kinase, tumor necrosis factor-α (TNFα), stromal cell-derived factor 1-α (SDF1α), and C-X-C chemokine receptor type 4 (CXCR4). However, the exact mechanisms of painful HIV sensory neuropathy are not known, which hinders our ability to develop effective treatments. In this study, we investigated whether inhibition of proinflammatory factors reduces the HIV-associated neuropathic pain state. Neuropathic pain was induced by peripheral HIV coat protein gp120 combined with 2',3'-dideoxycytidine (ddC, one of the nucleoside reverse transcriptase inhibitors (NRTIs)). Mechanical threshold was tested using von Frey filament fibers. Non-replicating herpes simplex virus (HSV) vectors expressing interleukin 10 (IL10) were inoculated into the hindpaws of rats. The expression of TNFα, SDF1α, and CXCR4 in the lumbar spinal cord and L4/5 dorsal root ganglia (DRG) was examined using western blots. IL-10 expression mediated by the HSV vectors resulted in a significant elevation of mechanical threshold. The anti-allodynic effect of IL-10 expression mediated by the HSV vectors lasted more than 3 weeks. The area under the effect-time curves (AUC) in mechanical threshold in rats inoculated with the HSV vectors expressing IL-10, was increased compared with the control vectors, indicating antinociceptive effect of the IL-10 vectors. The HSV vectors expressing IL-10 also concomitantly reversed the upregulation of p-p38, TNFα, SDF1α, and CXCR4 induced by gp120 in the lumbar spinal dorsal horn and/or the DRG at 2 and/or 4 weeks. The blocking of the signaling of these proinflammatory molecules is able to reduce HIV-related neuropathic pain, which provide a novel

  19. Determinants of a GP visit and cervical cancer screening examination in Great Britain.

    Directory of Open Access Journals (Sweden)

    Alexander Michael Labeit

    Full Text Available In the UK, women are requested to attend a cervical cancer test every 3 years as part of the NHS Cervical Screening Programme. This analysis compares the determinants of a cervical cancer screening examination with the determinants of a GP visit in the same year and investigates if cervical cancer screening participation is more likely for women who visit their GP.A recursive probit model was used to analyse the determinants of GP visits and cervical cancer screening examinations. GP visits were considered to be endogenous in the cervical cancer screening examination. The analysed sample consisted of 52,551 observations from 8,386 women of the British Household Panel Survey.The analysis showed that a higher education level and a worsening self-perceived health status increased the probability of a GP visit, whereas smoking decreased the probability of a GP visit. GP visits enhanced the uptake of a cervical cancer screening examination in the same period. The only variables which had the same positive effect on both dependent variables were higher education and living with a partner. The probability of a cervical cancer screening examination increased also with previous cervical cancer screening examinations and being in the recommended age groups. All other variables had different results for the uptake of a GP visit or a cervical cancer screening examination.Most of the determinants of visiting a GP and cervical cancer screening examination differ from each other and a GP visit enhances the uptake of a smear test.

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

    International Nuclear Information System (INIS)

    Bienz, D.; Clemetson, K.J.

    1989-01-01

    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 125 I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/[ 3 H]NaBH 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

  1. Fitting the elementary rate constants of the P-gp transporter network in the hMDR1-MDCK confluent cell monolayer using a particle swarm algorithm.

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    Deep Agnani

    Full Text Available P-glycoprotein, a human multidrug resistance transporter, has been extensively studied due to its importance to human health and disease. In order to understand transport kinetics via P-gp, confluent cell monolayers overexpressing P-gp are widely used. The purpose of this study is to obtain the mass action elementary rate constants for P-gp's transport and to functionally characterize members of P-gp's network, i.e., other transporters that transport P-gp substrates in hMDR1-MDCKII confluent cell monolayers and are essential to the net substrate flux. Transport of a range of concentrations of amprenavir, loperamide, quinidine and digoxin across the confluent monolayer of cells was measured in both directions, apical to basolateral and basolateral to apical. We developed a global optimization algorithm using the Particle Swarm method that can simultaneously fit all datasets to yield accurate and exhaustive fits of these elementary rate constants. The statistical sensitivity of the fitted values was determined by using 24 identical replicate fits, yielding simple averages and standard deviations for all of the kinetic parameters, including the efflux active P-gp surface density. Digoxin required additional basolateral and apical transporters, while loperamide required just a basolateral tranporter. The data were better fit by assuming bidirectional transporters, rather than active importers, suggesting that they are not MRP or active OATP transporters. The P-gp efflux rate constants for quinidine and digoxin were about 3-fold smaller than reported ATP hydrolysis rate constants from P-gp proteoliposomes. This suggests a roughly 3∶1 stoichiometry between ATP hydrolysis and P-gp transport for these two drugs. The fitted values of the elementary rate constants for these P-gp substrates support the hypotheses that the selective pressures on P-gp are to maintain a broad substrate range and to keep xenobiotics out of the cytosol, but not out of the

  2. The conserved residue Arg46 in the N-terminal heptad repeat domain of HIV-1 gp41 is critical for viral fusion and entry.

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    Xiaoyi Wang

    Full Text Available During the process of HIV-1 fusion with the target cell, the N-terminal heptad repeat (NHR of gp41 interacts with the C-terminal heptad repeat (CHR to form fusogenic six-helix bundle (6-HB core. We previously identified a crucial residue for 6-HB formation and virus entry--Lys63 (K63 in the C-terminal region of NHR (aa 54-70, which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121 in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46, in the N-terminal region of NHR (aa 35-53, which forms a hydrogen bond with a polar residue, Asn43 (N43, in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137, in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A or the negatively charged residue Glu (R46E resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB's stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A or Arg (E137R also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.

  3. Tamoxifen reduces P-gp-mediated multidrug resistance via inhibiting the PI3K/Akt signaling pathway in ER-negative human gastric cancer cells.

    Science.gov (United States)

    Mao, Zonglei; Zhou, Jin; Luan, Junwei; Sheng, Weihua; Shen, Xiaochun; Dong, Xiaoqiang

    2014-03-01

    Multidrug resistance (MDR), mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem limiting successful chemotherapy of gastric cancer. Tamoxifen (TAM), a triphenylethylene nonsteroidal antiestrogen agent, shows broad-spectrum antitumor properties. Emerging studies demonstrated that TAM could significantly reduce the MDR in a variety of human cancers. Here we investigated the effects and possible underlying mechanisms of action of TAM on the reversion of MDR in ER-negative human gastric cancer cells. Our results demonstrated that in MDR phenotype SGC7901/CDDP gastric cancer cells TAM dramatically lowered the IC50 of CDDP, 5-FU and ADM, increased the intracellular Rhodamine123 accumulation and induced G0/G1 phase arrest, while G2/M phase decreased accordingly. Furthermore, at the molecular level, TAM substantially decreased the expression of P-gp, p-Akt and the Akt-regulated downstream effectors such as p-GSK-3β, p-BAD, Bcl-XL and cyclinD1 proteins without affecting the expression of t-Akt, t-GSK-3β, t-BAD proteins in SGC7901/CDDP cells. Thus, our findings demonstrate that TAM reverses P-gp-mediated gastric cancer cell MDR via inhibiting the PI3K/Akt signaling pathway. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  4. Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

    International Nuclear Information System (INIS)

    Shang Liang; Hunter, Eric

    2010-01-01

    The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.

  5. Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

    Science.gov (United States)

    Prakobphol, A; Xu, F; Hoang, V M; Larsson, T; Bergstrom, J; Johansson, I; Frängsmyr, L; Holmskov, U; Leffler, H; Nilsson, C; Borén, T; Wright, J R; Strömberg, N; Fisher, S J

    2000-12-22

    Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

  6. 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.

  7. GP and pharmacist inter-professional learning - a grounded theory study.

    Science.gov (United States)

    Cunningham, David E; Ferguson, Julie; Wakeling, Judy; Zlotos, Leon; Power, Ailsa

    2016-05-01

    Practice Based Small Group Learning (PBSGL) is an established learning resource for primary care clinicians in Scotland and is used by one-third of general practitioners (GPs). Scottish Government and UK professional bodies have called for GPs and pharmacists to work more closely together to improve care. To gain GPs' and pharmacists' perceptions and experiences of learning together in an inter-professional PBSGL pilot. Qualitative research methods involving established GP PBSGL groups in NHS Scotland recruiting one or two pharmacists to join them. A grounded theory method was used. GPs were interviewed in focus groups by a fellow GP, and pharmacists were interviewed individually by two researchers, neither being a GP or a pharmacist. Interviews were audio-recorded, transcribed and analysed using grounded theory methods. Data saturation was achieved and confirmed. Three themes were identified: GPs' and pharmacists' perceptions and experiences of inter-professional learning; Inter-professional relationships and team-working; Group identity and purpose of existing GP groups. Pharmacists were welcomed into GP groups and both professions valued inter-professional PBSGL learning. Participants learned from each other and both professions gained a wider perspective of the NHS and of each others' roles in the organisation. Inter-professional relationships, communication and team-working were strengthened and professionals regarded each other as peers and friends.

  8. Enhancing field GP engagement in hospital-based studies. Rationale, design, main results and participation in the diagest 3-GP motivation study

    Directory of Open Access Journals (Sweden)

    Berkhout Christophe

    2012-06-01

    Full Text Available Abstract Background Diagest 3 was a study aimed at lowering the risk of developing type 2 diabetes within 3 years after childbirth. Women with gestational diabetes were enrolled in the study. After childbirth, the subjects showed little interest in the structured education programme and did not attend workshops. Their general practitioners (GPs were approached to help motivate the subjects to participate in Diagest 3, but the GPs were reluctant. The present study aimed to understand field GPs’ attitudes towards hospital-based studies, and to develop strategies to enhance their involvement and reduce subject drop-out rates. Methods We used a three-step process: step one used a phenomenological approach exploring the beliefs, attitudes, motivations and environmental factors contributing to the GPs’ level of interest in the study. Data were collected in face-to-face interviews and coded by hand and with hermeneutic software to develop distinct GP profiles. Step two was a cross-sectional survey by questionnaire to determine the distribution of the profiles in the GP study population and whether completion of an attached case report form (CRF was associated with a particular GP profile. In step three, we assessed the impact of the motivation study on participation rates in the main study. Results Fifteen interviews were conducted to achieve data saturation. Theorisation led to the definition of 4 distinct GP profiles. The response rate to the questionnaire was 73%, but dropped to 52% when a CRF was attached. The link between GP profiles and the rate of CRF completion remains to be verified. The GPs provided data on the CRF that was of comparable quality to those collected in the main trial. Our analysis showed that the motivation study increased overall participation in the main study by 23%, accounting for 16% (24/152 of all final visits for 536 patients who were initially enrolled in the Diagest 3 study. Conclusions When a hospital-led study

  9. Enhancing field GP engagement in hospital-based studies. Rationale, design, main results and participation in the Diagest 3-GP motivation study.

    Science.gov (United States)

    Berkhout, Christophe; Vandaele-Bétancourt, Marie; Robert, Stéphane; Lespinasse, Solène; Mitha, Gamil; Bradier, Quentin; Vambergue, Anne; Fontaine, Pierre

    2012-06-21

    Diagest 3 was a study aimed at lowering the risk of developing type 2 diabetes within 3 years after childbirth. Women with gestational diabetes were enrolled in the study. After childbirth, the subjects showed little interest in the structured education programme and did not attend workshops. Their general practitioners (GPs) were approached to help motivate the subjects to participate in Diagest 3, but the GPs were reluctant. The present study aimed to understand field GPs' attitudes towards hospital-based studies, and to develop strategies to enhance their involvement and reduce subject drop-out rates. We used a three-step process: step one used a phenomenological approach exploring the beliefs, attitudes, motivations and environmental factors contributing to the GPs' level of interest in the study. Data were collected in face-to-face interviews and coded by hand and with hermeneutic software to develop distinct GP profiles. Step two was a cross-sectional survey by questionnaire to determine the distribution of the profiles in the GP study population and whether completion of an attached case report form (CRF) was associated with a particular GP profile. In step three, we assessed the impact of the motivation study on participation rates in the main study. Fifteen interviews were conducted to achieve data saturation. Theorisation led to the definition of 4 distinct GP profiles. The response rate to the questionnaire was 73%, but dropped to 52% when a CRF was attached. The link between GP profiles and the rate of CRF completion remains to be verified. The GPs provided data on the CRF that was of comparable quality to those collected in the main trial. Our analysis showed that the motivation study increased overall participation in the main study by 23%, accounting for 16% (24/152) of all final visits for 536 patients who were initially enrolled in the Diagest 3 study. When a hospital-led study explores issues in primary care, its design must anticipate GP

  10. Use of λgt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    International Nuclear Information System (INIS)

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

    1986-01-01

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector λgt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the λgt11 vector, the cloned proteins were expressed in Escherichia coli as β-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [ 14 C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved

  11. Are there practical opportunities for developing leadership skills during GP training and beyond? A survey of GP trainees and trainers in South East Scotland.

    Science.gov (United States)

    Curry, Nicola; Denney, MeiLing

    2016-01-01

    There is currently a lack of formal training in leadership skills, particularly during GP training. This study aimed to explore the current training and practical opportunities which exist, specifically exploring the views of GP trainees and trainers. An electronic questionnaire was sent to 266 GP trainees and trainers in south-east Scotland. Questions focused on respondents' experience of leadership-specific training and opportunities to engage with leadership roles. There were a total of 76 respondents (28.6% response rate). Response rate was 19.0% in trainees and 34.6% in trainers. A majority of respondents (80.0%) were established GPs. Of those who had received training in leadership, most (72.1%) underwent this after qualifying as a GP. Respondents identified a range of leadership roles within and outside the practice covering clinical and non-clinical areas. Most were interested in future leadership roles (46.7% moderately interested; 28% very interested). More time, training opportunities and the presence of GP role models were motivating factors in terms of participants' readiness to take on future leadership roles. Signposting trainees, trainers and general practitioners to leadership opportunities and training would be relatively easy but addressing a lack of motivating factors at a local level is essential. The effectiveness of such training and opportunities for experiential learning in leadership roles requires further research.

  12. The effect of gender medicine education in GP training: a prospective cohort study.

    Science.gov (United States)

    Dielissen, Patrick; Verdonk, Petra; Waard, Magreet Wieringa-de; Bottema, Ben; Lagro-Janssen, Toine

    2014-11-01

    The purpose of this study is to compare the change in general practitioner (GP) trainees' gender awareness following a modular gender medicine programme or a mainstream gender medicine programme. In 2007, a prospective study was conducted in three cohorts of in total 207 GP trainees who entered GP training in the Netherlands. The outcome measure was the Nijmegen Gender Awareness in Medicine Scale and a 16-item gender knowledge questionnaire. Two gender medicine teaching methods were compared: a modular approach (n = 75) versus a mainstream approach (n = 72). Both strategies were compared with a control cohort (n = 60). Statistical analysis included analysis of variance and t-tests. The overall response rates for the modular, mainstream and control cohort were 78, 72 and 82 %, respectively. There was a significant difference in change in gender knowledge scores between the modular cohort compared with the mainstream and control cohort (p = 0.049). There were no statistical differences between the cohorts on gender sensitivity and gender role ideology. At entry and end, female GP trainees demonstrated significantly higher gender awareness than male GP trainees. A modular teaching method is not a more favourable educational method to teach gender medicine in GP training. Female GP trainees are more gender aware, but male GP trainees are not unaware of gender-related issues.

  13. Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

    Energy Technology Data Exchange (ETDEWEB)

    Cingolani, Gino, E-mail: cingolag@upstate.edu; Andrews, Dewan [Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210 (United States); Casjens, Sherwood [Department of Pathology, Division of Cell Biology and Immunology, University of Utah Medical School, Salt Lake City, UT 84112 (United States); Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210 (United States)

    2006-05-01

    The crystallogenesis of bacteriophage P22 tail-fiber gp26 is described. To study possible pH-induced conformational changes in gp26 structure, native trimeric gp26 has been crystallized at acidic pH (4.6) and a chimera of gp26 fused to maltose-binding protein (MBP-gp26) has been crystallized at neutral and alkaline pH (7-10). Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26 forms an ∼220 Å extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell’s outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 Å resolution and belong to space group P2{sub 1}, with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman–Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 Å resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.

  14. The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

    Directory of Open Access Journals (Sweden)

    Lowe Anson W

    2009-07-01

    Full Text Available Abstract Background GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria. Methods An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding. Results GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues. Conclusion GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.

  15. 77 FR 73635 - Northwest Storage GP, LLC; Notice of Application

    Science.gov (United States)

    2012-12-11

    ...) 1254 to a proposed 346-megawatt (MW) power plant located within the north industrial area of the Port...] Northwest Storage GP, LLC; Notice of Application Take notice that on November 21, 2012, Northwest Storage GP, LLC. (Northwest) filed with the Federal Energy Regulatory Commission an application under section 7 of...

  16. A single amino-acid change in a highly conserved motif of gp41 elicits HIV-1 neutralization and protects against CD4 depletion.

    Science.gov (United States)

    Petitdemange, Caroline; Achour, Abla; Dispinseri, Stefania; Malet, Isabelle; Sennepin, Alexis; Ho Tsong Fang, Raphaël; Crouzet, Joël; Marcelin, Anne-Geneviève; Calvez, Vincent; Scarlatti, Gabriella; Debré, Patrice; Vieillard, Vincent

    2013-09-01

    The induction of neutralizing antibodies against conserved regions of the human immunodeficiency virus type 1 (HIV-1) envelope protein is a major goal of vaccine strategies. We previously identified 3S, a critical conserved motif of gp41 that induces the NKp44L ligand of an activating NK receptor. In vivo, anti-3S antibodies protect against the natural killer (NK) cell-mediated CD4 depletion that occurs without efficient viral neutralization. Specific substitutions within the 3S peptide motif were prepared by directed mutagenesis. Virus production was monitored by measuring the p24 production. Neutralization assays were performed with immune-purified antibodies from immunized mice and a cohort of HIV-infected patients. Expression of NKp44L on CD4(+) T cells and degranulation assay on activating NK cells were both performed by flow cytometry. Here, we show that specific substitutions in the 3S motif reduce viral infection without affecting gp41 production, while decreasing both its capacity to induce NKp44L expression on CD4(+) T cells and its sensitivity to autologous NK cells. Generation of antibodies in mice against the W614 specific position in the 3S motif elicited a capacity to neutralize cross-clade viruses, notable in its magnitude, breadth, and durability. Antibodies against this 3S variant were also detected in sera from some HIV-1-infected patients, demonstrating both neutralization activity and protection against CD4 depletion. These findings suggest that a specific substitution in a 3S-based immunogen might allow the generation of specific antibodies, providing a foundation for a rational vaccine that combine a capacity to neutralize HIV-1 and to protect CD4(+) T cells.

  17. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    Science.gov (United States)

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Co-treatment with grapefruit juice inhibits while chronic administration activates intestinal P-glycoprotein-mediated drug efflux.

    Science.gov (United States)

    Panchagnula, R; Bansal, T; Varma, M V S; Kaul, C L

    2005-12-01

    P-Glycoprotein (P-gp) mediated efflux is recognized as a significant biochemical barrier affecting oral absorption for a number of drugs. Various conflicting reports have been published regarding the effects of grapefruit juice (GFJ) on P-gp-mediated drug efflux, in which GFJ has been shown both to inhibit and activate it. Hence, the present study adopted a two-way approach, involving both co-treatment and chronic administration. Bi-directional transport of paclitaxel (PCL) was carried out in the absence and presence of GFJ extract, in rat everted ileum sac. Further, the effect of chronic administration of GFJ to rats was characterized by permeability studies with indinavir (INDI). Co-treatment of GFJ extract at 100% concentration reduced the asymmetric transport of PCL (efflux ratio = 20.8) by increasing absorptive (A --> B) transport by 921% and reducing secretory (B --> A) transport by 41%. Further, GFJ showed a concentration dependent effect on PCL permeability. Imipramine, a passive permeability marker with absorptive permeability of 15.33 +/- 4.26 x 10(-6) cm/s showed no asymmetric transport and also no significant (P extract inhibited P-gp-mediated efflux in co-treatment, whereas chronic administration led to increased levels of P-gp expression, thus having a profound effect on intestinal absorption and GFJ-drug interactions in vivo.

  19. In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan.

    Science.gov (United States)

    Föger, Florian; Schmitz, Thierry; Bernkop-Schnürch, Andreas

    2006-08-01

    Recently, thiolated polymers, so called thiomers, have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a proof-of-principle for a delivery system based on thiolated chitosan in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. In vitro, the permeation enhancing effect of unmodified chitosan, chitosan-4 thiobutylamidine (Ch-TBA) and the combination of Ch-TBA with reduced glutathione (GSH) was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to buffer only, Rho-123 transport in presence of 0.5% (w/v) chitosan, 0.5% (w/v) Ch-TBA and the combination of 0.5% (w/v) Ch-TBA/0.5% (w/v) GSH, was 1.8-fold, 2.6-fold, 3.8-fold improved, respectively. Furthermore, enteric-coated tablets based on unmodified chitosan or Ch-TBA/GSH, were investigated in vivo. In rats, the Ch-TBA/GSH tablets increased the area under the plasma concentration time curve (AUC0-12) of Rho-123 by 217% in comparison to buffer control and by 58% in comparison to unmodified chitosan. This in vivo study showed that a delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.

  20. Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

    Science.gov (United States)

    Balasubramanian, Preetha; Williams, Constance; Shapiro, Mariya B; Sinangil, Faruk; Higgins, Keith; Nádas, Arthur; Totrov, Maxim; Kong, Xiang-Peng; Fiore-Gartland, Andrew J; Haigwood, Nancy L; Zolla-Pazner, Susan; Hioe, Catarina E

    2018-01-11

    Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 β-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

  1. EMERGE: A Randomized Phase II Study of the Antibody-Drug Conjugate Glembatumumab Vedotin in Advanced Glycoprotein NMB-Expressing Breast Cancer.

    Science.gov (United States)

    Yardley, Denise A; Weaver, Robert; Melisko, Michelle E; Saleh, Mansoor N; Arena, Francis P; Forero, Andres; Cigler, Tessa; Stopeck, Alison; Citrin, Dennis; Oliff, Ira; Bechhold, Rebecca; Loutfi, Randa; Garcia, Agustin A; Cruickshank, Scott; Crowley, Elizabeth; Green, Jennifer; Hawthorne, Thomas; Yellin, Michael J; Davis, Thomas A; Vahdat, Linda T

    2015-05-10

    Glycoprotein NMB (gpNMB), a negative prognostic marker, is overexpressed in multiple tumor types. Glembatumumab vedotin is a gpNMB-specific monoclonal antibody conjugated to the potent cytotoxin monomethyl auristatin E. This phase II study investigated the activity of glembatumumab vedotin in advanced breast cancer by gpNMB expression. Patients (n = 124) with refractory breast cancer that expressed gpNMB in ≥ 5% of epithelial or stromal cells by central immunohistochemistry were stratified by gpNMB expression (tumor, low stromal intensity, high stromal intensity) and were randomly assigned 2:1 to glembatumumab vedotin (n = 83) or investigator's choice (IC) chemotherapy (n = 41). The study was powered to detect overall objective response rate (ORR) in the glembatumumab vedotin arm between 10% (null) and 22.5% (alternative hypothesis) with preplanned investigation of activity by gpNMB distribution and/or intensity (Stratum 1 to Stratum 3). Glembatumumab vedotin was well tolerated as compared with IC chemotherapy (less hematologic toxicity; more rash, pruritus, neuropathy, and alopecia). ORR was 6% (five of 83) for glembatumumab vedotin versus 7% (three of 41) for IC, without significant intertreatment differences for predefined strata. Secondary end point revealed ORR of 12% (10 of 83) versus 12% (five of 41) overall, and 30% (seven of 23) versus 9% (one of 11) for gpNMB overexpression (≥ 25% of tumor cells). Unplanned analysis showed ORR of 18% (five of 28) versus 0% (0 of 11) in patients with triple-negative breast cancer (TNBC), and 40% (four of 10) versus 0% (zero of six) in gpNMB-overexpressing TNBC. Glembatumumab vedotin is well tolerated in heavily pretreated patients with breast cancer. Although the primary end point in advanced gpNMB-expressing breast cancer was not met for all enrolled patients (median tumor gpNMB expression, 5%), activity may be enhanced in patients with gpNMB-overexpressing tumors and/or TNBC. A pivotal phase II trial (METRIC

  2. Multimeric scaffolds displaying the HIV-1 envelope MPER induce MPER-specific antibodies and cross-neutralizing antibodies when co-immunized with gp160 DNA.

    Directory of Open Access Journals (Sweden)

    Shelly J Krebs

    Full Text Available Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab responses toward conserved regions of the viral Envelope (Env. However, the generation of neutralizing Abs (NAbs targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

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

    Science.gov (United States)

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

    2017-01-01

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

  4. Introducing a GP copayment in Australia: Who would carry the cost burden?

    Science.gov (United States)

    Elkins, Rosemary Kate; Schurer, Stefanie

    2017-05-01

    Recent policy changes designed to contain unsustainable health expenditure growth imply that many more Australians may soon be charged a copayment to consult a GP. We explore the distributional consequences associated with a range of hypothetical GP copayment scenarios using nationally-representative Australian survey data. For each scenario, we estimate the cost burden that individuals and households across the income distribution would need to absorb to maintain their current GP service utilisation. Even when concessional patients are charged a third or a quarter of the non-concessional copayment rate, the average estimated cost burden in the lowest income quartile is typically between three and six times that of the highest, and the average cost burden for women is significantly higher than for men within every income quartile. These disparities are intensified for those with a chronic illness. We conclude that the widespread implementation of GP copayments would disproportionately burden lower-income families, who experience higher rates of chronic illness, higher demand for GP services, and lower capacity to absorb price increases. The regressive nature of GP copayments is reduced when concessional and child patients are exempted entirely, highlighting the importance of supporting GPs-particularly in disadvantaged areas-to maintain bulk-billing arrangements for vulnerable patient groups. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Computer-Aided Approaches for Targeting HIVgp41

    Directory of Open Access Journals (Sweden)

    William J. Allen

    2012-08-01

    Full Text Available Virus-cell fusion is the primary means by which the human immunodeficiency virus-1 (HIV delivers its genetic material into the human T-cell host. Fusion is mediated in large part by the viral glycoprotein 41 (gp41 which advances through four distinct conformational states: (i native, (ii pre-hairpin intermediate, (iii fusion active (fusogenic, and (iv post-fusion. The pre-hairpin intermediate is a particularly attractive step for therapeutic intervention given that gp41 N-terminal heptad repeat (NHR and C‑terminal heptad repeat (CHR domains are transiently exposed prior to the formation of a six-helix bundle required for fusion. Most peptide-based inhibitors, including the FDA‑approved drug T20, target the intermediate and there are significant efforts to develop small molecule alternatives. Here, we review current approaches to studying interactions of inhibitors with gp41 with an emphasis on atomic-level computer modeling methods including molecular dynamics, free energy analysis, and docking. Atomistic modeling yields a unique level of structural and energetic detail, complementary to experimental approaches, which will be important for the design of improved next generation anti-HIV drugs.

  6. Synergistic cooperation between methamphetamine and HIV-1 gsp120 through the P13K/Akt pathway induces IL-6 but not IL-8 expression in astrocytes.

    Directory of Open Access Journals (Sweden)

    Ankit Shah

    Full Text Available HIV-1 envelope protein gp120 has been extensively studied for neurotoxic effects that have been attributed to the increased expression of various proinflammatory cytokines in the CNS. Recently we have shown that methamphetamine (MA also increases expression of proinflammatory cytokines in astrocytes. However, combined effect of gp120 and MA is not known. The present study was undertaken to determine cumulative effect and the mechanism(s/pathways involved in the functional interaction between gp120 and MA in SVGA astrocytes. Our results clearly suggest that gp120 and MA affect IL-6 but not IL-8 in a synergistic manner and this synergy was mediated by PI3K/Akt and NF-κB pathways. Inhibition of either of these pathways could abrogate the increased expression of IL-6 due to MA or gp120 alone, as well as the increased expression of IL-6 when the astrocytes were treated with both gp120 and MA. These results were confirmed by both, using chemical inhibitors/siRNA as well as western blotting. This study therefore provides novel information regarding the interaction between MA and gp120 in terms of the expression of IL-6 and the mechanisms underlying potential synergy between MA and gp120 in astrocytes.

  7. P-glycoprotein activity and biological response

    International Nuclear Information System (INIS)

    Vaalburg, W.; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

    2005-01-01

    P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators

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

    Science.gov (United States)

    Branco, Luis M; Grove, Jessica N; Moses, Lina M; Goba, Augustine; Fullah, Mohammed; Momoh, Mambu; Schoepp, Randal J; Bausch, Daniel G; Garry, Robert F

    2010-11-09

    Lassa hemorrhagic fever (LHF) is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. With a high rate of infection that may lead to morbidity and mortality, understanding how the virus interacts with the host's immune system is of great importance for generating vaccines and therapeutics. Previous work by our group identified a soluble isoform of the Lassa virus (LASV) GP1 (sGP1) in vitro resulting from the expression of the glycoprotein complex (GPC) gene [1, 2]. Though no work has directly been done to demonstrate the function of this soluble isoform in arenaviral infections, evidence points to immunomodulatory effects against the host's immune system mediated by a secreted glycoprotein component in filoviruses, another class of hemorrhagic fever-causing viruses. A significant fraction of shed glycoprotein isoforms during viral infection and biogenesis may attenuate the host's inflammatory response, thereby enhancing viral replication and tissue damage. Such shed glycoprotein mediated effects were previously reported for Ebola virus (EBOV), a filovirus that also causes hemorrhagic fever with nearly 90 percent fatality rates [3 - 5]. The identification of an analogous phenomenon in vivo could establish a new correlate of LHF infection leading to the development of sensitive diagnostics targeting the earliest molecular events of the disease. Additionally, the reversal of potentially untoward immunomodulatory functions mediated by sGP1 could potentiate the development of novel therapeutic intervention. To this end, we investigated the presence of sGP1 in the serum of suspected LASV patients admitted to the Kenema Government Hospital (KGH) Lassa Fever Ward (LFW), in Kenema, Sierra Leone that tested positive for viral antigen or displayed classical signs of Lassa fever. It is reasonable to expect that a narrow window exists for detection of sGP1 as the sole

  9. Exploring resilience in rural GP registrars ? implications for training

    OpenAIRE

    Walters, Lucie; Laurence, Caroline O.; Dollard, Joanne; Elliott, Taryn; Eley, Diann S.

    2015-01-01

    Background Resilience can be defined as the ability to rebound from adversity and overcome difficult circumstances. General Practice (GP) registrars face many challenges in transitioning into general practice, and additional stressors and pressures apply for those choosing a career in rural practice. At this time of international rural generalist medical workforce shortages, it is important to focus on the needs of rural GP registrars and how to support them to become resilient health care pr...

  10. Randomized Phase I: Safety, Immunogenicity and Mucosal Antiviral Activity in Young Healthy Women Vaccinated with HIV-1 Gp41 P1 Peptide on Virosomes.

    Directory of Open Access Journals (Sweden)

    Geert Leroux-Roels

    Full Text Available Mucosal antibodies harboring various antiviral activities may best protect mucosal surfaces against early HIV-1 entry at mucosal sites and they should be ideally induced by prophylactic HIV-1 vaccines for optimal prevention of sexually transmitted HIV-1. A phase I, double-blind, randomized, placebo-controlled trial was conducted in twenty-four healthy HIV-uninfected young women. The study objectives were to assess the safety, tolerability and immunogenicity of virosomes harboring surface HIV-1 gp41-derived P1 lipidated peptides (MYM-V101. Participants received placebo or MYM-V101 vaccine at 10 μg/dose or 50 μg/dose intramuscularly at week 0 and 8, and intranasally at week 16 and 24. MYM-V101 was safe and well-tolerated at both doses administered by the intramuscular and intranasal routes, with the majority of subjects remaining free of local and general symptoms. P1-specific serum IgGs and IgAs were induced in all high dose recipients after the first injection. After the last vaccination, vaginal and rectal P1-specific IgGs could be detected in all high dose recipients. Approximately 63% and 43% of the low and high dose recipients were respectively tested positive for vaginal P1-IgAs, while 29% of the subjects from the high dose group tested positive for rectal IgAs. Serum samples had total specific IgG and IgA antibody concentrations ≥ 0.4 μg/mL, while mucosal samples were usually below 0.01 μg/mL. Vaginal secretions from MYM-V101 vaccinated subjects were inhibiting HIV-1 transcytosis but had no detectable neutralizing activity. P1-specific Th1 responses could not be detected on PBMC. This study demonstrates the excellent safety and tolerability of MYM-V101, eliciting systemic and mucosal antibodies in the majority of subjects. Vaccine-induced mucosal anti-gp41 antibodies toward conserved gp41 motifs were harboring HIV-1 transcytosis inhibition activity and may contribute to reduce sexually-transmitted HIV-1.ClinicalTrials.gov NCT01084343.

  11. Curcumin as a Modulator of P-Glycoprotein in Cancer: Challenges and Perspectives

    Directory of Open Access Journals (Sweden)

    Vanessa Lopes-Rodrigues

    2016-11-01

    Full Text Available Multidrug resistance (MDR presents a serious challenge to the efficiency of cancer treatment, and may be associated with the overexpression of drug efflux pumps. P-glycoprotein (P-gp is a drug efflux pump often found overexpressed in cases of acquired MDR. Nevertheless, there are no P-gp inhibitors being used in the current clinical practice, due to toxicity problems, drug interactions, or pharmacokinetic issues. Therefore, it is important to identify novel inhibitors of P-gp activity or expression. Curcumin is a secondary metabolite isolated from the turmeric of Curcuma longa L. which has been associated with several biological activities, particularly P-gp modulatory activity (by inhibiting both P-gp function and expression. However, curcumin shows extensive metabolism and instability, which has justified the recent and intensive search for analogs of curcumin that maintain the P-gp modulatory activity but have enhanced stability. This review summarizes and compares the effects of curcumin and several curcumin analogs on P-glycoprotein function and expression, emphasizing the potential of these molecules for the possible development of safe and effective inhibitors of P-gp to overcome MDR in human cancer.

  12. A new strategy for full-length Ebola virus glycoprotein expression in E.coli.

    Science.gov (United States)

    Zai, Junjie; Yi, Yinhua; Xia, Han; Zhang, Bo; Yuan, Zhiming

    2016-12-01

    Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

  13. Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

    Directory of Open Access Journals (Sweden)

    Kuhn Andreas

    2011-09-01

    Full Text Available Abstract Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

  14. Etanercept overcomes P-glycoprotein-induced drug resistance in lymphocytes of patients with intractable rheumatoid arthritis.

    Science.gov (United States)

    Tsujimura, Shizuyo; Saito, Kazuyoshi; Nakayamada, Shingo; Tanaka, Yoshiya

    2010-04-01

    P-glycoprotein (P-gp) on activated lymphocytes is an adenosine triphosphate (ATP)-binding cassette transporter that causes drug resistance by exclusion of intracellular drugs in patients with active rheumatoid arthritis (RA). However, infliximab with methotrexate (MTX) can overcome P-gp-mediated drug resistance. We encounter patients who cannot continue infliximab or MTX. Here we tested how etanercept affected P-gp-mediated drug resistance in such intractable RA patients. Peripheral lymphocytes of 11 RA patients (3 switched from infliximab and 8 who could not be treated with MTX) were analyzed for P-gp expression by flow cytometry and for drug exclusion using radioisotope-labeled dexamethasone. Activated lymphocytes of RA patients overexpressed P-gp and coexpressed CD69. Incubation of these lymphocytes with dexamethasone in vitro reduced intracellular dexamethasone levels. Two-week etanercept therapy significantly reduced P-gp expression and eliminated such P-gp- and CD69-high-expressing subgroup. The reduction in P-gp resulted in recovery of intracellular dexamethasone levels in lymphocytes and improvement of disease activity, thus allowing tapering of corticosteroids. None of the patients experienced any severe adverse effects. Etanercept is useful for overcoming P-gp-mediated treatment resistance in intractable RA patients who have to discontinue infliximab or are intolerant to MTX.

  15. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    OpenAIRE

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

  16. Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements

    Energy Technology Data Exchange (ETDEWEB)

    Balbach, John J. [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Yang Jun; Weliky, David P. [Michigan State University, Department of Chemistry (United States); Steinbach, Peter J. [National Institutes of Health, Center for Molecular Modeling, Center for Information Technology (United States); Tugarinov, Vitali; Anglister, Jacob [Weizmann Institute of Science, Department of Structural Biology (Israel); Tycko, Robert [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2000-04-15

    We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5{beta}, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with {sup 15}N labels at the {eta} nitrogen positions of arginine side chains and {sup 13}C labels at glycine carbonyl positions and {sup 13}C-detected {sup 13}C-{sup 15}N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5{beta} complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of {phi} and {psi} backbone dihedral angles in the RP135/0.5{beta} complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect {sup 13}C-{sup 15}N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

  17. Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements

    International Nuclear Information System (INIS)

    Balbach, John J.; Yang Jun; Weliky, David P.; Steinbach, Peter J.; Tugarinov, Vitali; Anglister, Jacob; Tycko, Robert

    2000-01-01

    We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5β, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15 N labels at the η nitrogen positions of arginine side chains and 13 C labels at glycine carbonyl positions and 13 C-detected 13 C- 15 N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5β complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of φ and Ψ backbone dihedral angles in the RP135/0.5β complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13 C- 15 N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations

  18. Activation of erythropoietin receptors by Friend viral gp55 and by erythropoietin and down-modulation by the murine Fv-2r resistance gene

    International Nuclear Information System (INIS)

    Hoatlin, M.E.; Kozak, S.L.; Kabat, D.; Lilly, F.; Chakraborti, A.; Kozak, C.A.

    1990-01-01

    The leukemogenic membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus appears to bind to erythropoietin receptors (EpoR) to stimulate erythroblastosis. To directly compare the effects of gp55 with erythropoietin (Epo), the authors produced retrovirions that encode either gp55, Epo, or EpoR. After infection with EpoR virus, interleukin 3-dependent DA-3 cells bound 125 I-labeled Epo and grew without interleukin 3 in the presence of Epo. These latter cells, but not parental DA-3 cells, became factor-independent after superinfection either with Epo virus or with Friend spleen focus-forming virus. In addition, Epo virus caused a disease in mice that mimicked Friend erythroleukemia. Although Fv-2 r homozygotes are susceptible to all other retroviral diseases, they are resistant to both Epo viral and Friend viral erythroleukemia. These results indicate that both gp55 and Epo stimulate EpoR and that the Fv-2 gene encodes a protein that controls response to these ligands. However, the Fv-2 protein is not EpoR because the corresponding genes map to opposite ends of mouse chromosome 9. These results have important implications for understanding signal transduction by EpoR and the role of host genetic variation in controlling susceptibility to an oncogenic protein

  19. Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery Into Human Breast Cancer Cells and Ultrasound Cellular Imaging.

    Science.gov (United States)

    Yang, Hong; Deng, Liwei; Li, Tingting; Shen, Xue; Yan, Jie; Zuo, Liangming; Wu, Chunhui; Liu, Yiyao

    2015-12-01

    Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated

  20. Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

    International Nuclear Information System (INIS)

    Hoetzel, Isidro; Cheevers, William P.

    2005-01-01

    The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain β-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding

  1. Induction of regulatory T cells by high-dose gp96 suppresses murine liver immune hyperactivation.

    Directory of Open Access Journals (Sweden)

    Xinghui Li

    Full Text Available Immunization with high-dose heat shock protein gp96, an endoplasmic reticulum counterpart of the Hsp90 family, significantly enhances regulatory T cell (Treg frequency and suppressive function. Here, we examined the potential role and mechanism of gp96 in regulating immune-mediated hepatic injury in mice. High-dose gp96 immunization elicited rapid and long-lasting protection of mice against concanavalin A (Con A-and anti-CD137-induced liver injury, as evidenced by decreased alanine aminotransaminase (ALT levels, hepatic necrosis, serum pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6, and number of IFN-γ (+ CD4(+ and IFN-γ (+ CD8(+ T cells in the spleen and liver. In contrast, CD4(+CD25(+Foxp3(+ Treg frequency and suppressive function were both increased, and the protective effect of gp96 could be generated by adoptive transfer of Treg cells from gp96-immunized mice. In vitro co-culture experiments demonstrated that gp96 stimulation enhanced Treg proliferation and suppressive function, and up-regulation of Foxp3, IL-10, and TGF-β1 induced by gp96 was dependent on TLR2- and TLR4-mediated NF-κB activation. Our work shows that activation of Tregs by high-dose gp96 immunization protects against Con A- and anti-CD137-induced T cell-hepatitis and provides therapeutic potential for the development of a gp96-based anti-immune hyperactivation vaccine against immune-mediated liver destruction.

  2. Why are some patients in treatment for advanced cancer reluctant to consult their GP?

    DEFF Research Database (Denmark)

    Åbom, Birgit; Pfeiffer, Per

    2009-01-01

    and therefore consulted the doctor or the staff at the cancer treatment centre before seeking advice from their GP. Some patients found that the GP was not familiar enough with the treatments given; others that they did not want to inconvenience the busy GP with what they perceived to be minor non...

  3. 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

  4. Impact of Leishmania metalloprotease GP63 on macrophage signaling

    Science.gov (United States)

    Isnard, Amandine; Shio, Marina T.; Olivier, Martin

    2012-01-01

    The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions. PMID:22919663

  5. Incorporating and evaluating an integrated gender-specific medicine curriculum: a survey study in Dutch GP training

    Science.gov (United States)

    Dielissen, Patrick W; Bottema, Ben JAM; Verdonk, Petra; Lagro-Janssen, Toine LM

    2009-01-01

    Background We recently set standards for gender-specific medicine training as an integrated part of the GP training curriculum. This paper describes the programme and evaluation of this training. Methods The programme is designed for GP registrars throughout the 3-year GP training. The modules emphasize interaction, application, and clinically integrated learning and teaching methods in peer groups. In 2005 - 2008, after completion of each tutorial, GP registrars were asked to fill in a questionnaire on a 5-point Likert scale to assess the programme's methods and content. GP registrars were also asked to identify two learning points related to the programme. Results The teaching programme consists of five 3-hour modules that include gender themes related to and frequently seen by GPs such as in doctor-patient communication and cardiovascular disease. GP registrars evaluated the training course positively. The written learning points suggest that GP registrars have increased their awareness of why attention to gender-specific information is relevant. Conclusion In summary, gender-specific medicine training has been successfully integrated into an existing GP training curriculum. The modules and teaching methods are transferable to other training institutes for postgraduate training. The evaluation of the teaching programme shows a positive impact on GP registrars' gender awareness. PMID:19737396

  6. Incorporating and evaluating an integrated gender-specific medicine curriculum: a survey study in Dutch GP training

    Directory of Open Access Journals (Sweden)

    Lagro-Janssen Toine LM

    2009-09-01

    Full Text Available Abstract Background We recently set standards for gender-specific medicine training as an integrated part of the GP training curriculum. This paper describes the programme and evaluation of this training. Methods The programme is designed for GP registrars throughout the 3-year GP training. The modules emphasize interaction, application, and clinically integrated learning and teaching methods in peer groups. In 2005 - 2008, after completion of each tutorial, GP registrars were asked to fill in a questionnaire on a 5-point Likert scale to assess the programme's methods and content. GP registrars were also asked to identify two learning points related to the programme. Results The teaching programme consists of five 3-hour modules that include gender themes related to and frequently seen by GPs such as in doctor-patient communication and cardiovascular disease. GP registrars evaluated the training course positively. The written learning points suggest that GP registrars have increased their awareness of why attention to gender-specific information is relevant. Conclusion In summary, gender-specific medicine training has been successfully integrated into an existing GP training curriculum. The modules and teaching methods are transferable to other training institutes for postgraduate training. The evaluation of the teaching programme shows a positive impact on GP registrars' gender awareness.

  7. 65 nm LP/GP mix low cost platform for multi-media wireless and consumer applications

    Science.gov (United States)

    Tavel, B.; Duriez, B.; Gwoziecki, R.; Basso, M. T.; Julien, C.; Ortolland, C.; Laplanche, Y.; Fox, R.; Sabouret, E.; Detcheverry, C.; Boeuf, F.; Morin, P.; Barge, D.; Bidaud, M.; Biénacel, J.; Garnier, P.; Cooper, K.; Chapon, J. D.; Trouiller, Y.; Belledent, J.; Broekaart, M.; Gouraud, P.; Denais, M.; Huard, V.; Rochereau, K.; Difrenza, R.; Planes, N.; Marin, M.; Boret, S.; Gloria, D.; Vanbergue, S.; Abramowitz, P.; Vishnubhotla, L.; Reber, D.; Stolk, P.; Woo, M.; Arnaud, F.

    2006-04-01

    A complete 65 nm CMOS platform, called LP/GP Mix, has been developed employing thick oxide transistor (IO), Low Power (LP) and General Purpose (GP) devices on the same chip. Dedicated to wireless multi-media and consumer applications, this new triple gate oxide platform is low cost (+1mask only) and saves over 35% of dynamic power with the use of the low operating voltage GP. The LP/GP mix shows competitive digital performance with a ring oscillator (FO = 1) speed equal to 7 ps per stage (GP) and 6T-SRAM static power lower than 10 pA/cell (LP). Compatible with mixed-signal design requirements, transistors show high voltage gain, low mismatch factor and low flicker noise. Moreover, to address mobile phone demands, excellent RF performance has been achieved with FT = 160 GHz for LP and 280 GHz for GP nMOS transistors.

  8. Pumping of drugs by P-glycoprotein

    DEFF Research Database (Denmark)

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

    2003-01-01

    The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different...... levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows...... but rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin...

  9. Irradiation of rat brain reduces P-glycoprotein expression and function

    NARCIS (Netherlands)

    Bart, J.; Nagengast, W. B.; Coppes, R. P.; Wegman, T. D.; van der Graaf, W. T. A.; Groen, H. J. M.; Vaalburg, W.; de Vries, E. G. E.; Hendrikse, N. H.

    2007-01-01

    The blood - brain barrier ( BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P- glycoprotein ( P- gp), expressed on brain capillary endothelial cells, is part of the BBB. P- gp expression on capillary endothelium decreases 5 days after brain

  10. The Astringency of the GP Algorithm for Forecasting Software Failure Data Series

    Directory of Open Access Journals (Sweden)

    Yong-qiang Zhang

    2007-05-01

    Full Text Available The forecasting of software failure data series by Genetic Programming (GP can be realized without any assumptions before modeling. This discovery has transformed traditional statistical modeling methods as well as improved consistency for model applicability. The individuals' different characteristics during the evolution of generations, which are randomly changeable, are treated as Markov random processes. This paper also proposes that a GP algorithm with "optimal individuals reserved strategy" is the best solution to this problem, and therefore the adaptive individuals finally will be evolved. This will allow practical applications in software reliability modeling analysis and forecasting for failure behaviors. Moreover it can verify the feasibility and availability of the GP algorithm, which is applied to software failure data series forecasting on a theoretical basis. The results show that the GP algorithm is the best solution for software failure behaviors in a variety of disciplines.

  11. Synthesis and Preclinical Evaluation of Novel PET Probes for P-Glycoprotein Function and Expression

    NARCIS (Netherlands)

    van Waarde, Aren; Ramakrishnan, Nisha K.; Rybczynska, Anna A.; Elsinga, Philip H.; Berardi, Francesco; de Jong, Johan R.; Kwizera, Chantal; Perrone, Roberto; Cantore, Mariangela; Sijbesma, Jurgen W. A.; Dierckx, Rudi A.; Colabufo, Nicola A.

    2009-01-01

    P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. A P-gp substrate (7) and an inhibitor (6) were labeled with (11)C, resulting in potential tracers of P-gp function and expression. Methods: 6 and 7 were labeled using (11)CH(3)I. (11)C-verapamil was

  12. [Research progress on ebola virus glycoprotein].

    Science.gov (United States)

    Ding, Guo-Yong; Wang, Zhi-Yu; Gao, Lu; Jiang, Bao-Fa

    2013-03-01

    Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.

  13. Definition of novel GP6 polymorphisms and major difference in haplotype frequencies between populations by a combination of in-depth exon resequencing and genotyping with tag single nucleotide polymorphisms.

    Science.gov (United States)

    Watkins, N A; O'Connor, M N; Rankin, A; Jennings, N; Wilson, E; Harmer, I J; Davies, L; Smethurst, P A; Dudbridge, F; Farndale, R W; Ouwehand, W H

    2006-06-01

    Common genetic variants of cell surface receptors contribute to differences in functional responses and disease susceptibility. We have previously shown that single nucleotide polymorphisms (SNPs) in platelet glycoprotein VI (GP6) determine the extent of response to agonist. In addition, SNPs in the GP6 gene have been proposed as risk factors for coronary artery disease. To completely characterize genetic variation in the GP6 gene we generated a high-resolution SNP map by sequencing the promoter, exons and consensus splice sequences in 94 non-related Caucasoids. In addition, we sequenced DNA encoding the ligand-binding domains of GP6 from non-human primates to determine the level of evolutionary conservation. Eighteen SNPs were identified, six of which encoded amino acid substitutions in the mature form of the protein. The single non-synonymous SNP identified in the exons encoding the ligand-binding domains, encoding for a 103Leu > Val substitution, resulted in reduced ligand binding. Two common protein isoforms were confirmed in Caucasoid with frequencies of 0.82 and 0.15. Variation at the GP6 locus was characterized further by determining SNP frequency in over 2000 individuals from different ethnic backgrounds. The SNPs were polymorphic in all populations studied although significant differences in allele frequencies were observed. Twelve additional GP6 protein isoforms were identified from the genotyping results and, despite extensive variation in GP6, the sequence of the ligand-binding domains is conserved. Sequences from non-human primates confirmed this observation. These data provide valuable information for the optimal selection of genetic variants for use in future association studies.

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

    Directory of Open Access Journals (Sweden)

    Anna Maisa

    2009-06-01

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

  15. 5-HT2A receptor SPECT imaging with [123I]R91150 under P-gp inhibition with tariquidar: More is better?

    International Nuclear Information System (INIS)

    Tsartsalis, Stergios; Tournier, Benjamin B.; Huynh-Gatz, Trinh; Dumas, Noé; Ginovart, Nathalie; Moulin-Sallanon, Marcelle; Millet, Philippe

    2016-01-01

    Introduction: Pharmacological P-glycoprotein (P-gp) inhibition with tariquidar (TQD) is considered a promising strategy for the augmentation of radiotracer brain uptake. However, a region-dependent effect may compromise the robustness of quantitative studies. For this reason, we studied the effect of a TQD pretreatment on 5-HT2A imaging with [ 123 I]R91150 and compared results with those obtained in Mdr1a knock-out (KO) rats. Methods: Ex vivo autoradiography was performed in TQD (15 mg/kg) pretreated wild-type (WT-TQD), Mdr1a knock-out (KO) and untreated WT rats for Specific Binding Ratio (SBR) estimation. In vivo dynamic SPECT imaging with serial arterial blood sampling was performed in the former two groups of rats and kinetic analysis was performed with a one tissue-compartment (1TC) model and the Specific Uptake Ratio (SUR). Results were analyzed statistically using repeated measures ANOVA. Results: SBR values differed between WT-TQD, Mdr1a KO and WT rats in a region-dependent manner (p < 0.0001). In vivo brain uptake of radiotracer did not differ between groups. Similarly, kinetic analysis provided distribution volume (V T ) values that did not differ significantly between groups. SUR binding potential (BP ND ) values from both groups highly correlated with corresponding V T (r = 0.970, p < 0.0001 and r = 0.962, p < 0.0001, respectively). However, SUR measured over averaged images between 100 and 120 min, using cerebellum as reference region, demonstrated values that were, by average, 2.99 ± 0.53 times higher in the WT-TQD group, with the difference between groups being region-dependent (p < 0.001). In addition, coefficient of variation of the SUR BP ND values across brain regions was significantly higher in the WT-TQD rats (41.25% ± 9.63% versus 11.13% ± 5.59%, p < 0.0001). Conclusion: P-gp inhibition with TQD leads to region-dependent effect in the rat brain, with probably sub-optimal effect in cerebellum. This warrants attention when it is used as a

  16. Family effects on the rurality of GP's work location: a longitudinal panel study.

    Science.gov (United States)

    McGrail, Matthew R; Russell, Deborah J; O'Sullivan, Belinda G

    2017-10-19

    Reduced opportunities for children's schooling and spouse's/partner's employment are identified internationally as key barriers to general practitioners (GPs) working rurally. This paper aims to measure longitudinal associations between the rurality of GP work location and having (i) school-aged children and (ii) a spouse/partner in the workforce. Participants included 4377 GPs responding to at least two consecutive annual surveys of the Medicine in Australia: Balancing Employment and Life (MABEL) national longitudinal study between 2008 and 2014. The main outcome, GP work location, was categorised by remoteness and population size. Five sequential binary school-age groupings were defined according to whether a GP had no children, only preschool children (aged 0-4 years), at least one primary-school child (aged 5-11 years), at least one child in secondary school (aged 12-18 years), and all children older than secondary school (aged ≥ 19). Partner in the workforce was defined by whether a GP had a partner who was either currently working or looking for work, or not. Separate generalised estimating equation models, which aggregated consecutive annual observations per GP, tested associations between work location and (i) educational stages and (ii) partner employment, after adjusting for key covariates. Male GPs with children in secondary school were significantly less likely to work rurally (inclusive of > 50 000 regional centres through to the smallest rural towns of locations were not significantly associated with the educational stage of their children. Having a partner in the workforce was not associated with work location for male GPs, whereas female GPs with a partner in the workforce were significantly less likely to work in smaller rural/remote communities (location is related to key family needs which differ according to GP gender and educational stages of children. Such non-professional factors are likely to be dynamic across the GP's lifespan and

  17. Homologous and heterologous antibody responses of mice immunized with purified feline herpesvirus type 1 and canine herpesvirus glycoproteins.

    Science.gov (United States)

    Limcumpao, J A; Horimoto, T; Xuan, X N; Tohya, Y; Azetaka, M; Takahashi, E; Mikami, T

    1991-06-01

    The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.

  18. Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

    Directory of Open Access Journals (Sweden)

    Luping Du

    2018-01-01

    Full Text Available Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide (PLGA nanoparticles (NPs with Ulex europaeus agglutinin 1 (UEA-1 and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5] or subunit vaccine ORF5-encoded glycoprotein (GP5 from exposure to the gastrointestinal (GI tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05. Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system.

  19. Targeted Delivery of GP5 Antigen of PRRSV to M Cells Enhances the Antigen-Specific Systemic and Mucosal Immune Responses

    Science.gov (United States)

    Du, Luping; Yu, Zhengyu; Pang, Fengjiao; Xu, Xiangwei; Mao, Aihua; Yuan, Wanzhe; He, Kongwang; Li, Bin

    2018-01-01

    Efficient delivery of antigens through oral immunization is a first and critical step for successful induction of mucosal immunity, which can provide protection against pathogens invading the mucosa. Membranous/microfold cells (M cells) within the mucosa can transcytose internalized antigen without degradation and thus play an important role in initiating antigen-specific mucosal immune responses through inducing secretory IgA production. In this research, we modified poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) with Ulex europaeus agglutinin 1 (UEA-1) and successfully prepared an oral vaccine delivery system, UEA-1/PLGA NPs. PLGA NPs were prepared using a standard double emulsion solvent evaporation technique, which can protect the entrapped PRRSV DNA vaccine [pcDNA3.1-SynORF5 (synthetic ORF5)] or subunit vaccine ORF5-encoded glycoprotein (GP5) from exposure to the gastrointestinal (GI) tract and release the plasmids in a controlled manner. With UEA-1 modification, the UEA-1/PLGA NPs can be effectively transported by M-cells. We investigated immune response induced by UEA-1/PLGA-SynORF5 or UEA-1/PLGA-GP5 following inoculation in mice and piglets. Compared with PLGA-SynORF5 or PLGA-GP5 NPs, UEA-1/PLGA-SynORF5, or UEA-1/PLGA-GP5 NPs stimulated significantly increased serum IgG levels and augmented intestinal IgA levels in mice and piglets (P < 0.05). Our findings indicate UEA-1/PLGA NPs can be applied as a promising and universally robust oral vaccine delivery system. PMID:29423381

  20. Immunodiagnosis of paracoccidioidomycosis due to Paracoccidioides brasiliensis using a latex test: detection of specific antibody anti-gp43 and specific antigen gp43.

    Directory of Open Access Journals (Sweden)

    Priscila Oliveira Dos Santos

    2015-02-01

    Full Text Available Paracoccidioidomycosis (PCM is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations.A latex immunoassay using sensitized latex particles (SLPs with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43 was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF, and bronchoalveolar lavage (BAL from patients with PCM due to P. brasiliensis. The gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7-100.0, specificity (93.94%, 95% CI 87.3-97.7, and positive (91.4% and negative (98.9% predictive values. In addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3-99.6, specificity (88.89%, 95% CI 81.0-94.3, and positive (85.1% and negative (97.8% predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924 and mAb17c-SLPs (k = 0.850, which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. In addition, the latex test was useful for monitoring PCM patients receiving therapy.The high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but mainly in remote locations with limited laboratory infrastructure

  1. Which positive factors determine the GP satisfaction in clinical practice? A systematic literature review.

    Science.gov (United States)

    Le Floch, B; Bastiaens, H; Le Reste, J Y; Lingner, H; Hoffman, R D; Czachowski, S; Assenova, R; Koskela, T H; Klemenc-Ketis, Z; Nabbe, P; Sowinska, A; Montier, T; Peremans, L

    2016-09-13

    Looking at what makes General Practitioners (GPs) happy in their profession, may be important in increasing the GP workforce in the future. The European General Practice Research Network (EGPRN) created a research team (eight national groups) in order to clarify the factors involved in GP job satisfaction throughout Europe. The first step of this study was a literature review to explore how the satisfaction of GPs had been studied before. The research question was "Which factors are related to GP satisfaction in Clinical Practice?" Systematic literature review according to the PRISMA statement. The databases searched were Pubmed, Embase and Cochrane. All articles were identified, screened and included by two separate research teams, according to inclusion or exclusion criteria. Then, a qualitative appraisal was undertaken. Next, a thematic analysis process was undertaken to capture any issue relevant to the research question. The number of records screened was 458. One hundred four were eligible. Finally, 17 articles were included. The data revealed 13 subthemes, which were grouped into three major themes for GP satisfaction. First there were general profession-related themes, applicable to many professions. A second group of issues related specifically to a GP setting. Finally, a third group was related to professional life and personal issues. A number of factors leading to GP job satisfaction, exist in literature They should be used by policy makers within Europe to increase the GP workforce. The research team needs to undertake qualitative studies to confirm or enhance those results.

  2. 'Just a GP': a mixed method study of undermining of general practice as a career choice in the UK.

    Science.gov (United States)

    Alberti, Hugh; Banner, Kimberley; Collingwood, Helen; Merritt, Kymberlee

    2017-11-03

    Failure to recruit sufficient applicants to general practice (GP) training has been a problem both nationally and internationally for many years and undermining of GP is one possible contributing factor. The aim of our study was to ascertain what comments, both negative and positive, are being made in UK clinical settings to GP trainees about GP and to further explore these comments and their influence on career choice. We conducted a mixed methods study. We surveyed all foundation doctors and GP trainees within one region of Health Education England regarding any comments they experienced relating to a career in GP. We also conducted six focus groups with early GP trainees to discuss any comments that they experienced and whether these comments had any influence on their or others career choice. Positive comments reported by trainees centred around the concept that choosing GP is a positive, family-focused choice which facilities a good work-life balance. Workload was the most common negative comment, alongside the notion of being 'just a GP'; the belief that GP is boring, a waste of training and a second-class career choice. The reasons for and origin of the comments are multifactorial in nature. Thematic analysis of the focus groups identified key factors such as previous exposure to and experience of GP, family members who were GPs, GP role models, demographics of the clinician and referral behaviour. Trainees perceived that negative comments may be discouraging others from choosing GP as a career. Our study demonstrates that negative comments towards GP as a career do exist within clinical settings and are having a potential impact on poor recruitment rates to GP training. We have identified areas in which further negative comments could be prevented by changing perceptions of GP as a career. Additional time spent in GP as undergraduates and postgraduates, and positive GP role models, could particularly benefit recruitment. We recommend that undermining of GP

  3. Triage of febrile children at a GP cooperative : determinants of a consultation

    NARCIS (Netherlands)

    Monteny, Miriam; Berger, Marjolein Y.; van der Wouden, Johannes C.; Broekman, Berth J.; Koes, Bart W.

    Background Most febrile children contacting a GP cooperative are seen by a GP, although the incidence of serious illness is low. The guidelines for triage might not be suitable in primary care. Aim To investigate the determinants related to the outcome of triage in febrile children. Design of study

  4. A new in vivo method to study P-glycoprotein transport in tumors and the blood-brain barrier

    NARCIS (Netherlands)

    Hendrikse, NH; de Vries, EGE; Eriks-Fluks, L; van der Graaf, WTA; Hospers, GAP; Willemsen, ATM; Vaalburg, W; Franssen, EJF

    1999-01-01

    Drug resistance is a major cause of chemotherapy failure in cancer treatment, One reason is the overexpression of the drug efflux pump P-glycoprotein (P-gp), involved in multidrug resistance (MDR), In vivo pharmacokinetic analysis of P-gp transport might identify the capacity of modulation by P-gp

  5. Influences on GP coping and resilience: a qualitative study in primary care.

    Science.gov (United States)

    Cheshire, Anna; Ridge, Damien; Hughes, John; Peters, David; Panagioti, Maria; Simon, Chantal; Lewith, George

    2017-06-01

    'Neoliberal' work policies, austerity, NHS restructuring, and increased GP consultation rates provide the backdrop against increasing reports of GP burnout and an impending shortage of GPs. To explore GPs' experiences of workplace challenges and stresses, and their coping strategies, particularly focusing on understanding the impact of recent NHS workplace change. Study design was qualitative, with data collected from two focus groups and seven one-to-one telephone interviews. Focus groups and one-to-one telephone interviews explored the experiences of GPs currently practising in England, recruited through convenience sampling. Data were collected using a semi-structured interview approach and analysed using thematic analysis. There were 22 GP participants recruited: focus groups ( n = 15) and interviews ( n = 7). Interviewees understood GPs to be under intense and historically unprecedented pressures, which were tied to the contexts in which they work, with important moral implications for 'good' doctoring. Many reported that being a full-time GP was too stressful: work-related stress led to mood changes, sleep disruption, increases in anxiety, and tensions with loved ones. Some had subsequently sought ways to downsize their clinical workload. Workplace change resulted in little time for the things that helped GP resilience: a good work-life balance and better contact with colleagues. Although some GPs were coping better than others, GPs acknowledged that there was only so much an individual GP could do to manage their stress, given the external work issues they faced. GPs experience their emotional lives and stresses as being meaningfully shaped by NHS factors. To support GPs to provide effective care, resilience building should move beyond the individual to include systemic work issues. © British Journal of General Practice 2017.

  6. Blended learning in CME: the perception of GP trainers.

    Science.gov (United States)

    Te Pas, E; Meinema, J G; Visser, M R M; van Dijk, N

    2016-05-01

    Blended learning (the combination of electronic methods with traditional teaching methods) has the potential to combine the best of traditional education with the best of computer-mediated training. We chose to develop such an intervention for GP trainers who were undertaking a Continuing Medical Education (CME) course in evidence-based medicine (EBM). This study reports on our experience and investigated the factors influencing the perception on usefulness and logistics of blended learning for learners in CME. In total, 170 GP trainers participated in the intervention. We used questionnaires, observations during the four face-to-face meetings and evaluations in the e-course over one year. Additionally we organised focus groups to gain insight in some of the outcomes of the questionnaires and interpretations of the observations. The GP trainers found the design and the educational method (e-course in combination with meetings) attractive, instructive and complementary. Factors influencing their learning were (1) educational design, (2) educational method, (3) topic of the intervention, (4) time (planning), (5) time (intervention), (6) learning style, (7) technical issues, (8) preconditions and (9) level of difficulty. A close link between daily practice and the educational intervention was considered an important precondition for the success of the intervention in this group of learners. GP trainers were positive about blended learning: they found e-learning a useful way to gain knowledge and the meetings a pleasant way of transferring the knowledge into practice. Although some preconditions should be taken into consideration during its development and implementation, they would participate in similarly designed learning in the future.

  7. Mechanistic understanding of N-glycosylation in Ebola virus glycoprotein maturation and function.

    Science.gov (United States)

    Wang, Bin; Wang, Yujie; Frabutt, Dylan A; Zhang, Xihe; Yao, Xiaoyu; Hu, Dan; Zhang, Zhuo; Liu, Chaonan; Zheng, Shimin; Xiang, Shi-Hua; Zheng, Yong-Hui

    2017-04-07

    The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP 1 and the fusion-mediating GP 2 subunits and incorporated into virions to initiate infection. GP 1 and GP 2 form heterodimers that have 15 or two N -glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N -glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP 1 NGSs are not critical, the two GP 2 NGSs, Asn 563 and Asn 618 , are essential for GP function. Further analysis uncovered that Asn 563 and Asn 618 regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that both CNX and CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn 563 and Asn 618 are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Platelet glycoprotein IIb/IIIa polymorphism HPA-3 b/b is associated with increased risk of ischemic stroke in patients under 60 years of age.

    Science.gov (United States)

    Duan, Hao; Cai, Yan; Sun, Xiaojiang

    2012-01-01

    The role of genetic risk factors in ischemic stroke is unclear. Platelet glycoprotein IIb/IIIa (GpIIb-IIIa) has been implicated in the pathogenesis of ischemic stroke. We sought to evaluate the relationship between the GpIIb/IIIa complex gene polymorphism and ischemic stroke. We investigated the association of the GpIIb/IIIa complex gene polymorphism with stroke risk in 306 patients with acute ischemic stroke and 266 control subjects by determining the GpIIb and GpIIIa genotype from leukocyte DNA by polymerase chain reaction (PCR) followed by FokI and ScrFI digestion, respectively. Compared with controls, more patients presented with coronary heart disease, hypertension, smoking history, and diabetes. In addition, the patients had higher levels of cholesterol and glucose compared with the control subjects. All donors in the GpIIIa (n=572) group expressed the GpIIIa PlA1 (HPA-1 aa) phenotype. There were no significant differences between the HPA-3 genotype (GpIIb) patient distribution (aa=39.9%, ab=41.4%, bb=28.7%) and healthy control subjects (aa=36.1%, ab=35.0%, bb=28.9%) (P=0.580). Among study participants ischemic stroke >2-fold (P=0.008). The GpIIb Ile/Ser843 gene polymorphism is associated with ischemic stroke among young and middle-aged adults (ischemic stroke.

  9. Age-related P-glycoprotein expression in the intestine and affecting the pharmacokinetics of orally administered enrofloxacin in broilers.

    Science.gov (United States)

    Guo, Mengjie; Bughio, Shamsuddin; Sun, Yong; Zhang, Yu; Dong, Lingling; Dai, Xiaohua; Wang, Liping

    2013-01-01

    Bioavailability is the most important factor for the efficacy of any drug and it is determined by P- glycoprotein (P-gp) expression. Confirmation of P-gp expression during ontogeny is needed for understanding the differences in therapeutic efficacy of any drug in juvenile and adult animals. In this study, Abcb1 mRNA levels in the liver and intestine of broilers during ontogeny were analysed by RT qPCR. Cellular distribution of P-gp was detected by immunohistochemstry. Age-related differences of enrofloxacin pharmacokinetics were also studied. It was found that broilers aged 4 week-old expressed significantly (P0.05) age-related difference in the duodenum. Furthermore, the highest and lowest levels of Abcb1 mRNA expression were observed in the jejunum, and duodenum, respectively. P-gp immunoreactivity was detected on the apical surface of the enterocytes and in the bile canalicular membranes of the hepatocytes. Pharmacokinetic analysis revealed that the 8 week-old broilers, when orally administrated enrofloxacin, exhibited significantly higher Cmax (1.97 vs. 0.98 μg • ml(-1), P=0.009), AUC(14.54 vs. 9.35 μg • ml(-1) • h, P=0.005) and Ka (1.38 vs. 0.43 h(-1), P=0.032), as well as lower Tpeak (1.78 vs. 3.28 h, P=0.048) and T1/2 ka (0.6 vs. 1.64 h, P=0.012) than the 4 week-old broilers. The bioavailability of enrofloxacin in 8 week-old broilers was increased by 15.9%, compared with that in 4 week-old birds. Interestingly, combining verapamil, a P-gp modulator, significantly improved pharmacokinetic behaviour of enrofloxacin in all birds. The results indicate juvenile broilers had a higher expression of P-gp in the intestine, affecting the pharmacokinetics and reducing the bioavailability of oral enrofloxacin in broilers. On the basis of our results, it is recommended that alternative dose regimes are necessary for different ages of broilers for effective therapy.

  10. How can educators support general practice (GP) trainees to develop resilience to prevent burnout?

    Science.gov (United States)

    Sales, Bryony; Macdonald, Alexandra; Scallan, Samantha; Crane, Sue

    2016-11-01

    Burnout impacts adversely on professional and personal life, and holds implications for patient care. Current research on burnout mainly focuses on established general practitioners but it is unclear how early the signs of burnout really start. This work seeks to identify whether specific GP trainee groups are particularly at risk of burnout and the aspects of training they find stressful. A longitudinal cohort study, collecting qualitative and quantitative data through a single mode of data collection (questionnaire) took place with trainees from all GP training years (ST1-3), across a vocational training scheme (n = 48). Data gathered included the Oldenburg Burnout Inventory (OLBI). Higher than anticipated levels of burnout were displayed by all trainees. A sub-group self reporting higher levels of burnout comprised all-female, UK-trained-at-undergraduate GP trainees, with a partner but no children. Top reported stressors included knowledge/uncertainty, workload/time pressures and ePortfolio. Less than 50% of trainees perceived their burnout levels to be as high as their OLBI showing potential lack of insight. This research demonstrates that high levels of burnout are experienced in GP trainees as early as the first year of training. Early identification of burnout amongst trainees is essential by GP educators to help protect the future GP workforce.

  11. Clinicopathological correlations of podoplanin (gp38 expression in rheumatoid synovium and its potential contribution to fibroblast platelet crosstalk.

    Directory of Open Access Journals (Sweden)

    Manuel J Del Rey

    Full Text Available Synovial fibroblasts (SF undergo phenotypic changes in rheumatoid arthritis (RA that contribute to inflammatory joint destruction. This study was undertaken to evaluate the clinical and functional significance of ectopic podoplanin (gp38 expression by RA SF.Expression of gp38 and its CLEC2 receptor was analyzed by immunohistochemistry in synovial arthroscopic biopsies from RA patients and normal and osteoarthritic controls. Correlation between gp38 expression and RA clinicopathological variables was analyzed. In patients rebiopsied after anti-TNF-α therapy, changes in gp38 expression were determined. Platelet-SF coculture and gp38 silencing in SF were used to analyze the functional contribution of gp38 to SF migratory and invasive properties, and to SF platelet crosstalk.gp38 was abundantly but variably expressed in RA, and it was undetectable in normal synovial tissues. Among clinicopathologigal RA variables, significantly increased gp38 expression was only found in patients with lymphoid neogenesis (LN, and RF or ACPA autoantibodies. Cultured synovial but not dermal fibroblasts showed strong constitutive gp38 expression that was further induced by TNF-α. In RA patients, anti-TNF-α therapy significantly reduced synovial gp38 expression. In RA synovium, CLEC2 receptor expression was only observed in platelets. gp38 silencing in cultured SF did not modify their migratory and invasive properties but reduced the expression of IL-6 and IL-8 genes induced by SF-platelet interaction.In RA, synovial expression of gp38 is strongly associated to LN and it is reduced after anti-TNF-α therapy. Interaction between gp38 and CLEC2 platelet receptor is feasible in RA synovium in vivo and can specifically contribute to gene expression by SF.

  12. Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

    Science.gov (United States)

    Muro, Claudio; Gomez-Puerta, Luis A; Flecker, Robert H; Gamboa, Ricardo; Barreto, Percy Vilchez; Dorny, Pierre; Tsang, Victor C W; Gilman, Robert H; Gonzalez, Armando E; Garcia, Hector H; O'Neal, Seth E; For The Cysticercosis Working Group In Peru

    2017-12-01

    The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena- infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

  13. Immunization with Hexon modified adenoviral vectors integrated with gp83 epitope provides protection against Trypanosoma cruzi infection.

    Directory of Open Access Journals (Sweden)

    Anitra L Farrow

    2014-08-01

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.The "antigen capsid-incorporation" strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His6 were incorporated into the Ad serotype 5 (Ad5 vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83. This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes

  14. Patients' perceptions of GP non-verbal communication: a qualitative study.

    Science.gov (United States)

    Marcinowicz, Ludmila; Konstantynowicz, Jerzy; Godlewski, Cezary

    2010-02-01

    During doctor-patient interactions, many messages are transmitted without words, through non-verbal communication. To elucidate the types of non-verbal behaviours perceived by patients interacting with family GPs and to determine which cues are perceived most frequently. In-depth interviews with patients of family GPs. Nine family practices in different regions of Poland. At each practice site, interviews were performed with four patients who were scheduled consecutively to see their family doctor. Twenty-four of 36 studied patients spontaneously perceived non-verbal behaviours of the family GP during patient-doctor encounters. They reported a total of 48 non-verbal cues. The most frequent features were tone of voice, eye contact, and facial expressions. Less frequent were examination room characteristics, touch, interpersonal distance, GP clothing, gestures, and posture. Non-verbal communication is an important factor by which patients spontaneously describe and evaluate their interactions with a GP. Family GPs should be trained to better understand and monitor their own non-verbal behaviours towards patients.

  15. Overview of P-glycoprotein inhibitors: a rational outlook

    Directory of Open Access Journals (Sweden)

    Kale Mohana Raghava Srivalli

    2012-09-01

    Full Text Available P-glycoprotein (P-gp, a transmembrane permeability glycoprotein, is a member of ATP binding cassette (ABC super family that functions specifically as a carrier mediated primary active efflux transporter. It is widely distributed throughout the body and has a diverse range of substrates. Several vital therapeutic agents are substrates to P-gp and their bioavailability is lowered or a resistance is induced because of the protein efflux. Hence P-gp inhibitors were explored for overcoming multidrug resistance and poor bioavailability problems of the therapeutic P-gp substrates. The sensitivity of drug moieties to P-gp and vice versa can be established by various experimental models in silico, in vitro and in vivo. Ever since the discovery of P-gp, the research plethora identified several chemical structures as P-gp inhibitors. The aim of this review was to emphasize on the discovery and development of newer, inert, non-toxic, and more efficient, specifically targeting P-gp inhibitors, like those among the natural herb extracts, pharmaceutical excipients and formulations, and other rational drug moieties. The applications of cellular and molecular biology knowledge, in silico designed structural databases, molecular modeling studies and quantitative structure-activity relationship (QSAR analyses in the development of novel rational P-gp inhibitors have also been mentioned.Glicoproteína-p (P-gp, uma glicoproteína de transmembrana permeável, é um membro da superfamília (ABC de cassete de gene de ligação de ATP que funciona especificamente como um carreador mediado pelo transportador de efluxo ativo primário. É amplamente distribuído por todo o corpo e apresenta uma gama diversificada de substratos. Diversos agentes terapêuticos vitais são substratos para P-gp e sua biodisponibilidade é reduzida ou a resistência é induzida devido ao efluxo de proteínas. Portanto, os inibidores da P-gp foram explorados para a superação da resistência a

  16. 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.

  17. 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 kb......, 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...

  18. A review of a GP registrar-run mobile health clinic for homeless people.

    Science.gov (United States)

    O'Carroll, A; Irving, N; O'Neill, J; Flanagan, E

    2017-08-01

    Homeless people have excessively high morbidity and mortality rates, yet they face barriers accessing primary care. A mobile health clinic, staffed by GP registrars, was developed to provide services to homeless people, particularly rough sleepers and sex workers. The aims were to improve access to primary care and to challenge the stereotypes and prejudices of GP registrars through direct contact with homeless people. This was a qualitative study; questionnaires were completed on the mobile health clinic and two focus groups were conducted. All service users were asked to complete a questionnaire over a 3 month period. Two focus groups were conducted with 6 and 14 GP registrars who had worked on the bus. There was an 80% response rate (116 of 145). Fifty-two percent had no Medical Card meaning that they had no way to access the free primary care to which they are entitled. Had the clinic not been available, over half would not have sought further treatment and 16% would have gone to an Emergency Department. Ninety-one percent of users rated the service 10/10. The focus groups found that GP registrars who worked on the mobile health clinic had decreased negative stereotypes, increased empathy, and more knowledge of homeless issues. Furthermore, they intended to ensure that homeless people will not face discrimination in their future practice. A GP Registrar-run Mobile Health Clinic achieved its aims of improving access to primary care for rough sleepers and sex workers, and challenging stereotypes of GP Registrars.

  19. N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa.

    Science.gov (United States)

    Pierce, M W; Remold-O'Donnell, E; Todd, R F; Arnaout, M A

    1986-12-12

    Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.

  20. Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults.

    Science.gov (United States)

    Sokal, Etienne M; Hoppenbrouwers, Karel; Vandermeulen, Corinne; Moutschen, Michel; Léonard, Philippe; Moreels, Andre; Haumont, Michèle; Bollen, Alex; Smets, Françoise; Denis, Martine

    2007-12-15

    To date, there is no commercially available vaccine to prevent infectious mononucleosis, a disease frequently induced by Epstein-Barr virus (EBV) infection in adolescents or adults devoid of preexisting immunity to the virus. A total of 181 EBV-seronegative, healthy, young adult volunteers were randomized in a double-blind fashion to receive either placebo or a recombinant EBV subunit glycoprotein 350 (gp350)/aluminum hydroxide and 3-O-desacyl-4'-monophosphoryl lipid A (AS04) candidate vaccine in a 3-dose regimen. The vaccine had demonstrable efficacy (mean efficacy rate, 78.0% [95% confidence interval {CI}, 1.0%-96.0%]) in preventing the development of infectious mononucleosis induced by EBV infection, but it had no efficacy in preventing asymptomatic EBV infection. One month after receipt of the final dose of gp350 vaccine, 98.7% of subjects showed seroconversion to anti-gp350 antibodies (95% CI, 85.5%-97.9%), and they remained anti-gp350 antibody positive for >18 months. Furthermore, there were no concerns regarding the safety or reactogenicity of the gp350/AS04 vaccine. These data support the clinical feasibility of using an EBV vaccine to prevent infectious mononucleosis. ClinicalTrials.gov identifier: NCT00430534.

  1. Expression of P-gp, MRP, LRP, GST-π and TopoIIα and intrinsic resistance in human lung cancer cell lines.

    Science.gov (United States)

    Wang, Jiarui; Zhang, Jinhui; Zhang, Lichuan; Zhao, Long; Fan, Sufang; Yang, Zhonghai; Gao, Fei; Kong, Ying; Xiao, Gary Guishan; Wang, Qi

    2011-11-01

    This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST‑π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.

  2. Social relationships and GP use of middle-aged and older adults in Europe: a moderator analysis.

    Science.gov (United States)

    Bremer, Daniel; Lüdecke, Daniel; Vonneilich, Nico; von dem Knesebeck, Olaf

    2018-04-07

    This paper investigates (1) how social relationships (SRs) relate to the frequency of general practitioner (GP) visits among middle-aged and older adults in Europe, (2) if SRs moderate the association between self-rated health and GP visits, and (3) how the associations vary regarding employment status. Data stem from the Survey of Health, Ageing and Retirement in Europe project (wave 4, 56 989 respondents, 50 years or older). GP use was assessed by frequency of contacts with GPs in the last 12 months. Predictors were self-rated health and structural (Social Integration Index (SII), social contact frequency) and functional (emotional closeness) aspects of SR. Regressions were used to measure the associations between GP use and those predictors. Sociodemographic and socioeconomic factors were used as covariates. Additional models were computed with interactions. Analyses did not reveal significant associations of functional and structural aspects of SR with frequency of GP visits (SII: incidence rate ratio (IRR)=0.99, 95% CI 0.97 to 1.01, social contact frequency: IRR=1.04, 95% CI 1.00 to 1.07, emotional closeness: IRR=1.02, 95% CI 1.00 to 1.04). Moderator analyses showed that 'high social contact frequency people' with better health had more statistically significant GP visits than 'low social contact frequency people' with better health. Furthermore, people with poor health and an emotionally close network showed a significantly higher number of GP visits compared with people with same health, but less close networks. Three-way interaction analyses indicated employment status specific behavioural patterns with regard to SR and GP use, but coefficients were mostly not significant. All in all, the not employed groups showed a higher number of GP visits. Different indicators of SR showed statistically insignificantly associations with GP visits. Consequently, the relevance of SR may be rated rather low in quantitative terms for investigating GP use behaviour

  3. Patient satisfaction with out-of-hours GP cooperatives: a longitudinal study.

    Science.gov (United States)

    Smits, Marleen; Huibers, Linda; Oude Bos, Anita; Giesen, Paul

    2012-12-01

    For over a decade, out-of-hours primary care in the Netherlands has been provided by general practitioner (GP) cooperatives. In the past years, quality improvements have been made and patients have become acquainted with the service. This may have increased patient satisfaction. The objective of this study was to examine changes in patient satisfaction with GP cooperatives over time. Longitudinal observational study. A validated patient satisfaction questionnaire was distributed in 2003-2004 (T1) and 2007-2008 (T2). Items were rated on a scale from 0 to 10 (1 = very bad; 10 = excellent). Eight GP cooperatives in the Netherlands. Stratified sample of 9600 patients. Response was 55% at T1 (n = 2634) and 51% at T2 (n = 2462). Expectations met; satisfaction with triage nurses, GPs, and organization. For most patients the care received at the GP cooperative met their expectations (T1: 86.1% and T2: 88.4%). Patients were satisfied with the triage nurses (overall grade T1: 7.73 and T2: 7.99), GPs (T1: 8.04 and T2: 8.25), and organization (overall grade T1: 7.60 and T2: 7.78). Satisfaction with triage nurses showed the largest increase over time. The quality and effectiveness of advice or treatment were given relatively low grades. Of all organizational aspects, the lowest grades were given for waiting times and information about the cooperative. In general, patients were initially satisfied with GP cooperatives and satisfaction had even increased four years later. However, there is room for improvement in the content of the advice, waiting times, and information supply. More research is needed into satisfaction of specific patient groups.

  4. Using photography to enhance GP trainees' reflective practice and professional development.

    Science.gov (United States)

    Rutherford; Forde, Emer; Priego-Hernandez, Jacqueline; Butcher, Aurelia; Wedderburn, Clare

    2018-02-08

    The capacity and the commitment to reflect are integral to the practice of medicine and are core components of most general practitioners (GP) training programmes. Teaching through the humanities is a growing area within medical education, but one which is often considered a voluntary 'add-on' for the interested doctor. This article describes an evaluation of a highly innovative pedagogical project which used photography as a means to enhance GP trainees' reflective capacity, self-awareness and professional development. Photography was used as a tool to develop GP trainees' skills in recognising and articulating the attitudes, feelings and values that might impact on their clinical work and to enhance their confidence in their ability to deal with these concerns/issues. We submit that photography is uniquely well suited for facilitating insight and self-reflection because it provides the ability to record 'at the touch of a button' those scenes and images to which our attention is intuitively drawn without the need for-or the interference of-conscious decisions. This allows us the opportunity to reflect later on the reasons for our intuitive attraction to these scenes. These photography workshops were a compulsory part of the GP training programme and, despite the participants' traditional scientific backgrounds, the results clearly demonstrate the willingness of participants to accept-even embrace-the use of art as a tool for learning. The GP trainees who took part in this project acknowledged it to be beneficial for both their personal and professional development. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  5. Patient influences on satisfaction and loyalty for GP services.

    Science.gov (United States)

    Rundle-Thiele, Sharyn; Russell-Bennett, Rebekah

    2010-04-01

    Little is known about the influence that patients themselves have on their loyalty to a general practitioner (GP). Consequently, a theoretical framework that draws on diverse literature is proposed to suggest that along with satisfaction, patient loyalty is an important outcome for GPs. Comprising 174 Australian patients, this study identified that knowledgeable patients reported lower levels of loyalty while older patients and patients visiting a GP more frequently reported higher levels of loyalty. The results suggest that extending patient-centered care practices to encompass all patients may be warranted in order to improve patient satisfaction and loyalty. Further, future research opportunities abound, with intervention and dyadic research methodologies recommended.

  6. Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D

    DEFF Research Database (Denmark)

    Holmskov, U; Mollenhauer, J; Madsen, J

    1999-01-01

    in a soluble form and in association with the membranes of alveolar macrophages. The primary structure of gp-340 has been established by molecular cloning, which yielded a 7,686-bp cDNA sequence encoding a polypeptide chain of 2, 413 amino acids. The domain organization features 13 scavenger receptor cysteine...... in a way that suggested capping, whereas other macrophages showed strong intracellular staining within the phagosome/phagolysosome compartments. In some macrophages, SP-D and gp-340 were located in the same cellular compartment. Immunoreactive gp-340 was also found in epithelial cells of the small...

  7. Crystal Structure of the Carboxy-Terminal Region of the Bacteriophage T4 Proximal Long Tail Fiber Protein Gp34

    Directory of Open Access Journals (Sweden)

    Meritxell Granell

    2017-06-01

    Full Text Available Long tail fibers of bacteriophage T4 are formed by proteins gp34, gp35, gp36, and gp37, with gp34 located at the phage-proximal end and gp37 at the phage-distal, receptor-binding end. We have solved the structure of the carboxy-terminal region of gp34, consisting of amino acids 894–1289, by single-wavelength anomalous diffraction and extended the structure to amino acids 744–1289 using data collected from crystals containing longer gp34-fragments. The structure reveals three repeats of a mixed α-β fibrous domain in residues 744 to 877. A triple-helical neck connects to an extended triple β-helix domain (amino acids 900–1127 punctuated by two β-prism domains. Next, a β-prism domain decorated with short helices and extended β-helices is present (residues 1146–1238, while the C-terminal end is capped with another short β-helical region and three β-hairpins. The structure provides insight into the stability of the fibrous gp34 protein.

  8. The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity.

    Science.gov (United States)

    Scirè, Andrea; Tanfani, Fabio; Bertoli, Enrico; Furlani, Emiliano; Nadozie, Hope-Onyekwere N; Cerutti, Helena; Cortelazzo, Alessio; Bini, Luca; Guerranti, Roberto

    2011-07-15

    In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds. Copyright © 2011 Elsevier GmbH. All rights reserved.

  9. P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes.

    Science.gov (United States)

    Valton, Emeline; Amblard, Christian; Wawrzyniak, Ivan; Penault-Llorca, Frederique; Bamdad, Mahchid

    2013-12-05

    Blood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous "membrane detoxification proteins" implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality.

  10. In silico screening for inhibitors of p-glycoprotein that target the nucleotide binding domains.

    Science.gov (United States)

    Brewer, Frances K; Follit, Courtney A; Vogel, Pia D; Wise, John G

    2014-12-01

    Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  11. GP50 as a promising early diagnostic antigen for Taenia multiceps infection in goats by indirect ELISA.

    Science.gov (United States)

    Huang, Xing; Xu, Jing; Wang, Yu; Guo, Cheng; Chen, Lin; Gu, Xiaobin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

    2016-12-01

    Coenurosis is caused by coenurus, the metacestode of Taenia multiceps, which mainly parasitizes the brain and spinal cord of cattle, sheep and goats. To date, no widely-approved methods are available to identify early coenurus infection. In this study, we identified a full-length cDNA that encodes GP50 (TmGP50) from the transcriptome of T. multiceps, and then cloned and expressed in E. coli. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of recombinant TmGP50 protein (rTmGP50) for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, we orally infected 20 goats with mature T. multiceps eggs. Praziquantel (10%) was given to 10 of the goats 45 days post-infection (p.i.). Blood samples were collected for 17 weeks p.i. from the 20 goats and anti-rTmGP50 antibodies were evaluated using the indirect ELISA established here. The TmGP50 contains an 897 bp open reading frame, in which signal sequence resides in 1 ~ 48 sites and mature polypeptide consists of 282 amino acid residues. Immunofluorescence staining showed that native TmGP50 was localized to the microthrix and parenchymatous zone of the adult parasite and coenurus, and the coenurus cystic wall. The indirect ELISA based on rTmGP50 exhibited a sensitivity of 95.0% and a specificity of 92.6% when detecting GP50 antibodies in sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmGP50 antibody was detectable from 2 to 17 weeks p.i. in the control group, while the antibody fell below the cut-off value about 3 weeks after praziquantel treatment. Our results indicate that recombinant TmGP50 is a suitable early diagnostic antigen for coenurus infection in goats.

  12. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... towards the chemotactic peptide f-met-leu-phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan-activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63...... in a concentration-dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished...

  13. Using photography to enhance GP trainees’ reflective practice and professional development.

    OpenAIRE

    Rutherford,; Forde, Emer; Priego-Hernández, J.; Butcher, Aurelia; Wedderburn, Clare

    2018-01-01

    The capacity and the commitment to reflect are integral to the practice of medicine and are core components of most GP training programmes. Teaching through the Humanities is a growing area within medical education, but one which is often considered a voluntary ‘add on’ for the interested doctor. This article describes an evaluation of a highly innovative pedagogical project which used photography as a means to enhance GP trainees’ reflective capacity, self awareness and professional developm...

  14. The Pathogenicity of Anti-β2GP1-IgG Autoantibodies Depends on Fc Glycosylation

    Directory of Open Access Journals (Sweden)

    Christoph Fickentscher

    2015-01-01

    Full Text Available To analyze the glycosylation of anti-β2GP1, we investigated purified IgG from healthy children, patients with APS, and asymptomatic adult carriers of antiphospholipid antibodies. We observed that in the sera of healthy children and of patients with APS, IgG3 and IgG2 were predominant, respectively. The potentially protective anti-β2GP1-IgM was lower in the sera of healthy children. Although anti-β2GP1-associated C1q did not differ between children and patients with antiphospholipid syndrome, the associated C3c was significantly higher in the sera of healthy children. This indicates a more efficient clearance of anti-β2GP1 immune complexes in the healthy children. This clearance is not accompanied by inflammation or coagulatory events. It is likely that the most important pathogenic factor of the anti-β2GP1-IgG is related to the different glycosylation observed in healthy and diseased individuals. We detected a significantly higher sialylation of anti-β2GP1-IgG isolated from the sera of healthy children and asymptomatic adults when compared with that of patients with clinically apparent antiphospholipid syndrome. Low sialylated IgG reportedly ameliorates inflammation and inflammation promotes hyposialylation. Thus, both reactions create a vicious circle that precipitates the pathology of the antiphospholipid syndrome including thrombus-formation. We conclude that the increased sialylation of anti-β2GP1-IgG of sera of healthy individuals limits their pathogenicity.

  15. Genome profiling (GP method based classification of insects: congruence with that of classical phenotype-based one.

    Directory of Open Access Journals (Sweden)

    Shamim Ahmed

    Full Text Available Ribosomal RNAs have been widely used for identification and classification of species, and have produced data giving new insights into phylogenetic relationships. Recently, multilocus genotyping and even whole genome sequencing-based technologies have been adopted in ambitious comparative biology studies. However, such technologies are still far from routine-use in species classification studies due to their high costs in terms of labor, equipment and consumables.Here, we describe a simple and powerful approach for species classification called genome profiling (GP. The GP method composed of random PCR, temperature gradient gel electrophoresis (TGGE and computer-aided gel image processing is highly informative and less laborious. For demonstration, we classified 26 species of insects using GP and 18S rDNA-sequencing approaches. The GP method was found to give a better correspondence to the classical phenotype-based approach than did 18S rDNA sequencing employing a congruence value. To our surprise, use of a single probe in GP was sufficient to identify the relationships between the insect species, making this approach more straightforward.The data gathered here, together with those of previous studies show that GP is a simple and powerful method that can be applied for actually universally identifying and classifying species. The current success supported our previous proposal that GP-based web database can be constructible and effective for the global identification/classification of species.

  16. Correlation between the GP78 Gene Polymorphism and Coronary Atherosclerotic Heart Disease

    Directory of Open Access Journals (Sweden)

    Long Wang

    2018-01-01

    Full Text Available Objective: To study the correlation between the GP78 gene polymorphism and blood fat, blood glucose, blood pressure and coronary atherosclerotic heart disease. Methods: A total of 72 patients with coronary atherosclerotic heart disease were selected as the observation group, and 68 healthy participants were selected as the control group. The gp78 gene polymorphism of both groups was studied via polymerase chain reaction-restriction fragment length polymorphism (RFLP. At the same time, the multiple expression quantities of the GP78 gene in the tissues of both groups were tested via fluorogenic quantitative PCR, enzyme-linked immunosorbent assay (ELISA and Western-blotting assay. Furthermore, the blood fat, blood glucose and blood pressure of subjects in both groups were tested. Results: The percentages of the gp78 gene polymorphisms of Arg/Arg, Arg/Gly and Gly/Gly at the 145 locus of the study subjects in the observation group were 12.3%, 43.2% and 44.5%, respectively, while those in the control group were 74.3%, 11.2% and 14.5%, respectively, and there were significant differences between both groups. Based on the test results of the blood fat, blood glucose and blood pressure of the objects in the observation group and control group, significant differences were found between the two groups (P<0.05. Conclusion: There was a significant correlation between the 145 locus of the gp89 gene and coronary atherosclerotic heart disease, indexes of blood fat, blood glucose and blood pressure. Keywords: blood fat, blood glucose, blood pressure, coronary sclerosis, heart disease

  17. CRP-ductin, the mouse homologue of gp-340/deleted in malignant brain tumors 1 (DMBT1), binds gram-positive and gram-negative bacteria and interacts with lung surfactant protein D

    DEFF Research Database (Denmark)

    Madsen, Jens; Tornøe, Ida; Nielsen, Ole

    2003-01-01

    CRP-ductin is a protein expressed mainly by mucosal epithelial cells in the mouse. Sequence homologies indicate that CRP-ductin is the mouse homologue of human gp-340, a glycoprotein that agglutinates microorganisms and binds the lung mucosal collectin surfactant protein-D (SP-D). Here we report...... that purified CRP-ductin binds human SP-D in a calcium-dependent manner and that the binding is not inhibited by maltose. The same properties have previously been observed for gp-340 binding of SP-D. CRP-ductin also showed calcium-dependent binding to both gram-positive and -negative bacteria. A polyclonal...... antibody raised against gp-340 reacted specifically with CRP-ductin in Western blots. Immunoreactivity to CRP-ductin was found in the exocrine pancreas, in epithelial cells throughout the gastrointestinal tract and in the parotid ducts. A panel of RNA preparations from mouse tissues was screened for CRP...

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

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Suchita

    2011-01-01

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

  19. Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

    Science.gov (United States)

    Xuan, X; Maeda, K; Mikami, T; Otsuka, H

    1996-12-01

    The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

  20. The learner’s perspective in GP teaching practices with multi-level learners: a qualitative study

    Science.gov (United States)

    2014-01-01

    Background Medical students, junior hospital doctors on rotation and general practice (GP) registrars are undertaking their training in clinical general practices in increasing numbers in Australia. Some practices have four levels of learner. This study aimed to explore how multi-level teaching (also called vertical integration of GP education and training) is occurring in clinical general practice and the impact of such teaching on the learner. Methods A qualitative research methodology was used with face-to-face, semi-structured interviews of medical students, junior hospital doctors, GP registrars and GP teachers in eight training practices in the region that taught all levels of learners. Interviews were audio-recorded and transcribed. Qualitative analysis was conducted using thematic analysis techniques aided by the use of the software package N-Vivo 9. Primary themes were identified and categorised by the co-investigators. Results 52 interviews were completed and analysed. Themes were identified relating to both the practice learning environment and teaching methods used. A practice environment where there is a strong teaching culture, enjoyment of learning, and flexible learning methods, as well as learning spaces and organised teaching arrangements, all contribute to positive learning from a learners’ perspective. Learners identified a number of innovative teaching methods and viewed them as positive. These included multi-level learner group tutorials in the practice, being taught by a team of teachers, including GP registrars and other health professionals, and access to a supernumerary GP supervisor (also termed “GP consultant teacher”). Other teaching methods that were viewed positively were parallel consulting, informal learning and rural hospital context integrated learning. Conclusions Vertical integration of GP education and training generally impacted positively on all levels of learner. This research has provided further evidence about the

  1. ABCB1 (P-glycoprotein) reduces bacterial attachment to human gastrointestinal LS174T epithelial cells.

    Science.gov (United States)

    Crowe, Andrew; Bebawy, Mary

    2012-08-15

    The aim of this project was to show elevated P-glycoprotein (P-gp) expression decreasing bacterial association with LS174T human gastrointestinal cells, and that this effect could be reversed upon blocking functional P-gp efflux. Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Lactobacillus acidophilus and numerous strains of Escherichia coli, from commensal to enteropathogenic and enterohaemorrhagic strains (O157:H7) were fluorescently labelled and incubated on LS174T cultures either with or without P-gp amplification using rifampicin. PSC-833 was used as a potent functional P-gp blocking agent. Staphylococcus and Pseudomonas displayed the greatest association with the LS174T cells. Surprisingly, lactobacilli retained more fluorescence than enteropathogenic-E. coli in this system. Irrespective of attachment differences between the bacterial species, the increase in P-gp protein expression decreased bacterial fluorescence by 25-30%. This included the GFP-labelled E. coli, and enterohaemorrhagic E. coli (O157:H7). Blocking P-gp function through the co-administration of PSC-833 increased the amount of bacteria associated with P-gp expressing LS174T cells back to control levels. As most bacteria were affected to the same degree, irrespective of pathogenicity, it is unlikely that P-gp has a direct influence on adhesion of bacteria, and instead P-gp may be playing an indirect role by secreting a bank of endogenous factors or changing the local environment to one less suited to bacterial growth in general. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  2. Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

    Science.gov (United States)

    Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

    2016-05-01

    Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

  3. Lessons learned from an Internet GP information system.

    Science.gov (United States)

    Briggs, J S; Bradley, M P

    1998-01-01

    We describe the prototype of an application that in actual use would allow GPs to find out more information about consultants at hospitals. This would aid the GP in making the decision about which consultant a patient should be referred to. The requirements of the application from the GP's perspective are described, together with some of the issues that have to be resolved before hospitals can provide the necessary information in a standard format. The application is implemented as a client--server system using standard Internet technologies such as Java and HTML. This architecture has a number of advantages but also revealed some issues concerning security and the format of data, among other things. The project showed that there is a desire for such a system and that that desire can be fulfilled at a relatively low cost.

  4. Regional increase in P-glycoprotein function in the blood-brain barrier of patients with chronic schizophrenia : A PET study with [C-11]verapamil as a probe for P-glycoprotein function

    NARCIS (Netherlands)

    de Klerk, Onno L.; Willemsen, Antoon T. M.; Bosker, Fokko J.; Bartels, Anna L.; Hendrikse, N. Harry; den Boer, Johan A.; Dierckx, Rudy A.

    2010-01-01

    P-glycoprotein (P-gp), a major efflux pump in the blood-brain barrier (BBB) has a profound effect on entry of drugs, peptides and other substances into the central nervous system (CNS). The brain's permeability can be negatively influenced by modulation of the transport function of P-gp.

  5. Oncostatin M induces heat hypersensitivity by gp130-dependent sensitization of TRPV1 in sensory neurons

    Directory of Open Access Journals (Sweden)

    Langeslag Michiel

    2011-12-01

    Full Text Available Abstract Oncostatin M (OSM is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130-/- and gp130 floxed (gp130fl/fl mice. Subcutaneous injection of pathophysiologically relevant concentrations of OSM into the hind-paw of C57BL6J wild type mice significantly reduced paw withdrawal latencies to heat stimulation. In contrast to gp130fl/fl mice, OSM did not induce heat hypersensitivity in vivo in SNS-gp130-/- mice. OSM applied at the receptive fields of sensory neurons in in vitro skin-nerve preparations showed that OSM significantly increased the discharge rate during a standard ramp-shaped heat stimulus. The capsaicin- and heat-sensitive ion channel TRPV1, expressed on a subpopulation of nociceptive neurons, has been shown to play an important role in inflammation-induced heat hypersensitivity. Stimulation of cultured dorsal root ganglion neurons with OSM resulted in potentiation of capsaicin induced ionic currents. In line with these recordings, mice with a null mutation of the TRPV1 gene did not show any signs of OSM-induced heat hypersensitivity in vivo. The present data suggest that OSM induces thermal hypersensitivity by directly sensitizing nociceptors via OSMR-gp130 receptor mediated potentiation of TRPV1.

  6. Adult T-cell leukemia-associated antigen (ATLA): detection of a glycoprotein in cell- and virus-free supernatant.

    Science.gov (United States)

    Yamamoto, N; Schneider, J; Hinuma, Y; Hunsmann, G

    1982-01-01

    A glycoprotein of an apparent molecular mass of 46,000, gp 46, was enriched by affinity chromatography from the virus- and cell-free culture medium of adult T-cell leukemia virus (ATLV) infected cells. gp 46 was specifically precipitated with sera from patients with adult T-cell leukemia associated antigen (ATLA). Thus, gp 46 is a novel component of the ATLA antigen complex.

  7. Admission and poor performance of trainees in the postgraduate GP training in the Netherlands

    NARCIS (Netherlands)

    Vermeulen, M.I.

    2014-01-01

    Until 2014 the selection for the Dutch postgraduate GP training was conducted locally, on the eight GP departments. The procedure consisted of a letter of application and a semi-structured interview. We investigated to what extend department of choice, candidates’ characteristics and qualities

  8. 'I need her to be a doctor': patients' experiences of presenting health information from the internet in GP consultations.

    Science.gov (United States)

    Bowes, Parvathy; Stevenson, Fiona; Ahluwalia, Sanjiv; Murray, Elizabeth

    2012-11-01

    Patients are increasingly using the internet for health-related information and may bring this to a GP consultation. There is scant information about why patients do this and what they expect from their GP. The aim was to explore patients' motivation in presenting information, their perception of the GP's response and what they wanted from their doctor. Qualitative study based in North London involving patients with experience of bringing health information from the internet to their GP. Semi-structured face-to-face and telephone interviews using a critical incident technique, recorded, transcribed verbatim, and subjected to thematic analysis by a multidisciplinary team of researchers. Twenty-six interviews were completed. Participants reported using the internet to become better informed about their health and hence make best use of the limited time available with the GP and to enable the GP to take their problem more seriously. Patients expected their GP to acknowledge the information; discuss, explain, or contextualise it; and offer a professional opinion. Patients tended to prioritise the GP opinion over the internet information. However, if the GP appeared disinterested, dismissive or patronising patients reported damage to the doctor-patient relationship, occasionally to the extent of seeking a second opinion or changing their doctor. This is the first in-depth qualitative study to explore why patients present internet information to their GP within the consultation and what they want when they do this. This information should help GPs respond appropriately in such circumstances.

  9. GPs' experiences with out-of-hours GP cooperatives: a survey study from the Netherlands.

    Science.gov (United States)

    Smits, Marleen; Keizer, Ellen; Huibers, Linda; Giesen, Paul

    2014-09-01

    Out-of-hours primary care has been provided by general practitioner (GP) cooperatives since the year 2000 in the Netherlands. Early studies in countries with similar organizational structures showed positive GP experiences. However, nowadays it is said that GPs experience a high workload at the cooperative and that they outsource a considerable part of their shifts. To examine positive and negative experiences of GPs providing out-of-hours primary care, and the frequency and reasons for outsourcing shifts. A cross-sectional observational survey among 688 GPs connected to six GP cooperatives in the Netherlands, using a web-based questionnaire. The response was 55% (n = 378). The main reasons for working in GP cooperatives were to retain registration as GP (79%) and remain experienced in acute care (74%). GPs considered the peak hours (81%) and the high number of patients (73%) as the most negative aspects. Most GPs chose to provide the out-of-hours shifts themselves: 85% outsourced maximally 25% of their shifts. The percentage of outsourced shifts increased with age. Main reasons for outsourcing were the desire to have more private time (76%); the high workload in daytime practice (71%); and less the workload during out-of-hours (46%). GPs are motivated to work in out-of-hours GP cooperatives, and they outsource few shifts. GPs consider the peak load and the large number of (non-urgent) help requests as the most negative aspects. To motivate and involve GPs for 7 × 24-h primary care, it is important to set limits on their workload.

  10. Critical review and hydrologic application of threshold detection methods for the generalized Pareto (GP) distribution

    Science.gov (United States)

    Mamalakis, Antonios; Langousis, Andreas; Deidda, Roberto

    2016-04-01

    Estimation of extreme rainfall from data constitutes one of the most important issues in statistical hydrology, as it is associated with the design of hydraulic structures and flood water management. To that extent, based on asymptotic arguments from Extreme Excess (EE) theory, several studies have focused on developing new, or improving existing methods to fit a generalized Pareto (GP) distribution model to rainfall excesses above a properly selected threshold u. The latter is generally determined using various approaches, such as non-parametric methods that are intended to locate the changing point between extreme and non-extreme regions of the data, graphical methods where one studies the dependence of GP distribution parameters (or related metrics) on the threshold level u, and Goodness of Fit (GoF) metrics that, for a certain level of significance, locate the lowest threshold u that a GP distribution model is applicable. In this work, we review representative methods for GP threshold detection, discuss fundamental differences in their theoretical bases, and apply them to 1714 daily rainfall records from the NOAA-NCDC open-access database, with more than 110 years of data. We find that non-parametric methods that are intended to locate the changing point between extreme and non-extreme regions of the data are generally not reliable, while methods that are based on asymptotic properties of the upper distribution tail lead to unrealistically high threshold and shape parameter estimates. The latter is justified by theoretical arguments, and it is especially the case in rainfall applications, where the shape parameter of the GP distribution is low; i.e. on the order of 0.1 ÷ 0.2. Better performance is demonstrated by graphical methods and GoF metrics that rely on pre-asymptotic properties of the GP distribution. For daily rainfall, we find that GP threshold estimates range between 2÷12 mm/d with a mean value of 6.5 mm/d, while the existence of quantization in the

  11. Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells

    DEFF Research Database (Denmark)

    Stavru, Fabrizia; Nautrup-Pedersen, Gitte; Cordes, Volker C

    2006-01-01

    So far, POM121 and gp210 are the only known anchoring sites of vertebrate nuclear pore complexes (NPCs) within the lipid bilayer of the nuclear envelope (NE) and, thus, are excellent candidates for initiating the NPC assembly process. Indeed, we demonstrate that POM121 can recruit several...... as depletion of POM121 from human fibroblasts, which do not express gp210, further suggest that NPCs can assemble or at least persist in a POM121- and gp210-free form. This points to extensive redundancies in protein-protein interactions within NPCs and suggests that vertebrate NPCs contain additional membrane...

  12. Comb-like amphiphilic polypeptide-based copolymer nanomicelles for co-delivery of doxorubicin and P-gp siRNA into MCF-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Suo, Aili, E-mail: ailisuo@mail.xjtu.edu.cn [Department of Oncology, The First Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710061 (China); Qian, Junmin, E-mail: jmqian@mail.xjtu.edu.cn [State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China); Zhang, Yaping; Liu, Rongrong; Xu, Weijun [State Key Laboratory for Mechanical Behavior of Materials, Xi' an Jiaotong University, Xi' an 710049 (China); Wang, Hejing [Department of Oncology, The First Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710061 (China)

    2016-05-01

    A comb-like amphiphilic copolymer methoxypolyethylene glycol-graft-poly(L-lysine)-block-poly(L-phenylalanine) (mPEG-g-PLL-b-Phe) was successfully synthesized. To synthesize mPEG-g-PLL-b-Phe, diblock copolymer PLL-b-Phe was first synthesized by successive ring-opening polymerization of α-amino acid N-carboxyanhydrides followed by the removal of benzyloxycarbonyl protecting groups, and then mPEG was grafted onto PLL-b-Phe by reductive amination via Schiff's base formation. The chemical structures of the copolymers were identified by {sup 1}H NMR. mPEG-g-PLL-b-Phe copolymer had a critical micelle concentration of 6.0 mg/L and could self-assemble in an aqueous solution into multicompartment nanomicelles with a mean diameter of approximately 78 nm. The nanomicelles could encapsulate doxorubicin (DOX) through hydrophobic and π–π stacking interactions between DOX molecules and Phe blocks and simultaneously complex P-gp siRNA with cationic PLL blocks via electrostatic interactions. The DOX/P-gp siRNA-loaded nanomicelles showed spherical morphology, possessed narrow particle size distribution and had a mean particle size of 120 nm. The DOX/P-gp siRNA-loaded nanomicelles exhibited pH-responsive release behaviors and displayed accelerated release under acidic conditions. The DOX/P-gp siRNA-loaded nanomicelles were efficiently internalized into MCF-7 cells, and DOX released could successfully reach nuclei. In vitro cytotoxicity assay demonstrated that the DOX/P-gp siRNA-loaded nanomicelles showed a much higher cytotoxicity in MCF-7 cells than DOX-loaded nanomicelles due to their synergistic killing effect and that the blank nanomicelles had good biocompatibility. Thus, the novel comb-like mPEG-g-PLL-b-Phe nanomicelles could be a promising vehicle for co-delivery of chemotherapeutic drug and genetic material. - Highlights: • Comb-like amphiphilic copolymer mPEG-g-PLL-b-Phe was successfully synthesized. • Polypeptide-based copolymer could self-assemble into

  13. Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing.

    Directory of Open Access Journals (Sweden)

    Zhitao Wan

    Full Text Available The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline. Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.

  14. The Role of Conserved N-Linked Glycans on Ebola Virus Glycoprotein 2.

    Science.gov (United States)

    Lennemann, Nicholas J; Walkner, Madeline; Berkebile, Abigail R; Patel, Neil; Maury, Wendy

    2015-10-01

    N-linked glycosylation is a common posttranslational modification found on viral glycoproteins (GPs) and involved in promoting expression, cellular attachment, protection from proteases, and antibody evasion. The GP subunit GP2 of filoviruses contains 2 completely conserved N-linked glycosylation sites (NGSs) at N563 and N618, suggesting that they have been maintained through selective pressures. We assessed mutants lacking these glycans for expression and function to understand the role of these sites during Ebola virus entry. Elimination of either GP2 glycan individually had a modest effect on GP expression and no impact on antibody neutralization of vesicular stomatitis virus pseudotyped with Ebola virus GP. However, loss of the N563 glycan enhanced entry by 2-fold and eliminated GP detection by a well-characterized monoclonal antibody KZ52. Loss of both sites dramatically decreased GP expression and abolished entry. Surprisingly, a GP that retained a single NGS at N563, eliminating the remaining 16 NGSs from GP1 and GP2, had detectable expression, a modest increase in entry, and pronounced sensitivity to antibody neutralization. Our findings support the importance of the GP2 glycans in GP expression/structure, transduction efficiency, and antibody neutralization, particularly when N-linked glycans are also removed from GP1. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Anti-HIV-1 activity of anionic polymers: a comparative study of candidate microbicides

    Directory of Open Access Journals (Sweden)

    Li Yun-Yao

    2002-11-01

    Full Text Available Abstract Background Cellulose acetate phthalate (CAP in soluble form blocks coreceptor binding sites on the virus envelope glycoprotein gp120 and elicits gp41 six-helix bundle formation, processes involved in virus inactivation. CAP is not soluble at pH Methods Enzyme linked immunosorbent assays (ELISA were used to (1 study HIV-1 IIIB and BaL binding to micronized CAP; (2 detect virus disintegration; and (3 measure gp41 six-helix bundle formation. Cells containing integrated HIV-1 LTR linked to the β-gal gene and expressing CD4 and coreceptors CXCR4 or CCR5 were used to measure virus infectivity. Results 1 HIV-1 IIIB and BaL, respectively, effectively bound to micronized CAP. 2 The interaction between HIV-1 and micronized CAP led to: (a gp41 six-helix bundle formation; (b virus disintegration and shedding of envelope glycoproteins; and (c rapid loss of infectivity. Polymers other than CAP, except Carbomer 974P, elicited gp41 six-helix bundle formation in HIV-1 IIIB but only poly(napthalene sulfonate, in addition to CAP, had this effect on HIV-1 BaL. These polymers differed with respect to their virucidal activities, the differences being more pronounced for HIV-1 BaL. Conclusions Micronized CAP is the only candidate topical microbicide with the capacity to remove rapidly by adsorption from physiological fluids HIV-1 of both the X4 and R5 biotypes and is likely to prevent virus contact with target cells. The interaction between micronized CAP and HIV-1 leads to rapid virus inactivation. Among other anionic polymers, cellulose sulfate, BufferGel and aryl sulfonates appear most effective in this respect.

  17. Binding of radiolabeled asbestos fibers to guinea pig (gp) alveolar macrophages (AM)

    International Nuclear Information System (INIS)

    Giannotti, M.A.; Tewson, T.J.; Francsechini, M.P.; Scheule, R.K.; Holian, A.

    1990-01-01

    The mechanism by which fibrogenic particulates cause pulmonary fibrosis in humans is not understood, but is likely to involve the AM. Using two fibrogenic particulates, namely, chrysotile (CHR) and crocidolite (CRO) asbestos and gpAM as components of an in vitro model system, the authors have shown that CHR stimulates the gpAM to release superoxide anion, but CRO does not. To examine whether this difference in stimulatory abilities is a result of differences in cell-asbestos binding they have developed an efficient procedure that radiolabels asbestos fibers while retaining their bioactivity. The fibers are labeled with 68 Ge. The 68 Ge decays into 68 Ga, which then can be detected by its characteristic position emission. Both CHR and CRO asbestos were radiolabled successfully. Mild reaction conditions and short reaction times were found under which >90% of the added 68 Ge and 68 Ga bound to the fibers. The radiolabel was retained even after washing the fibers extensively with physiologic buffers. A density gradient procedure was developed to quantitate the binding of asbestos to gpAM in suspension. The binding of both fibers increased with time over one hr. Thus, these results indicate that although both CHR and CRO interact with the gpAM, only CHR interacts productively to stimulate superoxide anion release

  18. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

    International Nuclear Information System (INIS)

    Papaneri, Amy B.; Wirblich, Christoph; Cann, Jennifer A.; Cooper, Kurt; Jahrling, Peter B.; Schnell, Matthias J.; Blaney, Joseph E.

    2012-01-01

    We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVΔG-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RVΔG-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RVΔG-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVΔG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

  19. 77 FR 20807 - Northwest Pipeline GP; Notice of Application

    Science.gov (United States)

    2012-04-06

    ... diameter pipelines away from an adjacent surface coal mine west of Kemmerer, Wyoming. Northwest also... directed to Pam Barnes, Manager Certificates and Tariffs, Northwest Pipeline GP, 295 Chipeta Way, Salt Lake...

  20. Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

    International Nuclear Information System (INIS)

    Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner . E-mail; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria

    2008-01-01

    The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo- and radiotherapy, particularly when radiation exposure precedes chemical treatment. (author)

  1. Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear]. E-mail; neylisantos@yahoo.com.br; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria [Fundacao de Hematologia e Hemoterapia de Pernambuco, Recife, PE (Brazil). Unidade de Laboratorios Especializados. Lab. de Imunofenotipagem

    2008-12-15

    The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo- and radiotherapy, particularly when radiation exposure precedes chemical treatment. (author)

  2. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    International Nuclear Information System (INIS)

    Walker, G.; Bourguignon, L.Y.

    1990-01-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation

  3. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    Energy Technology Data Exchange (ETDEWEB)

    Walker, G.; Bourguignon, L.Y. (Univ. of Miami Medical School, FL (USA))

    1990-08-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

  4. Commissioning of the iGp Feedback System at DAΦNE

    International Nuclear Information System (INIS)

    Drago, A.; Fox, J.D.; Teytelman, D.; Tobiyama, M.

    2011-01-01

    The iGp (Integrated Gigasample Processor) is an innovative digital bunch-by-bunch feedback system developed by a KEK / SLAC / INFN-LNF joint collaboration. The processing unit can sample at 500 MHz and compute the bunch-by-bunch output signal for up to ∼5000 bunches. The feedback gateware code is implemented inside just one FPGA (Field Programmable Gate Array) chip, a Xilinx Virtex-II. The FPGA implements two banks of 16-tap FIR (Finite Impulse Response) filters. Each filter is realtime programmable through the operator interface. At DAΦNE, the Frascati Φ-Factory, two iGp units have been commissioned in the April 2007. The iGp systems have substituted the previous betatron feedback systems. This insertion has been very fast and has shown no problems involving just a substitution of the old, less flexible, digital systems, letting unchanged the baseband analog frontend and backend. The commissioning has been very simple, due to the complete and powerful EPICS operator interface, working well in local and remote operations. The software includes also tools for analyzing post processor data. A description of the commissioning with the operations done is reported.

  5. Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates.

    Directory of Open Access Journals (Sweden)

    Chad E Mire

    Full Text Available The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV that expresses an individual filovirus glycoprotein (GP in place of the VSV glycoprotein (G. The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV GP; three animals received rVSV-wild type (wt vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.

  6. Values of serum AFP, GGTⅡ and GP73 in diagnosis of primary hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    ZHU Chen

    2014-10-01

    Full Text Available ObjectiveTo explore the early diagnostic values of serum alpha-fetoprotein (AFP, gamma-glutamyltransferase Ⅱ (GGTⅡ, and Golgi protein 73 (GP73 in patients with primary hepatocellular carcinoma (PHC. MethodsThe serum specimens of 100 patients with liver diseases (50 cases of hepatitis and liver cirrhosis and 50 cases of PHC and 50 healthy people were collected in our hospital from February 2013 to February 2014. Electrochemical luminescence technique, specific immuno-membrane adsorption assay, and enzyme-linked immunosorbent assay were used to measure the serum levels of AFP, GGTⅡ, and GP73. Comparison of continuous data between multiple groups was made by analysis of variance, and comparison between two groups was made by q test. The receiver operating characteristic (ROC curves of single or combined test results were made, and the areas under the ROC curves (AUCs were calculated. The sensitivity, specificity, and AUCs of AFP, GGTⅡ, GP73, and the combined test were analyzed and compared. ResultsThe level of serum GGTⅡ in the PHC group was significantly different compared with those in the other two groups (F=16.224, P<0.05, but there was no significant difference between the normal group and the hepatitis and liver cirrhosis group (P>0.05. Significant differences in serum levels of AFP and GP73 were observed by paired comparison between the PHC group, hepatitis and liver cirrhosis group, and normal group (F=193.128, F=20.231, P<0.05 for both. When assayed alone, the specificities of GP73, GGTⅡ, and AFP were 69%, 64% and 51%, respectively, and the sensitivities were 92%, 84%, and 76%, respectively. In combined test, the specificity was 94.6% and the sensitivity was 98.8%. ConclusionThe GP73 test is the best performer in the single assays. Combined test of serum AFP, GGTⅡ, and GP73 shows a good diagnostic value for PHC with greatly improved specificity and sensitivity.

  7. A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

    International Nuclear Information System (INIS)

    Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko; Kobayashi, Michihiro

    2010-01-01

    We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.

  8. P-gp Expression and Rh 123 Efflux Assay Have no Impact on Survival in Egyptian Pediatric Acute Lymphoblastic Leukemia Patients

    International Nuclear Information System (INIS)

    Kamel, A.M.; El-Sharkawy, N.; Yassin, D.; Shaaban, Kh.; Hussein, H.; Sidhom, I; Abo El-Naga, S.; Ameen, M.; El-Hattab, O.; Aly El-Din, N.H.

    2005-01-01

    In a previous work we have studied MDR status in terms of P-glycoprotein (P-gp) expression and Rhodamine 123 efflux assay in Egyptian acute leukemia patients. We have reported results comparable to the literature as regards ANLL both in pediatric and adult cases. However, higher figures were encountered for the functional assay in ALL. As our ALL cases especially in pediatric age group show worse prognosis compared to literature, we hypothesized that the higher percentage of cases with positive Rh 123 efflux assay might be a contributing factor. Material and Methods: A total of 108 cases were studied including 80 ALL and 28 ANLL. ALL cases included 48 male and 32 female with an age range of 6m to 18 yrs and a median of 7 yrs. ANLL cases included 18 male and 10 female with an age range of 6m to 18 yrs and a median of 8 yrs. P-gp expression was evaluated using 4E3 and DIC2 mAb, analyzed by Coulter XL flow cytometer and expressed as a ratio at a cut off of ≤1.1 and/or ≤5% positive cells. For the evaluation of MDR function Rh 123 efflux assay using cyclosporine as a blocker and expressed as a ratio at a cutoff of ≤1. 1 and/or ≤ 10% positive cells was performed. MDR expression and function were correlated to age, Hb, TLC, CD34 expression, immuno phenotype and DNA index in ALL, FAB subtypes in ANLL as well as to CR, DFS and EFS in ALL. In ALL, P-gp expression was encountered in 26.4% of cases. Positive Rh efflux was reported in 61.5%. No correlation was encountered between neither expression nor functional assay with age, Hb, TLC, CD34 expression or immuno phenotype. CR was achieved in 89.8%; neither P-gp expression nor Rh123 efflux had an impact on CR except for Rh 123 efflux in T-ALL where a cutoff of 1.25 could predict CR at a total accuracy of 70.6%. DFS was 92.3% while EFS was 72.2% for the whole group. No significant difference was encountered neither between cases expressing or lacking P-gp nor between cases with negative or positive Rh 123 efflux assay

  9. Differences in cell morphometry, cell wall topography and gp70 expression correlate with the virulence of Sporothrix brasiliensis clinical isolates.

    Directory of Open Access Journals (Sweden)

    Rafaela A Castro

    Full Text Available Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.

  10. Mechanisms of P-Glycoprotein Modulation by Semen Strychni Combined with Radix Paeoniae Alba

    Directory of Open Access Journals (Sweden)

    Li-Li Liu

    2017-01-01

    Full Text Available Semen Strychni has been extensively used as a Chinese herb, but its therapeutic window is narrowed by the strong toxicity of the compound, which limits its effectiveness. Radix Paeoniae Alba has been reported to reduce the toxic effects and increase the therapeutic effects of Semen Strychni, but the underlying mechanism remains unknown. This research aimed to explore the mechanism through which P-glycoprotein (P-gp is modulated by Semen Strychni combined with Radix Paeoniae Alba in vitro. An MTT assay was used to study cytotoxicity in an MDCK-MDR1 cell model. Rh123 efflux and accumulation were measured to assess P-gp function. The expression levels of MDR1 mRNA and P-gp protein in MDCK-MDR1 cells were investigated. A P-gp ATPase activity assay kit was applied to detect the effect on P-gp ATPase activity. Semen Strychni combined with Radix Paeoniae Alba could induce P-gp-mediated drug transport by inhibiting brucine and strychnine transport in MDCK-MDR1 cells, enhancing the P-gp efflux function, upregulating the P-gp expression and MDR1 mRNA levels, and stimulating P-gp ATPase activity.

  11. Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

    International Nuclear Information System (INIS)

    Kunz, Stefan; Edelmann, Kurt H.; Torre, Juan-Carlos de la; Gorney, Robert; Oldstone, Michael B.A.

    2003-01-01

    The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP

  12. Do practice nurse solve future GP capacity problems?

    NARCIS (Netherlands)

    Lamkaddem, M.; Haan, J. de; Bakker, D. de

    2003-01-01

    Background: Task delegation is viewed as an important policy instrument to counter foreseen future shortages in GP capacity in the Netherlands. Therefore, a national programme to introduce practice nurses in general practice was launched in 1998 by the National Association of General Practice. In

  13. Potable Water Treatment Facility General Permit (PWTF GP) ...

    Science.gov (United States)

    2017-08-28

    The Final PWTF GP establishes permit eligibility conditions, Notice of Intent (NOI) requirements, effluent limitations, standards, prohibitions, and best management practices for facilities that discharge to waters in the Commonwealth of Massachusetts (including both Commonwealth and Indian country lands) and the State of New Hampshire.

  14. P-glycoprotein Inhibition by the Agricultural Pesticide Propiconazole and Its Hydroxylated Metabolites: Implications for Pesticide-Drug Interactions.

    Science.gov (United States)

    The human efflux transporter P-glycoprotein (P-gp; MDR1) functions an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure ass...

  15. Triorganotin Derivatives Induce Cell Death Effects on L1210 Leukemia Cells at Submicromolar Concentrations Independently of P-glycoprotein Expression

    Directory of Open Access Journals (Sweden)

    Viera Bohacova

    2018-05-01

    Full Text Available The acceleration of drug efflux activity realized by plasma membrane transporters in neoplastic cells, particularly by P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family, represents a frequently observed molecular cause of multidrug resistance (MDR. This multiple resistance represents a real obstacle in the effective chemotherapy of neoplastic diseases. Therefore, identifying cytotoxic substances that are also effective in P-gp overexpressing cells may be useful for the rational design of substances for the treatment of malignancies with developed MDR. Here, we showed that triorganotin derivatives—tributyltin-chloride (TBT-Cl, tributyltin-bromide (TBT-Br, tributyltin-iodide (TBT-I and tributyltin-isothiocyanate (TBT-NCS or triphenyltin-chloride (TPT-Cl and triphenyltin-isothiocyanate (TPT-NCS—could induce the death of L1210 mice leukemia cells at a submicromolar concentration independently of P-gp overexpression. The median lethal concentration obtained for triorganotin derivatives did not exceed 0.5 µM in the induction of cell death of either P-gp negative or P-gp positive L1210 cells. Apoptosis related to regulatory pathway of Bcl-2 family proteins seems to be the predominant mode of cell death in either P-gp negative or P-gp positive L1210 cells. TBT-Cl and TBT-Br were more efficient with L1210 cells overexpressing P-gp than with their counterpart P-gp negative cells. In contrast, TBT-I and TPT-NCS induced a more pronounced cell death effect on P-gp negative cells than on P-gp positive cells. Triorganotin derivatives did not affect P-gp efflux in native cells measured by calcein retention within the cells. Taken together, we assumed that triorganotin derivatives represent substances suitable for suppressing the viability of P-gp positive malignant cells.

  16. Analysis of purified gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and tandem mass spectrometry.

    Science.gov (United States)

    Fairburn, B; Muthana, M; Hopkinson, K; Slack, L K; Mirza, S; Georgiou, A S; Espigares, E; Wong, C; Pockley, A G

    2006-09-01

    The stress protein gp96 exhibits a number of immunological activities, the majority of studies into which have used gp96 purified from a variety of tissues. On the basis of 1-D gel electrophoresis, the purity of these preparations has been reported to range between 70% and 99%. This study analyzed gp96 preparations from rat and mouse livers using 2-D gel electrophoresis and liquid chromatography electrospray ionization tandem mass spectrometry (MS-MS). The procedure for purifying gp96 was reproducible, as similar protein profiles were observed in replicate gels of gp96 preparations. The purity of the preparations was typically around 70%, with minor co-purified proteins of varying molecular weights and mobilities being present. Dominant bands at 95-100 kDa in preparations from Wistar rats and C57BL/6 mice were identified as gp96 by ECL Western blotting. Multiple bands having similar, yet distinct molecular weights and differing pI mobility on ECL Western blots were confirmed as being gp96 in preparations from Wistar rats using MS-MS. The most striking feature of the 2-D gel analysis was the presence of additional dominant bands at 55 kDa in preparations from Wistar rats, and at 75-90 kDa in preparations from C57BL/6 mice. These were identified as gp96 by ECL Western blotting and, in the case of preparations from Wistar rats, by MS-MS. Although the lower molecular weight, gp96-related molecules might be partially degraded gp96, their reproducible presence, definition and characteristics suggest that they are alternative, species-specific isoforms of the molecule. A 55 kDa protein which exhibited a lower pI value than gp96 was present in all preparations and this was identified as calreticulin, another putative immunoregulatory molecule. This study confirms the reproducibility of the gp96 purification protocol and reveals the presence of multiple gp96 isoforms, some of which likely result from post-translational modifications such as differential glycosylation and

  17. (11) C- and (18) F-Labeled Radioligands for P-Glycoprotein Imaging by Positron Emission Tomography.

    NARCIS (Netherlands)

    Cantore, Mariangela; Benadiba, Marcel; Elsinga, Philippus; Kwizera, Chantal; Dierckx, Rudi; Colabufo, Nicola A.; Luurtsema, Geert

    2016-01-01

    P-Glycoprotein (P-gp) is an efflux transporter widely expressed at the human blood-brain barrier. It is involved in xenobiotics efflux and in onset and progression of neurodegenerative disorders. For these reasons, there is great interest in the assessment of P-gp expression and function by

  18. P-glycoprotein-deficient mice have proximal tubule dysfunction but are protected against ischemic renal injury

    NARCIS (Netherlands)

    Huls, M.; Kramers, C.; Levtchenko, E.N.; Wilmer, M.J.G.; Dijkman, H.B.P.M.; Kluijtmans, L.A.J.; Hoorn, J.W.A. van der; Russel, F.G.M.; Masereeuw, R.

    2007-01-01

    The multidrug resistance gene 1 product, P-glycoprotein (P-gp), is expressed in several excretory organs, including the apical membrane of proximal tubules. After inducing acute renal failure, P-gp expression is upregulated and this might be a protective function by pumping out toxicants and harmful

  19. Relationship between recurrent spontaneous abortion and anti-β2-glycoprotein I antibody

    International Nuclear Information System (INIS)

    Liu Jingzhu; Hu Jing; Zhang Shiping; Zhang Shuang; Lin Li; Fan Jing

    2008-01-01

    Objective: To explore the relationship between recurrent spontaneous abortion (RSA) and anti-β 2 - glycoprotein I (anti-β 2 -GP I) antibody. Methods: The levels of anti-β 2 -GP I antibody in serum from 81 RSA patients and 39 normal women were detected by ELISA. Results: The positive rate of anti-β 2 -GP I in RSA patients (42.0%) was obviously higher than that in normal women (7.7 %) (P 2 -GP I IgG in statistics between RSA patients (40.8%) and normal women (7.7%) (P 2 -GP I IgM in statistics between RSA patients and normal women (P>0.05). There was no difference of the positive rate of anti-β 2 -GP I in statistics between early and late, as well as between 2 times and more than 2 times abortions of RSA (P>0.05). Conclusion: The anti-β 2 -GP I antibody is related to RSA, and it may be regarded as a immunological assistant diagnostic index for RSA. (authors)

  20. Study of coherence strain of GP II zones in an aged aluminum composite

    International Nuclear Information System (INIS)

    Hernández-Rivera, J.L.; Rivera, J.J. Cruz; Koch, C.T.; Özdöl, V.B.; Martínez-Sánchez, R.

    2012-01-01

    Highlights: ► Precipitation sequence was changed in 2024 Al alloy. ► Image artifacts ought to the mask size and shape increased the real strain value. ► The strain distribution around experimental GP area was non uniform and more complex than reference. ► The origin of this strain distribution are related to mechanisms by which GP precipitates lose coherence. - Abstract: Strain mapping using the geometric phase analysis (GPA) technique was applied to Al–GP II (Guinier–Preston) nanoscale precipitates, using both high resolution transmission electron microscopy (HRTEM) micrographs as well as the exit wave function (EWF) obtained by focal series reconstruction. The experimental strain results were compared with strain maps obtained from an atomic model which consisted of an Al supercell containing a GP II precipitate. It was built as a reference from literature data. The experimental results demonstrate a complex strain distribution and larger fluctuations than the reference strain maps. These differences were found to be partly a consequence of image artifacts produced by the technique as well as complex microstructural events which were present at the development stage studied.

  1. In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

    International Nuclear Information System (INIS)

    Han Yi; Chin Tan, Theresa May; Lim, Lee-Yong

    2008-01-01

    Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 μM, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [ 3 H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [ 3 H]-digoxin polarized transport attained at 50 μM of piperine. In contrast, prolonged (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 μM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [ 3 H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 μg/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper

  2. Structural and biochemical characterization of phage λ FI protein (gpFI) reveals a novel mechanism of DNA packaging chaperone activity.

    Science.gov (United States)

    Popovic, Ana; Wu, Bin; Arrowsmith, Cheryl H; Edwards, Aled M; Davidson, Alan R; Maxwell, Karen L

    2012-09-14

    One of the final steps in the morphogenetic pathway of phage λ is the packaging of a single genome into a preformed empty head structure. In addition to the terminase enzyme, the packaging chaperone, FI protein (gpFI), is required for efficient DNA packaging. In this study, we demonstrate an interaction between gpFI and the major head protein, gpE. Amino acid substitutions in gpFI that reduced the strength of this interaction also decreased the biological activity of gpFI, implying that this head binding activity is essential for the function of gpFI. We also show that gpFI is a two-domain protein, and the C-terminal domain is responsible for the head binding activity. Using nuclear magnetic resonance spectroscopy, we determined the three-dimensional structure of the C-terminal domain and characterized the helical nature of the N-terminal domain. Through structural comparisons, we were able to identify two previously unannotated prophage-encoded proteins with tertiary structures similar to gpFI, although they lack significant pairwise sequence identity. Sequence analysis of these diverse homologues led us to identify related proteins in a variety of myo- and siphophages, revealing that gpFI function has a more highly conserved role in phage morphogenesis than was previously appreciated. Finally, we present a novel model for the mechanism of gpFI chaperone activity in the DNA packaging reaction of phage λ.

  3. Zosuquidar restores drug sensitivity in P-glycoprotein expressing acute myeloid leukemia (AML)

    International Nuclear Information System (INIS)

    Tang, Ruoping; Faussat, Anne-Marie; Perrot, Jean-Yves; Marjanovic, Zora; Cohen, Simy; Storme, Thomas; Morjani, Hamid; Legrand, Ollivier; Marie, Jean-Pierre

    2008-01-01

    Chemotherapeutic drug efflux via the P-glycoprotein (P-gp) transporter encoded by the MDR1/ABCB1 gene is a significant cause of drug resistance in numerous malignancies, including acute leukemias, especially in older patients with acute myeloid leukemia (AML). Therefore, the P-gp modulators that block P-gp-mediated drug efflux have been developed, and used in combination with standard chemotherapy. In this paper, the capacity of zosuquidar, a specific P-gp modulator, to reverse chemoresistance was examined in both leukemia cell lines and primary AML blasts. The transporter protein expressions were analyzed by flow cytometry using their specific antibodies. The protein functionalities were assessed by the uptake of their fluorescence substrates in presence or absence their specific modulators. The drug cytotoxicity was evaluated by MTT test. Zosuquidar completely or partially restored drug sensitivity in all P-gp-expressing leukemia cell lines tested and enhanced the cytotoxicity of anthracyclines (daunorubicin, idarubicin, mitoxantrone) and gemtuzumab ozogamicin (Mylotarg) in primary AML blasts with active P-gp. In addition, P-gp inhibition by zosuquidar was found to be more potent than cyclosporine A in cells with highly active P-gp. These in vitro studies suggest that zosuquidar may be an effective adjunct to cytotoxic chemotherapy for AML patients whose blasts express P-gp, especially for older patients

  4. Leukemia: Derived heat shock protein gp96-peptide complex ...

    African Journals Online (AJOL)

    Jane

    2011-06-27

    Jun 27, 2011 ... Leukemia is a malignant clonal disease in hematopoietic stem cells that is typically treated with chemotherapy and radiotherapy. However ..... with autologous tumor-derived heatshock protein gp96 after liver resection for ...

  5. Studies on adsorptions of metallic ions in water by zirconium glyphosate (ZrGP): Behaviors and mechanisms

    International Nuclear Information System (INIS)

    Jia Yunjie; Zhang Yuejuan; Wang Runwei; Fan Faying; Xu Qinghong

    2012-01-01

    A new adsorbent named zirconium glyphosate [Zr(O 3 PCH 2 NHCH 2 COOH) 2 ·0.5H 2 O, denoted as ZrGP] and its selective adsorptions to Pb 2+ , Cd 2+ , Mg 2+ and Ca 2+ ions in water were reported in this paper. Compared to other zirconium adsorbents, such as zirconium phosphate [Zr(HPO 4 ) 2 ], ZrGP exhibited highly selective adsorption to Pb 2+ in solution which contained Pb 2+ , Cd 2+ , Mg 2+ and Ca 2+ ions. The loaded ZrGP with metallic ions can be efficaciously regenerated by aqueous solution of HCl (1.0 M) without any noticeable capacity loss, and almost all of it can be reused and recycled. The memory effect on structural regeneration of ZrGP was also found when Mg 2+ and Ca 2+ were adsorbed. To be specific, the structure of ZrGP was destroyed due to adsorbing these two ions, but it could be regenerated after the loaded materials were dipped in HCl solution (1.0 M) for several minutes to remove metallic ions.

  6. The characteristics of general practice and the attractiveness of working as a GP: medical students' views.

    Science.gov (United States)

    Landstrom, Bjorn; Mattsson, Bengt; Nordin, Per; Rudebeck, Carl E

    2014-03-15

    The aim of the study was to investigate medical students' views on general practice based on their experiences in training, and to find out whether there were certain views associated with the intention to become a GP. A questionnaire, based on our earlier studies about GP working behaviour, was handed out to medical students in terms 1, 3, 5, 7, 10 and 11 of undergraduate studies in Gothenburg, Sweden. The analysis comprised statistical descriptions and comparisons. The students regarded general practice positively. They found the work environment good, the GP's awareness of patients' living conditions necessary, and that GP work requires medical breadth. The status of the GP in the medical profession was not considered high. One-fourth of the students strongly agreed with the possibility of a future as a GP. This attitude was statistically associated with support to the statements that general practice offers a good work environment and should be a major component in undergraduate training. Students with a negative attitude to working as GPs were also negative to having a major component of general practice in undergraduate training. Medical students with a positive stated attitude towards becoming GPs support changes in undergraduate training to include more general practice. The risk of increasing a negative attitude should be considered when changes are discussed.

  7. β2-glycoprotein-1 autoantibodies from patients with antiphospholipid syndrome are sufficient to potentiate arterial thrombus formation in a mouse model

    Science.gov (United States)

    Arad, Ariela; Proulle, Valerie; Furie, Richard A.; Furie, Barbara C.

    2011-01-01

    Antiphospholipid syndrome is characterized by thrombosis, recurrent fetal loss, and the presence of the lupus anticoagulant, anticardiolipin antibodies, or anti–β2-glycoprotein-1 (anti–β2-GP1) antibodies. Although anti–β2-GP1 antibodies have been documented as a biomarker for diagnosis of antiphospholipid syndrome, their direct role in the pathogenesis of thrombosis is unknown. We have demonstrated using intravital microscopy that anti–β2-GP1 autoantibodies purified from the sera of patients with antiphospholipid syndrome complicated by thrombosis greatly amplify thrombus size after laser-induced vessel wall injury in live mice. Anti–β2-GP1 autoantibodies from 3 patients with antiphospholipid syndrome were affinity-purified using human β2-GP1 bound to agarose. The effects of purified anti–β2-GP1 IgG autoantibodies, of anti–β2-GP1–depleted IgG, and of IgG from normal human sera on thrombus formation were measured in mice after arterial injury in the cremaster muscle. Before injury, purified anti–β2-GP1 IgG autoantibodies, anti–β2-GP1 antibody–depleted IgG, or IgG from normal human sera were infused. Increasing amounts of purified anti–β2-GP1 autoantibodies increased thrombus size in a dose-dependent manner, whereas neither anti–β2-GP1 antibody-depleted IgG nor IgG from normal serum affected thrombus size. These results indicate that anti–β2-GP1 IgG autoantibodies in antiphospholipid syndrome patient sera are not only a marker of antiphospholipid syndrome but are directly involved in the pathogenesis of thrombosis. PMID:21245481

  8. A functional F analogue of AcMNPV GP64 is from the Agrotis segetum granulovirus

    NARCIS (Netherlands)

    Yin, F.; Wang, M.; Tan, Y.; Deng, F.; Vlak, J.M.; Hu, Z.H.; Wang, H.

    2008-01-01

    The envelope fusion protein F of Plutella xylostella granulovirus is a computational analogue of the GP64 envelope fusion protein of Autographa californica nucleopolyhedrovirus (AcMNPV). Granulovirus (GV) F proteins were thought to be unable to functionally replace GP64 in the AcMNPV pseudotyping

  9. The Role of Turmerones on Curcumin Transportation and P-Glycoprotein Activities in Intestinal Caco-2 Cells

    Science.gov (United States)

    Yue, Grace G.L.; Cheng, Sau-Wan; Yu, Hua; Xu, Zi-Sheng; Lee, Julia K.M.; Hon, Po-Ming; Lee, Mavis Y.H.; Kennelly, Edward J.; Deng, Gary; Yeung, Simon K.; Cassileth, Barrie R.; Fung, Kwok-Pui; Leung, Ping-Chung

    2012-01-01

    Abstract The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases. PMID:22181075

  10. The role of turmerones on curcumin transportation and P-glycoprotein activities in intestinal Caco-2 cells.

    Science.gov (United States)

    Yue, Grace G L; Cheng, Sau-Wan; Yu, Hua; Xu, Zi-Sheng; Lee, Julia K M; Hon, Po-Ming; Lee, Mavis Y H; Kennelly, Edward J; Deng, Gary; Yeung, Simon K; Cassileth, Barrie R; Fung, Kwok-Pui; Leung, Ping-Chung; Lau, Clara B S

    2012-03-01

    The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases.

  11. 25-Hydroxycholesterol Inhibition of Lassa Virus Infection through Aberrant GP1 Glycosylation

    Directory of Open Access Journals (Sweden)

    Punya Shrivastava-Ranjan

    2016-12-01

    Full Text Available Lassa virus (LASV infection is a major public health concern due to high fatality rates and limited effective treatment. The interferon-stimulated gene cholesterol 25-hydroxylase (CH25H encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC. 25HC is involved in regulating cholesterol biosynthesis and has recently been identified as a potent antiviral targeting enveloped virus entry. Here, we show a previously unrecognized role of CH25H in inhibiting LASV glycoprotein glycosylation and the production of infectious virus. Overexpression of CH25H or treatment with 25HC decreased LASV G1 glycoprotein N-glycan maturation and reduced the production of infectious LASV. Depletion of endogenous CH25H using small interfering RNA (siRNA enhanced the levels of fully glycosylated G1 and increased infectious LASV production. Finally, LASV particles produced from 25HC-treated cells were found to be less infectious, to incorporate aberrantly glycosylated GP1 species, and to be defective in binding alpha-dystroglycan, an attachment and entry receptor. Our findings identify a novel role for CH25H in controlling LASV propagation and indicate that manipulation of the expression of CH25H or the administration of 25HC may be a useful anti-LASV therapy.

  12. Selected Thermophysical Properties of 2,2 Dimethylcyclopentyl Methylphosphonofluoridate (GP) and 2,2 Dimethylcyclopentanol (DMCP)

    Science.gov (United States)

    2016-09-01

    chromatography using a thermal conductivity detector (GC-TCD). Table 1. Sample Information for GP and DMCP Chemical Name Mole Fraction Purity...Proving Ground, MD, 1983; UNCLASSIFIED Report (ADC033491). 23. Weast, R.C. CRC Handbook of Chemistry and Physics, 53rd ed.; CRC Press: Boca Raton, FL...gas chromatography GD pinacolyl methylphosphonofluoridate GF cyclohexyl methylphosphonofluoridate GP 2,2-dimethylcyclopentyl

  13. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...... recognizing gp63 can take part in the host defence against L. major infections....

  14. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

    Energy Technology Data Exchange (ETDEWEB)

    Papaneri, Amy B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Wirblich, Christoph [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Cann, Jennifer A.; Cooper, Kurt [Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Jahrling, Peter B. [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States); Integrated Research Facility, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick MD, 21702 (United States); Schnell, Matthias J., E-mail: matthias.schnell@jefferson.edu [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Jefferson Vaccine Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Blaney, Joseph E., E-mail: jblaney@niaid.nih.gov [Emerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, MD 21702 (United States)

    2012-12-05

    We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RV{Delta}G-GP) are both avirulent after intracerebral inoculation of adult mice. Furthermore, RV{Delta}G-GP is completely avirulent upon intracerebral inoculation of suckling mice unlike parental RABV vaccine or RV-GP. Analysis of RV{Delta}G-GP in the brain by quantitative PCR, determination of virus titer, and immunohistochemistry indicated greatly restricted virus replication. In summary, our findings indicate that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RV{Delta}G-GP would appear to be an even safer alternative for use in wildlife or consideration for human use.

  15. 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 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.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.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.

  16. Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

    International Nuclear Information System (INIS)

    Xiang Shihua; Wang Liping; Abreu, Mariam; Huang, C.-C.; Kwong, Peter D.; Rosenberg, Eric; Robinson, James E.; Sodroski, Joseph

    2003-01-01

    Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the β19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies

  17. Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry.

    Science.gov (United States)

    Wang, Jizhen; Manicassamy, Balaji; Caffrey, Michael; Rong, Lijun

    2011-06-01

    Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

  18. Synthesis and characterization of macromolecular rhodamine tethers and their interactions with P-glycoprotein.

    Science.gov (United States)

    Crawford, Lindsey; Putnam, David

    2014-08-20

    Rhodamine dyes are well-known P-glycoprotein (P-gp) substrates that have played an important role in the detection of inhibitors and other substrates of P-gp, as well as in the understanding of P-gp function. Macromolecular conjugates of rhodamines could prove useful as tethers for further probing of P-gp structure and function. Two macromolecular derivatives of rhodamine, methoxypolyethylene glycol-rhodamine6G and methoxypolyethylene glycol-rhodamine123, were synthesized through the 2'-position of rhodamine6G and rhodamine123, thoroughly characterized, and then evaluated by inhibition with verapamil for their ability to interact with P-gp and to act as efflux substrates. To put the results into context, the P-gp interactions of the new conjugates were compared to the commercially available methoxypolyethylene glycol-rhodamineB. FACS analysis confirmed that macromolecular tethers of rhodamine6G, rhodamine123, and rhodamineB were accumulated in P-gp expressing cells 5.2 ± 0.3%, 26.2 ± 4%, and 64.2 ± 6%, respectively, compared to a sensitive cell line that does not overexpress P-gp. Along with confocal imaging, the efflux analysis confirmed that the macromolecular rhodamine tethers remain P-gp substrates. These results open potential avenues for new ways to probe the function of P-gp both in vitro and in vivo.

  19. Studies on adsorptions of metallic ions in water by zirconium glyphosate (ZrGP): Behaviors and mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Jia Yunjie; Zhang Yuejuan [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Box. 98, No.15, Beisanhuan donglu, Beijing 100029 (China); Wang Runwei [State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, Jilin University, Changchun 130012 (China); Fan Faying [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Box. 98, No.15, Beisanhuan donglu, Beijing 100029 (China); Xu Qinghong, E-mail: xuqh@mail.buct.edu.cn [State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Box. 98, No.15, Beisanhuan donglu, Beijing 100029 (China)

    2012-01-15

    A new adsorbent named zirconium glyphosate [Zr(O{sub 3}PCH{sub 2}NHCH{sub 2}COOH){sub 2}{center_dot}0.5H{sub 2}O, denoted as ZrGP] and its selective adsorptions to Pb{sup 2+}, Cd{sup 2+}, Mg{sup 2+} and Ca{sup 2+} ions in water were reported in this paper. Compared to other zirconium adsorbents, such as zirconium phosphate [Zr(HPO{sub 4}){sub 2}], ZrGP exhibited highly selective adsorption to Pb{sup 2+} in solution which contained Pb{sup 2+}, Cd{sup 2+}, Mg{sup 2+} and Ca{sup 2+} ions. The loaded ZrGP with metallic ions can be efficaciously regenerated by aqueous solution of HCl (1.0 M) without any noticeable capacity loss, and almost all of it can be reused and recycled. The memory effect on structural regeneration of ZrGP was also found when Mg{sup 2+} and Ca{sup 2+} were adsorbed. To be specific, the structure of ZrGP was destroyed due to adsorbing these two ions, but it could be regenerated after the loaded materials were dipped in HCl solution (1.0 M) for several minutes to remove metallic ions.

  20. Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Hongping Min

    Full Text Available Development of multidrug resistance (MDR is a continuous clinical challenge partially due to the overexpression of P-glycoprotein (P-gp for chronic myelogenous leukemia (CML patients. Herein, we evaluated the inhibitory potency of emodin, a natural anthraquinone derivative isolated from Rheum palmatum L, on P-gp in P-gp positive K562/ADM cells. Competition experiments combined with molecular docking analysis were utilized to investigate the binding modes between emodin and binding sites of P-gp. Emodin reversed adriamycin resistance in K562/ADM cells accompanied with the decrease of P-gp protein expression, further increasing the uptake of rhodamine123 in both K562/ADM and Caco-2 cells, indicating the inhibition of P-gp efflux function. Moreover, when incubated with emodin under different conditions where P-gp was inhibited, K562/ADM cells displayed increasing intracellular uptake of emodin, suggesting that emodin may be the potential substrate of P-gp. Importantly, rhodamine 123 could increase the Kintrinsic (Ki value of emodin linearly, whereas, verapamil could not, implying that emodin competitively bound to the R site of P-gp and noncompetition existed between emodin and verapamil at the M site, in a good accordance with the results of molecular docking that emodin bound to the R site of P-gp with higher affinity. Based on our results, we suggest that emodin might be used to modulate P-gp function and expression.

  1. Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

    Directory of Open Access Journals (Sweden)

    Bingchun Zhao

    2018-05-01

    Full Text Available Epstein–Barr virus (EBV was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications.

  2. The GP problem: quantifying gene-to-phenotype relationships.

    Science.gov (United States)

    Cooper, Mark; Chapman, Scott C; Podlich, Dean W; Hammer, Graeme L

    2002-01-01

    In this paper we refer to the gene-to-phenotype modeling challenge as the GP problem. Integrating information across levels of organization within a genotype-environment system is a major challenge in computational biology. However, resolving the GP problem is a fundamental requirement if we are to understand and predict phenotypes given knowledge of the genome and model dynamic properties of biological systems. Organisms are consequences of this integration, and it is a major property of biological systems that underlies the responses we observe. We discuss the E(NK) model as a framework for investigation of the GP problem and the prediction of system properties at different levels of organization. We apply this quantitative framework to an investigation of the processes involved in genetic improvement of plants for agriculture. In our analysis, N genes determine the genetic variation for a set of traits that are responsible for plant adaptation to E environment-types within a target population of environments. The N genes can interact in epistatic NK gene-networks through the way that they influence plant growth and development processes within a dynamic crop growth model. We use a sorghum crop growth model, available within the APSIM agricultural production systems simulation model, to integrate the gene-environment interactions that occur during growth and development and to predict genotype-to-phenotype relationships for a given E(NK) model. Directional selection is then applied to the population of genotypes, based on their predicted phenotypes, to simulate the dynamic aspects of genetic improvement by a plant-breeding program. The outcomes of the simulated breeding are evaluated across cycles of selection in terms of the changes in allele frequencies for the N genes and the genotypic and phenotypic values of the populations of genotypes.

  3. Identification by Mass Spectrometry and Immune Response Analysis of Guinea Pig Cytomegalovirus (GPCMV Pentameric Complex Proteins GP129, 131 and 133

    Directory of Open Access Journals (Sweden)

    Josephine S. Gnanandarajah

    2014-02-01

    Full Text Available Development of a vaccine against congenital infection with human cytomegalovirus (HCMV is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV, consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.

  4. Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

    Science.gov (United States)

    Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

    2018-06-05

    Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

  5. Identification of gene products suppressed by human immunodeficiency virus type 1 infection or gp120 exposure of primary human astrocytes by rapid subtraction hybridization.

    Science.gov (United States)

    Su, Zao-Zhong; Kang, Dong-Chul; Chen, Yinming; Pekarskaya, Olga; Chao, Wei; Volsky, David J; Fisher, Paul B

    2003-06-01

    Neurodegeneration and human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are the major disease manifestations of HIV-1 colonization of the central nervous system (CNS). In the brain, HIV-1 replicates in microglial cells and infiltrating macrophages and it persists in a low-productive, noncytolytic state in astrocytes. Astrocytes play critical roles in the maintenance of the brain microenvironment, responses to injury, and in neuronal signal transmission, and disruption of these functions by HIV-1 could contribute to HAD. To better understand the potential effects of HIV-1 on astrocyte biology, the authors investigated changes in gene expression using an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue, low-passage cells were infected with HIV-1 or mock infected, and total cellular RNAs were isolated at multiple time points over a period of 1 week. This approach is designed to identify gene products modulated early and late after HIV-1 infection and limits the cloning of genes displaying normal cell-cycle fluctuations in astrocytes. By subtracting temporal cDNAs derived from HIV-1-infected astrocytes from temporal cDNAs made from uninfected cells, 10 genes displaying reduced expression in infected cells, termed astrocyte suppressed genes (ASGs), were identified and their suppression was confirmed by Northern blot hybridization. Both known and novel ASGs, not reported in current DNA databases, that are down-regulated by HIV-1 infection are described. Northern blotting confirms suppression of the same panel of ASGs by treatment of astrocytes with recombinant HIV-1 envelope glycoprotein, gp120. These results extend our previous analysis of astrocyte genes induced or enhanced by HIV-1 infection and together they suggest that HIV-1 and viral proteins have profound effects on astrocyte physiology, which may influence their function in the CNS.

  6. P-glycoprotein expression and DNA topoisomerase I and II activity in benign tumors of the ovary and in malignant tumors of the ovary, before and after platinum/cyclophosphamide chemotherapy

    NARCIS (Netherlands)

    van der Zee, A G; Hollema, H; de Jong, S; Boonstra, H; Gouw, A; Willemse, P H; Zijlstra, J G; de Vries, E G; de Jong, Steven

    1991-01-01

    P-glycoprotein (P-gp) expression and DNA topoisomerase (Topo) II are important variables in multidrug resistant tumor cell lines. The aim of this study was to evaluate P-gp expression and Topo I and II activity in benign and malignant epithelial ovarian tumors. P-gp expression was analyzed

  7. Frequency and persistency of DNA vaccine encoding GP25 by oral on common carp

    Directory of Open Access Journals (Sweden)

    Sri Nuryati

    2015-05-01

    Full Text Available ABSTRACT Koi herpesvirus (KHV is a major viral pathogen that infects common carp and koi. KHV disease outbreak is happened in almost all centre of common carp culture in Indonesia and caused mass mortality. Deoxyribonucleic acid (DNA vaccination method is one of ways to cope with KHV infection. Vaccines were commonly given by injection. The aim of this research was to get frequency and persistency of DNA vaccine encoding GP25 given by oral delivery method in common carp. This research would like to determine dose, frequency of vaccination, persistency of DNA vaccine and culture medium for the bacterial host. DNA vaccine persistency test was done by using polymerase chain reaction (PCR method with the specific primer for GP25 gene. The results showed that level of DNA vaccine that could be detected in feed was 7.56 ng (equal to 1.598×1010 copies. Efficient culture medium for Escherichia coli DH5α carrying DNA vaccine was LB triptone. Feeding fish with diet supplemented with 1 mL E. coli DH5α containing DNA vaccine for each fish and two times a week allowed persistence of DNA vaccine in kindney and spleen. Keywords: common carp, KHV, DNA vaccine, GP25, persistance  ABSTRAK Koi herpesvirus (KHV adalah virus patogen utama yang menginfeksi ikan mas dan ikan koi. Wabah penyakit KHV terjadi di hampir semua sentra budidaya ikan mas di Indonesia dan menyebabkan kematian massal ikan. Metode vaksinasi DNA merupakan salah satu cara yang dapat dilakukan untuk menanggulangi serangan KHV. Pemberian vaksin umumnya dilakukan dengan cara injeksi. Tujuan penelitian ini adalah untuk menguji frekuensi dan persistensi vaksin DNA GP25 antivirus KHV yang diberikan melalui oral pada ikan mas. Pada penelitian ini dilakukan uji dosis, frekuensi pemberian vaksin, persistensi vaksin DNA, dan media kultur bakteri inang. Persistensi vaksin DNA dianalisis menggunakan metode PCR dengan primer spesifik gen GP25. Hasil penelitian menunjukkan bahwa dosis vaksin DNA yang

  8. The Influences of Glycosylation on the Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines

    National Research Council Canada - National Science Library

    Dowling, William; Thompson, Elizabeth; Badger, Catherine; Mellquist, Jenny L; Garrison, Aura R; Smith, Jeffrey M; Paragas, Jason; Hogan, Robert J; Schmaljohn, Connie

    2006-01-01

    ... or with deletions in the central hypervariable mucin region. We showed that mutation of one of the two N-linked GP2 glycosylation sites was highly detrimental to the antigenicity and immunogenicity of GP...

  9. GP-9s are ubiquitous proteins unlikely involved in olfactory mediation of social organization in the red imported fire ant, Solenopsis invicta.

    Directory of Open Access Journals (Sweden)

    Walter S Leal

    Full Text Available The red imported fire ant (RIFA, Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9, identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla.

  10. Blended learning in CME: the perception of GP trainers

    NARCIS (Netherlands)

    te Pas, E.; Meinema, J. G.; Visser, M. R. M.; van Dijk, N.

    2016-01-01

    Blended learning (the combination of electronic methods with traditional teaching methods) has the potential to combine the best of traditional education with the best of computer-mediated training. We chose to develop such an intervention for GP trainers who were undertaking a Continuing Medical

  11. GP-initiated preconception counselling in a randomised controlled trial does not induce anxiety

    Directory of Open Access Journals (Sweden)

    Neven A Knuistingh

    2006-11-01

    Full Text Available Abstract Background Preconception counselling (PCC can reduce adverse pregnancy outcome by addressing risk factors prior to pregnancy. This study explores whether anxiety is induced in women either by the offer of PCC or by participation with GP-initiated PCC. Methods Randomised trial of usual care versus GP-initiated PCC for women aged 18–40, in 54 GP practices in the Netherlands. Women completed the six-item Spielberger State Trait Anxiety Inventory (STAI before PCC (STAI-1 and after (STAI-2. After pregnancy women completed a STAI focusing on the first trimester of pregnancy (STAI-3. Results The mean STAI-1-score (n = 466 was 36.4 (95% CI 35.4 – 37.3. Following PCC there was an average decrease of 3.6 points in anxiety-levels (95% CI, 2.4 – 4.8. Mean scores of the STAI-3 were 38.5 (95% CI 37.7 – 39.3 in the control group (n = 1090 and 38.7 (95% CI 37.9 – 39.5 in the intervention group (n = 1186. Conclusion PCC from one's own GP reduced anxiety after participation, without leading to an increase in anxiety among the intervention group during pregnancy. We therefore conclude that GPs can offer PCC to the general population without fear of causing anxiety. Trial Registration: ISRCTN53942912

  12. Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

    Science.gov (United States)

    Kanao, Megumi; Kanda, Hirotsugu; Huang, Wan; Liu, Shue; Yi, Hyun; Candiotti, Keith A; Lubarsky, David A; Levitt, Roy C; Hao, Shuanglin

    2015-06-01

    Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection-related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of γ-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBPβ was tested using immunohistochemistry. In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time-effect curves in the HSV vector expressing GAD67 was increased compared with that in the

  13. P-glycoprotein targeted nanoscale drug carriers

    KAUST Repository

    Li, Wengang; Abu Samra, Dina Bashir Kamil; Merzaban, Jasmeen; Khashab, Niveen M.

    2013-01-01

    Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug

  14. Effects of local application of methylprednisolone delivered by the C/GP-hydrogel on the recovery of facial nerves.

    Science.gov (United States)

    Chao, Xiuhua; Fan, Zhaomin; Han, Yuechen; Wang, Yan; Li, Jianfeng; Chai, Renjie; Xu, Lei; Wang, Haibo

    2015-01-01

    Local administration of MP delivered by the C/GP-MP-hydrogel can improve the recovery of facial nerve following crush injury. The findings suggested that locally injected MP delivered by C/GP-hydrogel might be a promising treatment for facial nerve damage. In this study, the aim is to assess the effectiveness of locally administrating methylprednisolone(MP) loaded by chitosan-β-glycerophosphate hydrogel (C/GP-hydrogel) on the regeneration of facial nerve crush injury. After the crush of left facial nerves, Wistar rats were randomly divided into four different groups. Then, four different therapies were used to treat the damaged facial nerves. At the 1(st), 2(nd), 3(rd), and 4(th) week after injury, the functional recovery of facial nerves and the morphological changes of facial nerves were assessed. The expression of growth associated protein-43 (GAP-43) protein in the facial nucleus were also evaluated. Locally injected MP delivered by C/GP-hydrogel effectively accelerated the facial functional recovery. In addition, the regenerated facial nerves in the C/GP-MP group were more mature than those in the other groups. The expression of GAP-43 protein was also improved by the MP, especially in the C/GP-MP group.

  15. Feasibility, acceptability and effectiveness of an online alternative to face-to-face consultation in general practice: a mixed-methods study of webGP in six Devon practices.

    Science.gov (United States)

    Carter, Mary; Fletcher, Emily; Sansom, Anna; Warren, Fiona C; Campbell, John L

    2018-02-15

    To evaluate the feasibility, acceptability and effectiveness of webGP as piloted by six general practices. Mixed-methods evaluation, including data extraction from practice databases, general practitioner (GP) completion of case reports, patient questionnaires and staff interviews. General practices in NHS Northern, Eastern and Western Devon Clinical Commissioning Group's area approximately 6 months after implementing webGP (February-July 2016). Six practices provided consultations data; 20 GPs completed case reports (regarding 61 e-consults); 81 patients completed questionnaires; 5 GPs and 5 administrators were interviewed. Attitudes and experiences of practice staff and patients regarding webGP. WebGP uptake during the evaluation was small, showing no discernible impact on practice workload. The completeness of cross-sectional data on consultation workload varied between practices.GPs judged 41/61 (72%) of webGP requests to require a face-to-face or telephone consultation. Introducing webGP appeared to be associated with shifts in responsibility and workload between practice staff and between practices and patients.81/231 patients completed a postal survey (35.1% response rate). E-Consulters were somewhat younger and more likely to be employed than face-to-face respondents. WebGP appeared broadly acceptable to patients regarding timeliness and quality/experience of care provided. Similar problems were presented by all respondents. Both groups appeared equally familiar with other practice online services; e-consulters were somewhat more likely to have used them.From semistructured staff interviews, it appeared that, while largely acceptable within practice, introducing e-consults had potential for adverse interactions with pre-existing practice systems. There is potential to assess the impact of new systems on consultation patterns by extracting routine data from practice databases. Staff and patients noticed subtle changes to responsibilities associated with

  16. Development of a novel, guinea pig-specific IFN-γ ELISPOT assay and characterization of guinea pig cytomegalovirus GP83-specific cellular immune responses following immunization with a modified vaccinia virus Ankara (MVA)-vectored GP83 vaccine.

    Science.gov (United States)

    Gillis, Peter A; Hernandez-Alvarado, Nelmary; Gnanandarajah, Josephine S; Wussow, Felix; Diamond, Don J; Schleiss, Mark R

    2014-06-30

    The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Effects of sertraline and fluoxetine on p-glycoprotein at barrier sites: in vivo and in vitro approaches.

    Directory of Open Access Journals (Sweden)

    Amita Kapoor

    Full Text Available Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.The P-gp substrate, tritiated digoxin ([(3H] digoxin, was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [(3H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.

  18. In vitro and in vivo evaluations of the P-glycoprotein-mediated efflux of dibenzoylhydrazines

    Energy Technology Data Exchange (ETDEWEB)

    Miyata, Ken-ichi, E-mail: Miyata.Kenichi@otsuka.jp [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan); Otsuka Pharmaceutical Co., Ltd., Tokushima 771-0182 (Japan); Nakagawa, Yoshiaki; Kimura, Yasuhisa [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan); Ueda, Kazumitsu [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8502 (Japan); Akamatsu, Miki, E-mail: akamatsu@kais.kyoto-u.ac.jp [Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 (Japan)

    2016-05-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter family. It actively transports a wide variety of compounds out of cells to protect humans from xenobiotics. Thus, determining whether chemicals are substrates and/or inhibitors of P-gp is important in risk assessments of pharmacokinetic interactions among chemicals because P-gp-mediated transport processes play a significant role in their absorption and disposition. We previously reported that dibenzoylhydrazines (DBHs) such as tebufenozide and methoxyfenozide (agrochemicals) stimulated P-gp ATPase activity. However, it currently remains unclear whether these derivatives are transport substrates of P-gp and inhibit transport of other chemicals by P-gp. In the present study, in order to evaluate the interactions of DBHs with other chemicals in humans, we determined whether DBHs are P-gp transport substrates using both the in vitro bidirectional transport assay and the in vivo study of rats. In the in vivo study, we investigated the influence of P-gp inhibitors on the brain to plasma ratio of methoxyfenozide in rats. We also examined the inhibitory effects of DBHs on quinidine (a P-gp substrate) transport by P-gp in order to ascertain whether these derivatives are inhibitors of P-gp. Based on the results, DBHs were concluded to be weak P-gp transport substrates and moderate P-gp inhibitors. However, the risk of DBHs caused by interaction with other chemicals including drugs was considered to be low by considering the DBHs' potential as the substrates and inhibitors of P-gp as well as their plasma concentrations as long as DBHs are properly used. - Highlights: • Transport of DBHs by P-gp was not detected in in vitro bidirectional transport assay. • DBHs were weak P-gp transport substrates based on in vivo studies in rats. • The in vivo studies are useful methods for evaluating P-gp transport substrates. • DBHs inhibit quinidine transport by P-gp in in vitro bidirectional transport

  19. P-glycoprotein Inhibition Increases the Brain Distribution and Antidepressant-Like Activity of Escitalopram in Rodents

    Science.gov (United States)

    O'Brien, Fionn E; O'Connor, Richard M; Clarke, Gerard; Dinan, Timothy G; Griffin, Brendan T; Cryan, John F

    2013-01-01

    Despite the clinical prevalence of the antidepressant escitalopram, over 30% of escitalopram-treated patients fail to respond to treatment. Recent gene association studies have highlighted a potential link between the drug efflux transporter P-glycoprotein (P-gp) and response to escitalopram. The present studies investigated pharmacokinetic and pharmacodynamic interactions between P-gp and escitalopram. In vitro bidirectional transport studies revealed that escitalopram is a transported substrate of human P-gp. Microdialysis-based pharmacokinetic studies demonstrated that administration of the P-gp inhibitor cyclosporin A resulted in increased brain levels of escitalopram without altering plasma escitalopram levels in the rat, thereby showing that P-gp restricts escitalopram transport across the blood–brain barrier (BBB) in vivo. The tail suspension test (TST) was carried out to elucidate the pharmacodynamic impact of P-gp inhibition on escitalopram effect in a mouse model of antidepressant activity. Pre-treatment with the P-gp inhibitor verapamil enhanced the response to escitalopram in the TST. Taken together, these data indicate that P-gp may restrict the BBB transport of escitalopram in humans, potentially resulting in subtherapeutic brain concentrations in certain patients. Moreover, by verifying that increasing escitalopram delivery to the brain by P-gp inhibition results in enhanced antidepressant-like activity, we suggest that adjunctive treatment with a P-gp inhibitor may represent a beneficial approach to augment escitalopram therapy in depression. PMID:23670590

  20. Membrane microparticles mediate transfer of P-glycoprotein to drug sensitive cancer cells.

    Science.gov (United States)

    Bebawy, M; Combes, V; Lee, E; Jaiswal, R; Gong, J; Bonhoure, A; Grau, G E R

    2009-09-01

    Multidrug resistance (MDR), a significant impediment to the successful treatment of cancer clinically, has been attributed to the overexpression of P-glycoprotein (P-gp), a plasma membrane multidrug efflux transporter. P-gp maintains sublethal intracellular drug concentrations by virtue of its drug efflux capacity. The cellular regulation of P-gp expression is currently known to occur at either pre- or post-transcriptional levels. In this study, we identify a 'non-genetic' mechanism whereby microparticles (MPs) serve as vectors in the acquisition and spread of MDR. MPs isolated from drug-resistant cancer cells (VLB(100)) were co-cultured with drug sensitive cells (CCRF-CEM) over a 4 h period to allow for MP binding and P-gp transfer. Presence of P-gp on MPs was established using flow cytometry (FCM) and western blotting. Whole-cell drug accumulation assays using rhodamine 123 and daunorubicin (DNR) were carried out to validate the transfer of functional P-gp after co-culture. We establish that MPs shed in vitro from drug-resistant cancer cells incorporate cell surface P-gp from their donor cells, effectively bind to drug-sensitive recipient cells and transfer functional P-gp to the latter. These findings serve to substantially advance our understanding of the molecular basis for the emergence of MDR in cancer clinically and lead to new treatment strategies which target and inhibit MP mediated transfer of P-gp during the course of treatment.

  1. Point mutations associated with HIV-1 drug resistance, evasion of the immune response and AIDS pathogenesis

    CSIR Research Space (South Africa)

    Khati, M

    2012-03-01

    Full Text Available RNA and Vpr, respectively pol Protease (PR) gag/pol cleavage pol Reverse Transcriptase (RT) Reverse transcription pol RNase H RNase H activity pol Integrase (IN) DNA provirus integration env Env (gp120 and gp41) gp120 binds CD4 receptor and CXCR4.../CCR5 co-receptors , gp41 mediates fusion tat Tat Viral transcription activator rev Rev RNA transport, stability and utilization factor (phosphoprotein) vif Vif Promotes virion maturation and infectivity vpr Vpr Promotes nuclear localization...

  2. Beta-Amyloid Downregulates MDR1-P-Glycoprotein (Abcb1 Expression at the Blood-Brain Barrier in Mice

    Directory of Open Access Journals (Sweden)

    Anja Brenn

    2011-01-01

    Full Text Available Neurovascular dysfunction is an important component of Alzheimer's disease, leading to reduced clearance across the blood-brain barrier and accumulation of neurotoxic β-amyloid (Aβ peptides in the brain. It has been shown that the ABC transport protein P-glycoprotein (P-gp, ABCB1 is involved in the export of Aβ from the brain into the blood. To determine whether Aβ influences the expression of key Aβ transporters, we studied the effects of 1-day subcutaneous Aβ1-40 and Aβ1-42 administration via Alzet mini-osmotic pumps on P-gp, BCRP, LRP1, and RAGE expression in the brain of 90-day-old male FVB mice. Our results demonstrate significantly reduced P-gp, LRP1, and RAGE mRNA expression in mice treated with Aβ1-42 compared to controls, while BCRP expression was not affected. The expression of the four proteins was unchanged in mice treated with Aβ1-40 or reverse-sequence peptides. These findings indicate that, in addition to the age-related decrease of P-gp expression, Aβ1-42 itself downregulates the expression of P-gp and other Aβ-transporters, which could exacerbate the intracerebral accumulation of Aβ and thereby accelerate neurodegeneration in Alzheimer's disease and cerebral β-amyloid angiopathy.

  3. GP2 is selectively expressed by small intestinal CD103+CD11b+ cDC

    DEFF Research Database (Denmark)

    Müller-Luda, Katarzyna; Ahmadi, Fatemeh; Ohno, Hiroshi

    DC in the small intestine. Moreover, GP2 expressing cDC in the small intestine were dramatically reduced in the setting of intestinal inflammation. We have previously shown that mice with an IRF4 deletion in CD11c+ cells (Cd11c-cre.Irf4 fl/fl mice) have reduced numbers of small intestinal CD103+CD11b+ c......DC. Interesting, we found that GP2+ CD103+CD11b+ cDC were dramatically reduced in these mice. Finally, to address the in vivo role of GP2 expression by cDC, we have generated mice with a selective deletion of GP2 in CD103+CD11b+ cDC (huLangerin-cre.gp2 fl/fl  mice). Results from these ongoing studies...

  4. Menadione serves as a substrate for P-glycoprotein: implication in chemosensitizing activity.

    Science.gov (United States)

    Oh, Seok-Jeong; Han, Hyo-Kyung; Kang, Keon-Wook; Lee, Young-Joo; Lee, Moo-Yeol

    2013-04-01

    Based on its chemosensitizing effect, we questioned whether menadione is an inhibitor or a substrate of P-glycoprotein (P-gp). To test this hypothesis, we assessed the effect of menadione on P-gp activity and examined the P-gp-dependency of cellular accumulation and cytotoxicity of menadione as well. Treatment with menadione resulted in the concentration-dependent increase of rhodamine 123 (Rh123) accumulation in P-gp-overexpressing MDCKII/MDR1 and NCI/ADR-RES cells, suggesting that menadione inhibits Rh123 extrusion by P-gp. Compared with MDCKII or MCF-7, intracellular distribution of [(3)H]-menadione was significantly lower in MDCKII/MDR1 or NCI/ADR-RES cells, which could be restored by the P-gp inhibitors, verapamil and quinidine. Consistent with these results, MDCKII/MDR1 or NCI/ADR-RES cells were more resistant to the cytotoxicity of menadione than MDCKII or MCF-7 cells, respectively. Such resistance was abolished by the combined treatment of verapamil and quinidine in NCI/ADR-RES cells. Our study identified menadione as a substrate of P-gp, which presumably, acts as the mechanism for the chemosensitizing effect. Menadione may be a promising chemotherapeutic enhancer by its ability of circumventing drug resistance, in addition to its own anti-cancer activity.

  5. Selection of GP. Mur antigen-negative RBC for blood recipients with anti-'Mia ' records decreases transfusion reaction rates in Taiwan.

    Science.gov (United States)

    Yang, C-A; Lin, J-A; Chang, C-W; Wu, K-H; Yeh, S-P; Ho, C-M; Chang, J-G

    2016-10-01

    To evaluate the clinical significance of GP. Mur antigen-negative blood selection for transfusion in patients with anti-'Mi a ' records. The GP. Mur RBC phenotype is prevalent (7·3%) in Taiwan. Antibodies against GP. Mur (anti-'Mi a ') are identified in 1·24% of our population, and anti-'Mi a ' screening using GP. Mur RBC has been routine for Taiwan's blood banks. However, due to the lack of commercial antibodies, only cross-matching was used to prevent transfusion of GP. Mur-positive blood to patients with anti-'Mi a ' in most hospitals. There is still a risk of GP. Mur-positive RBC exposure and subsequent anti-'Mi a '-related transfusion reactions. Since February 2014, GP. Mur antigen-negative RBCs identified by reaction with anti-'Mi a '-positive serum were selected for blood recipients with anti-'Mi a ' records. The transfusion reactions between January 2013 and January 2014 were compared with those that occurred between February 2014 and July 2015. The transfusion reaction rate was significantly higher in anti-'Mi a '-positive blood recipients compared to total subjects receiving an RBC transfusion before GP. Mur-negative donor RBC selection. After antigen-negative RBC selection, the transfusion reaction frequency in subjects with anti-'Mi a ' became similar to total blood recipients. IgG form anti-'Mi a ' antibodies were present in all cases of probable anti-'Mi a '-related transfusion reactions. The time required for anti-'Mi a ' boosting after transfusion was around 4-21 days. Selection of GP. Mur-negative RBC for transfusion to patients with anti-'Mi a ' records could decrease the rate of transfusion reaction and antibody boosting. This procedure should be incorporated into blood bank routines in areas where anti-'Mi a ' is prevalent. © 2016 British Blood Transfusion Society.

  6. GP supervisors--an investigation into their motivations and teaching activities.

    Science.gov (United States)

    Ingham, Gerard; O'Meara, Peter; Fry, Jennifer; Crothers, Natalie

    2014-11-01

    There is little empirical information on how general practitioner (GP) supervisors teach and the reasons for the variation in their teaching methods. Could the variation be due to differing motivations to teach? Supervisors from one regional training provider who attended educational workshops in 2013 were surveyed, seeking infor-mation on their motivation to become and remain a supervisor, and the frequency of use of selected teaching activities. The majority of respondents cited intrinsic motivators, including enjoying teaching (84%), contribution to the profession and community (82%), adding variety (78%) and workforce/succession planning (69%), as reasons for becoming GP supervisors. The expected relationships between motivations and teaching activities were not found. Variation in teaching activities used by supervisors does not appear to be associated with differing motivations. Measuring the use of teaching activities is not a mechanism to determine a supervisor's commitment to teaching.

  7. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    International Nuclear Information System (INIS)

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai

    2006-01-01

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV

  8. P-glycoprotein induction in Caco-2 cells by newly synthetized thioxanthones prevents paraquat cytotoxicity.

    Science.gov (United States)

    Silva, Renata; Palmeira, Andreia; Carmo, Helena; Barbosa, Daniel José; Gameiro, Mariline; Gomes, Ana; Paiva, Ana Mafalda; Sousa, Emília; Pinto, Madalena; Bastos, Maria de Lourdes; Remião, Fernando

    2015-10-01

    The induction of P-glycoprotein (P-gp), an ATP-dependent efflux pump, has been proposed as a strategy against the toxicity induced by P-gp substrates such as the herbicide paraquat (PQ). The aim of this study was to screen five newly synthetized thioxanthonic derivatives, a group known to interact with P-gp, as potential inducers of the pump's expression and/or activity and to evaluate whether they would afford protection against PQ-induced toxicity in Caco-2 cells. All five thioxanthones (20 µM) caused a significant increase in both P-gp expression and activity as evaluated by flow cytometry using the UIC2 antibody and rhodamine 123, respectively. Additionally, it was demonstrated that the tested compounds, when present only during the efflux of rhodamine 123, rapidly induced an activation of P-gp. The tested compounds also increased P-gp ATPase activity in MDR1-Sf9 membrane vesicles, indicating that all derivatives acted as P-gp substrates. PQ cytotoxicity was significantly reduced in the presence of four thioxanthone derivatives, and this protective effect was reversed upon incubation with a specific P-gp inhibitor. In silico studies showed that all the tested thioxanthones fitted onto a previously described three-feature P-gp induction pharmacophore. Moreover, in silico interactions between thioxanthones and P-gp in the presence of PQ suggested that a co-transport mechanism may be operating. Based on the in vitro activation results, a pharmacophore model for P-gp activation was built, which will be of further use in the screening for new P-gp activators. In conclusion, the study demonstrated the potential of the tested thioxanthonic compounds in protecting against toxic effects induced by P-gp substrates through P-gp induction and activation.

  9. Access to and utilisation of GP services among Burmese migrants in London: a cross-sectional descriptive study.

    Science.gov (United States)

    Aung, Nyein Chan; Rechel, Bernd; Odermatt, Peter

    2010-10-12

    An estimated 10,000 Burmese migrants are currently living in London. No studies have been conducted on their access to health services. Furthermore, most studies on migrants in the United Kingdom (UK) have been conducted at the point of service provision, carrying the risk of selection bias. Our cross-sectional study explored access to and utilisation of General Practice (GP) services by Burmese migrants residing in London. We used a mixed-method approach: a quantitative survey using self-administered questionnaires was complemented by qualitative in-depth interviews for developing the questionnaire and triangulating the findings of the survey. Overall, 137 questionnaires were received (a response rate of 57%) and 11 in-depth interviews conducted. The main outcome variables of the study included GP registration, barriers towards registration, GP consultations, barriers towards consultations, and knowledge on entitlements to health care. Quantitative data were analysed using descriptive statistics, association tests, and a multivariate analysis using logistic regression. The qualitative information was analysed using content analysis. The respondents were young, of roughly equal gender (51.5% female), well educated, and had a fair level of knowledge on health services in the UK. Although the GP registration rate was relatively high (80%, 109 out of 136), GP service utilisation during the last episode of illness, at 56.8% (54 out of 95), was low. The statistical analysis showed that age being younger than 35 years, lacking prior overseas experience, having an unstable immigration status, having a shorter duration of stay, and resorting to self-medication were the main barriers hindering Burmese migrants from accessing primary health care services. These findings were corroborated by the in-depth interviews. Our study found that having formal access to primary health care was not sufficient to ensure GP registration and health care utilisation. Some respondents faced

  10. Access to and utilisation of GP services among Burmese migrants in London: a cross-sectional descriptive study

    Directory of Open Access Journals (Sweden)

    Rechel Bernd

    2010-10-01

    Full Text Available Abstract Background An estimated 10,000 Burmese migrants are currently living in London. No studies have been conducted on their access to health services. Furthermore, most studies on migrants in the United Kingdom (UK have been conducted at the point of service provision, carrying the risk of selection bias. Our cross-sectional study explored access to and utilisation of General Practice (GP services by Burmese migrants residing in London. Methods We used a mixed-method approach: a quantitative survey using self-administered questionnaires was complemented by qualitative in-depth interviews for developing the questionnaire and triangulating the findings of the survey. Overall, 137 questionnaires were received (a response rate of 57% and 11 in-depth interviews conducted. The main outcome variables of the study included GP registration, barriers towards registration, GP consultations, barriers towards consultations, and knowledge on entitlements to health care. Quantitative data were analysed using descriptive statistics, association tests, and a multivariate analysis using logistic regression. The qualitative information was analysed using content analysis. Results The respondents were young, of roughly equal gender (51.5% female, well educated, and had a fair level of knowledge on health services in the UK. Although the GP registration rate was relatively high (80%, 109 out of 136, GP service utilisation during the last episode of illness, at 56.8% (54 out of 95, was low. The statistical analysis showed that age being younger than 35 years, lacking prior overseas experience, having an unstable immigration status, having a shorter duration of stay, and resorting to self-medication were the main barriers hindering Burmese migrants from accessing primary health care services. These findings were corroborated by the in-depth interviews. Conclusions Our study found that having formal access to primary health care was not sufficient to ensure GP

  11. How international medical graduates view their learning needs for UK GP training.

    Science.gov (United States)

    Warwick, Christopher

    2014-03-01

    International medical graduates (IMGs) form a vital group of general practitioners (GPs) in the NHS. They are known to face additional challenges above and beyond those faced by UK medical graduates in the course of their GP training. Whilst they are a heterogeneous group of professionals, their views on what they need to learn, and how they are supported, are often distant from those of the educators responsible for planning their education. This study was undertaken, through narrative-based focus groups, to explore the issues which matter to the IMGs, in an attempt to empower their voices about their experiences in GP training, and to see what lessons could be drawn from these views. The findings confirmed the central importance, and considerable challenge involved, in making an effective transition into the culture of the NHS and UK general practice. The IMGs felt that induction needed to be an on-going, iterative process of learning which continued throughout training, with a more effective individualised learning needs analysis at the start of GP training. Lack of sophisticated language skills was highlighted as a real concern. Recognition that their lack of knowledge about the NHS at the start of training should not be seen as an indicator of deficiency, but a clue to what they needed to learn were also key messages. IMGs also felt the earlier in their training they undertook a GP placement, the quicker they would start to understand the culture of general practice in the UK. Further work following on from this research should include how to manage change in the educational network for these barriers to be overcome.

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

    Directory of Open Access Journals (Sweden)

    Renske M. Raaphorst

    2017-09-01

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

  13. Development of the gas puff charge exchange recombination spectroscopy (GP-CXRS) technique for ion measurements in the plasma edge

    International Nuclear Information System (INIS)

    Churchill, R. M.; Theiler, C.; Lipschultz, B.; Dux, R.; Pütterich, T.; Viezzer, E.

    2013-01-01

    A novel charge-exchange recombination spectroscopy (CXRS) diagnostic method is presented, which uses a simple thermal gas puff for its donor neutral source, instead of the typical high-energy neutral beam. This diagnostic, named gas puff CXRS (GP-CXRS), is used to measure ion density, velocity, and temperature in the tokamak edge/pedestal region with excellent signal-background ratios, and has a number of advantages to conventional beam-based CXRS systems. Here we develop the physics basis for GP-CXRS, including the neutral transport, the charge-exchange process at low energies, and effects of energy-dependent rate coefficients on the measurements. The GP-CXRS hardware setup is described on two separate tokamaks, Alcator C-Mod and ASDEX Upgrade. Measured spectra and profiles are also presented. Profile comparisons of GP-CXRS and a beam based CXRS system show good agreement. Emphasis is given throughout to describing guiding principles for users interested in applying the GP-CXRS diagnostic technique

  14. Development of the gas puff charge exchange recombination spectroscopy (GP-CXRS) technique for ion measurements in the plasma edge

    Energy Technology Data Exchange (ETDEWEB)

    Churchill, R. M.; Theiler, C.; Lipschultz, B. [MIT Plasma Science and Fusion Center, Cambridge, Massachusetts 02139 (United States); Dux, R.; Pütterich, T.; Viezzer, E. [Max-Planck-Institut für Plasmaphysik, EURATOM Association, Boltzmannstrasse 2, D-85748 Garching (Germany); Collaboration: Alcator C-Mod Team; ASDEX Upgrade Team

    2013-09-15

    A novel charge-exchange recombination spectroscopy (CXRS) diagnostic method is presented, which uses a simple thermal gas puff for its donor neutral source, instead of the typical high-energy neutral beam. This diagnostic, named gas puff CXRS (GP-CXRS), is used to measure ion density, velocity, and temperature in the tokamak edge/pedestal region with excellent signal-background ratios, and has a number of advantages to conventional beam-based CXRS systems. Here we develop the physics basis for GP-CXRS, including the neutral transport, the charge-exchange process at low energies, and effects of energy-dependent rate coefficients on the measurements. The GP-CXRS hardware setup is described on two separate tokamaks, Alcator C-Mod and ASDEX Upgrade. Measured spectra and profiles are also presented. Profile comparisons of GP-CXRS and a beam based CXRS system show good agreement. Emphasis is given throughout to describing guiding principles for users interested in applying the GP-CXRS diagnostic technique.

  15. Gravity Probe-B (GP-B) Mission and Tracking, Telemetry and Control Subsystem Overview

    Science.gov (United States)

    Kennedy, Paul; Bell, Joseph L. (Technical Monitor)

    2001-01-01

    The National Aeronautics and Space Administration's (NASA) Marshall Space Flight Center (MSFC) in Huntsville, Alabama will launch the Gravity Probe B (GP-B) space experiment in the Fall of 2002. The GP-B spacecraft was developed to prove Einstein's theory of General Relativity. This paper will provide an overview of the GPB mission and will discuss the design, and test of the spacecraft Tracking, Telemetry and Control (TT&C) subsystem which incorporates NASA's latest generation standard transponder for use with the NASA Tracking and Data Relay Satellite System (TDRSS).

  16. Neutralization of X4- and R5-tropic HIV-1 NL4-3 variants by HOCl-modified serum albumins

    Directory of Open Access Journals (Sweden)

    Schwalbe Birco

    2010-06-01

    Full Text Available Abstract Background Myeloperoxidase (MPO, an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl from H2O2 and chloride, which is released into body fluids. Besides its direct microbicidal activity, HOCl can react with amino acid residues and HOCl-modified proteins can be detected in vivo. Findings This report is based on binding studies of HOCl-modified serum albumins to HIV-1 gp120 and three different neutralization assays using infectious virus. The binding studies were carried out by surface plasmon resonance spectroscopy and by standard ELISA techniques. Virus neutralization assays were carried out using HIV-1 NL4-3 virus and recombinant strains with CXCR4 and CCR5 coreceptor usage. Viral infection was monitored by a standard p24 or X-gal staining assay. Our data demonstrate that HOCl-modified mouse-, bovine- and human serum albumins all bind to the HIV-1 NL4-3 gp120 (LAV glycoprotein in contrast to non-modified albumin. Binding of HOCl-modified albumin to gp120 correlated to the blockade of CD4 as well as that of V3 loop specific monoclonal antibody binding. In neutralization experiments, HOCl-modified serum albumins inhibited replication and syncytium formation of the X4- and R5-tropic NL4-3 isolates in a dose dependent manner. Conclusions Our data indicate that HOCl-modified serum albumin veils the binding site for CD4 and the V3 loop on gp120. Such masking of the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when HOCl-modified serum albumin is applied, for example, as a topical microbicide.

  17. Small-angle scattering from GP zones in Al–Cu alloy

    Indian Academy of Sciences (India)

    Administrator

    ficial aging, which are extensively used in commercial practice. It is well established ... Cu system shows the variety of metastable states follow- ing the sequence SSS .... the model for spinodal decomposition of GP 1 zones in. Al–Cu system ...

  18. 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......·cm(2)) and its low endogenous expression of ABC-type efflux transporters. The IPEC-J2 cells were transfected with a plasmid that contained the sequence of the human MDR1 gene, which encodes P-gp, followed by a selection of successfully transfected cells with geneticin and puromycin. The resulting cell...

  19. Re-Audit of the Contents of GP Referral letters to General Adult Community Psychiatrists.

    Science.gov (United States)

    Odelola, Catherine; Jabbar, Farid

    2017-09-01

    The quality of information provided by referring general practitioners to secondary care mental health services are crucial elements in the effective management of patients. In order to establish effective communication, both primary and secondary care health professionals should contribute to planning and organising this process taking into account their different opinions and views. Anonymous questionnaire was designed to collect information on items that GPs and psychiatrist rated as most important items in GP referral letters to psychiatrists. The questionnaires were sent out electronically. Each item was scored using a rating scale where 0 was least important and 10 was most important. Items that scored 8 and above were agreed by all as the most important items. 76 GP letters were audited using a devised checklist of the identified most important items. Data was collected and analysed using a devised data collection tool. A re-audit was done 6months later. A response rate of 70% was obtained for both psychiatrists and GPs. Reasons for referral were described in almost all GP referral letters (95%). Only 24% referral letters had details about current physical health which improved to 59%. Concerns about risk were described in only 47% of letters and treatment provided by GP in 50% of letters. These improved in 79% and 71% of letters respectively in the re-audit. The involvement of professionals in devising a standardised approach for referral letters has improved communication in this re-audit between GPs and Psychiatrists. This is evident in the improvement in key aspects of the referral letters: past medical history, past psychiatric history, current physical health, treatment provided by GP. Efficient communication between GPs and psychiatrists improves the quality of health care for patients.

  20. Dose-response assessment of tariquidar and elacridar and regional quantification of P-glycoprotein inhibition at the rat blood-brain barrier using (R)-[11C]verapamil PET

    International Nuclear Information System (INIS)

    Kuntner, Claudia; Wanek, Thomas; Stundner, Gloria; Bankstahl, Jens P.; Bankstahl, Marion; Loescher, Wolfgang; Stanek, Johann; Mueller, Markus; Karch, Rudolf; Brauner, Rebecca; Meier, Martin; Ding, Xiaoqi; Langer, Oliver

    2010-01-01

    Overactivity of the multidrug efflux transporter P-glycoprotein (P-gp) at the blood-brain barrier (BBB) is believed to play an important role in resistance to central nervous system drug treatment. (R)-[ 11 C]verapamil (VPM) PET can be used to measure the function of P-gp at the BBB, but low brain uptake of VPM hampers the mapping of regional differences in cerebral P-gp function and expression. The aim of this study was to evaluate the dose-response relationship of two potent P-gp inhibitors and to investigate if increased brain uptake of VPM mediated by P-gp inhibition can be used to assess regional differences in P-gp activity. Two groups of Sprague-Dawley rats (n = 12) underwent single VPM PET scans at 120 min after administration of different doses of the P-gp inhibitors tariquidar and elacridar. In an additional six rats, paired VPM PET scans were performed before and after administration of 3 mg/kg tariquidar. Inhibitor administration resulted in an up to 11-fold increase in VPM brain distribution volumes (DV) with half-maximum effective dose (ED 50 ) values of 3.0 ± 0.2 and 1.2 ± 0.1 mg/kg for tariquidar and elacridar, respectively. In paired PET scans, 3 mg/kg tariquidar resulted in regionally different enhancement of brain activity distribution, with lowest DV in cerebellum and highest DV in thalamus. Our data show that tariquidar and elacridar are able to increase VPM brain distribution in rat brain up to 11-fold over baseline at maximum effective doses, with elacridar being about three times more potent than tariquidar. Regional differences in tariquidar-induced modulation of VPM brain uptake point to regional differences in cerebral P-gp function and expression in rat brain. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Luis Mario Rodríguez-Martínez

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

  2. Tumor p-glycoprotein correlates with efficacy of PF-3758309 in in vitro and in vivo models of colorectal cancer.

    OpenAIRE

    Erica Lynn Bradshaw-Pierce; Erica Lynn Bradshaw-Pierce; Todd M Pitts; Aik-Choon eTan; Kelly eMcPhillips; Mark eWest; Daniel L Gustafson; Charles eHalsey; Leslie eNguyen; Nathan V Lee; Julie LC Kan; Brion William Murray; S. Gail eEckhardt

    2013-01-01

    P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309), a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB...

  3. Are physical symptoms among survivors of a disaster presented to the general practitioner? A comparison between self-reports and GP data

    Directory of Open Access Journals (Sweden)

    Stellato Rebecca K

    2007-09-01

    Full Text Available Abstract Background Most studies examining medically unexplained symptoms (MUS have been performed in primary or secondary care and have examined symptoms for which patients sought medical attention. Disasters are often described as precipitating factors for MUS. However, health consequences of disasters are typically measured by means of questionnaires, and it is not known whether these self-reported physical symptoms are presented to the GP. It is also not known if the self-reported symptoms are related to a medical disorder or if they remain medically unexplained. In the present study, three research questions were addressed. Firstly, were self-reported symptoms among survivors presented to the GP? Secondly, were the symptoms presented to the GP associated with a high level of functional impairment and distress? Thirdly, what was the GP's clinical judgment of the presented symptoms, i.e. were the symptoms related to a medical diagnosis or could they be labeled MUS? Methods Survivors of a man-made disaster (N = 887 completed a questionnaire 3 weeks (T1 and 18 months (T2 post-disaster. This longitudinal health survey was combined with an ongoing surveillance program of health problems registered by GPs. Results The majority of self-reported symptoms was not presented to the GP and survivors were most likely to present persistent symptoms to the GP. For example, survivors with stomachache at both T1 and T2 were more likely to report stomachache to their GP (28% than survivors with stomachache at only T1 (6% or only T2 (13%. Presentation of individual symptoms to the GP was not consistently associated with functional impairment and distress. 56 – 91% of symptoms were labeled as MUS after clinical examination. Conclusion These results indicate that the majority of self-reported symptoms among survivors of a disaster are not presented to the GP and that the decision to consult with a GP for an individual symptom is not dependent on the level of

  4. Expression and Significance of gp96 and Immune-related Gene CTLA-4, CD8 in Lung Cancer Tissues

    Directory of Open Access Journals (Sweden)

    Haiyan ZHENG

    2010-08-01

    Full Text Available Background and objective It has been proven that gp96 plays an important role in specific cytotoxic immune response which is involved in anti-tumor effect in the body. The aim of this study is to investigate the biological significance of heat shock protein gp96 and immune-related gene CTLA-4, CD8 expressions in lung cancer tissues of different progressive stages. Methods We used Envision immunohistochemistry method to detect the levels of expression of gp96, CTLA-4, CD8 in tissue microarray, which contained 89 primary lung cancer tissues, 12 lymph node metastasis lung cancer tissues, 12 precancerous lesions and 10 normal lung tissues, and analyzed the relationship between their expressions and clinicopathological parameters. Results (1 The positive rate of gp96 in primary lung cancer was remarkably higher than that in precancerous lesion and normal lung tissue (P < 0.05. The positive rate of CTLA-4 in primary lung cancer tissue and precancerous lesion was significantly higher than that in normal lung tissue (P < 0.05. The positive rate of CD8 in primary lung cancer tissue was significantly higher than that in normal lung tissue (P < 0.05. The positive rate of gp96 in CD8-positive lymphocytes in the high expression group was less than that in the low group (P < 0.05. (2 The positive rate of gp96 was closely related to sex, differentiation and clinical stage (P < 0.05, but not to age, gross type, histological type and lymph node metastasis (P > 0.05. The positive rate of CTLA-4 was closely related to age and differentiation (P < 0.05, but not to sex, gross type, histological type, clinical stage and lymph node metastasis (P > 0.05. CD8 expression was related to clinical stage (P < 0.05, but not to sex, age, gross type, histological type, differentiation and lymph node metastasis (P > 0.05. The positive rates of gp96, CTLA-4 were higher than that of CD8 in squamous cell carcinoma and SCLC, respectively. (3 There was positive correlation between gp

  5. A combined AHP-GP model to allocate internal auditing time to ...

    African Journals Online (AJOL)

    Analytic hierarchy process, goal programming, internal audit, risk assessment. ..... Services area and are listed, together with their associated management ..... [2] Badri MA, 2001, A combined AHP-GP model for quality control systems, Interna-.

  6. The construction of a decision tool to analyse local demand and local supply for GP care using a synthetic estimation model.

    Science.gov (United States)

    de Graaf-Ruizendaal, Willemijn A; de Bakker, Dinny H

    2013-10-27

    This study addresses the growing academic and policy interest in the appropriate provision of local healthcare services to the healthcare needs of local populations to increase health status and decrease healthcare costs. However, for most local areas information on the demand for primary care and supply is missing. The research goal is to examine the construction of a decision tool which enables healthcare planners to analyse local supply and demand in order to arrive at a better match. National sample-based medical record data of general practitioners (GPs) were used to predict the local demand for GP care based on local populations using a synthetic estimation technique. Next, the surplus or deficit in local GP supply were calculated using the national GP registry. Subsequently, a dynamic internet tool was built to present demand, supply and the confrontation between supply and demand regarding GP care for local areas and their surroundings in the Netherlands. Regression analysis showed a significant relationship between sociodemographic predictors of postcode areas and GP consultation time (F [14, 269,467] = 2,852.24; P 1,000 inhabitants in the Netherlands covering 97% of the total population. Confronting these estimated demand figures with the actual GP supply resulted in the average GP workload and the number of full-time equivalent (FTE) GP too much/too few for local areas to cover the demand for GP care. An estimated shortage of one FTE GP or more was prevalent in about 19% of the postcode areas with >1,000 inhabitants if the surrounding postcode areas were taken into consideration. Underserved areas were mainly found in rural regions. The constructed decision tool is freely accessible on the Internet and can be used as a starting point in the discussion on primary care service provision in local communities and it can make a considerable contribution to a primary care system which provides care when and where people need it.

  7. Conceptualising GP teachers' knowledge: a pedagogical content knowledge perspective.

    Science.gov (United States)

    Cantillon, Peter; de Grave, Willem

    2012-05-01

    Most teacher development initiatives focus on enhancing knowledge of teaching (pedagogy), whilst largely ignoring other important features of teacher knowledge such as subject matter knowledge and awareness of the learning context. Furthermore, teachers' ability to learn from faculty development interventions is limited by their existing (often implicit) pedagogical knowledge and beliefs. Pedagogical content knowledge (PCK) represents a model of teacher knowledge incorporating what they know about subject matter, pedagogy and context. PCK can be used to explore teachers' prior knowledge and to structure faculty development programmes so that they take account of a broader range of teachers' knowledge. We set out to examine the application of a PCK model in a general practice education setting. This study is part of a larger study that employed a mixed method approach (concept mapping, phenomenological interviews and video-stimulated recall) to explore features of GP teachers' subject matter knowledge, pedagogical knowledge and knowledge of the learning environment in the context of a general practice tutorial. This paper presents data on GP teachers' pedagogical and context knowledge. There was considerable overlap between different GP teachers' knowledge and beliefs about learners and the clinical learning environment (i.e. knowledge of context). The teachers' beliefs about learners were largely based on assumptions derived from their own student experiences. There were stark differences, however, between teachers in terms of pedagogical knowledge, particularly in terms of their teaching orientations (i.e. transmission or facilitation orientation) and this was manifest in their teaching behaviours. PCK represents a useful model for conceptualising clinical teacher prior knowledge in three domains, namely subject matter, learning context and pedagogy. It can and should be used as a simple guiding framework by faculty developers to inform the design and delivery of

  8. P-glycoprotein Inhibition Increases the Brain Distribution and Antidepressant-Like Activity of Escitalopram in Rodents

    OpenAIRE

    O'Brien, Fionn E; O'Connor, Richard M; Clarke, Gerard; Dinan, Timothy G; Griffin, Brendan T; Cryan, John F

    2013-01-01

    Despite the clinical prevalence of the antidepressant escitalopram, over 30% of escitalopram-treated patients fail to respond to treatment. Recent gene association studies have highlighted a potential link between the drug efflux transporter P-glycoprotein (P-gp) and response to escitalopram. The present studies investigated pharmacokinetic and pharmacodynamic interactions between P-gp and escitalopram. In vitro bidirectional transport studies revealed that escitalopram is a transported subst...

  9. Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction

    International Nuclear Information System (INIS)

    Mo Hongmei; Konstantinidis, Alex K.; Stewart, Kent D.; Dekhtyar, Tatyana; Ng, Teresa; Swift, Kerry; Matayoshi, Edmund D.; Kati, Warren; Kohlbrenner, William; Molla, Akhteruzzaman

    2004-01-01

    The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry

  10. Inhibition of the multidrug resistance P-glycoprotein: time for a change of strategy?

    Science.gov (United States)

    Callaghan, Richard; Luk, Frederick; Bebawy, Mary

    2014-04-01

    P-glycoprotein (P-gp) is a key player in the multidrug-resistant phenotype in cancer. The protein confers resistance by mediating the ATP-dependent efflux of an astonishing array of anticancer drugs. Its broad specificity has been the subject of numerous attempts to inhibit the protein and restore the efficacy of anticancer drugs. The general strategy has been to develop compounds that either compete with anticancer drugs for transport or act as direct inhibitors of P-gp. Despite considerable in vitro success, there are no compounds currently available to "block" P-gp-mediated resistance in the clinic. The failure may be attributed to toxicity, adverse drug interaction, and numerous pharmacokinetic issues. This review provides a description of several alternative approaches to overcome the activity of P-gp in drug-resistant cells. These include 1) drugs that specifically target resistant cells, 2) novel nanotechnologies to provide high-dose, targeted delivery of anticancer drugs, 3) compounds that interfere with nongenomic transfer of resistance, and 4) approaches to reduce the expression of P-gp within tumors. Such approaches have been developed through the pursuit of greater understanding of resistance mediators such as P-gp, and they show considerable potential for further application.

  11. Epidemiology and resistance patterns in urinary pathogens from long-term care facilities and GP populations.

    LENUS (Irish Health Repository)

    Brabazon, E D

    2012-06-01

    Urinary tract infections (UTIs) are a major source of antimicrobial prescribing in the clinical setting and a potential reservoir for the emergence of resistant organisms. Although studies have been published on resistance rates for urinary pathogens from both hospital and general practitioner (GP) settings, there is little information from Long-Term Care Facilities (LTCFs) in Ireland. This study aimed to document the epidemiology and resistance rates in urinary isolates, in the LTCF and GP setting, from samples submitted to a typical microbiology laboratory. In 2010, there were 963 urinary isolates from LTCFs and 1,169 urinary isolates from GPs, identified from patients 65 years and over, with cytology suggestive of infection. E. coil was the most common causative organism identified. There were significantly higher levels of resistance to ampicillin, co-amoxiclav, ciprofloxacin, nitrofurantoin, trimethoprim, and piperacillin\\/tazobactam in the LTCF population compared to the GP population (e.g. for E. coli, 86%-v-69%; 30%-v- 21%; 58%-v-26%, 10%-v-3%, 68%-v-48%, 10%-v- 4% respectively). Isolates with resistance mechanisms to beta-lactams, were identified in both populations. Results presented in this paper demonstrate significant differences between resistance rates in LTCF and GP populations which suggest that there are implications for empiric antimicrobial prescribing for UTIs in the LTCF setting.

  12. IL-1β and TNFα inhibit GPR120 (FFAR4) and stimulate GPR84 (EX33) and GPR41 (FFAR3) fatty acid receptor expression in human adipocytes: implications for the anti-inflammatory action of n-3 fatty acids.

    Science.gov (United States)

    Muredda, Laura; Kępczyńska, Małgorzata A; Zaibi, Mohamed S; Alomar, Suliman Y; Trayhurn, Paul

    2018-05-01

    Regulation of the expression of GPCR fatty acid receptor genes has been examined in human adipocytes differentiated in culture. TNFα and IL-1β induced a marked reduction in GPR120 expression, mRNA level falling 17-fold at 24 h in adipocytes incubated with TNFα. In contrast, GPR84 mRNA was dramatically increased by these cytokines (>500-fold for IL-1β at 4 h); GPR41 expression was also stimulated. Rosiglitazone did not affect GPR84 expression, but GPR120 and GPR41 expression increased. Dexamethasone, insulin, linoleic and docosahexaenoic acids (DHA), and TUG891 (GPR120 agonist) had little effect on GPR120 and GPR84 expression. TUG891 did not attenuate the pro-inflammatory actions of TNFα and IL-1β. DHA slightly countered the actions of IL-1β on CCL2, IL6 and ADIPOQ expression, though not on secretion of these adipokines. GPR120 and GP84 gene expression in human adipocytes is highly sensitive to pro-inflammatory mediators; the inflammation-induced inhibition of GPR120 expression may compromise the anti-inflammatory action of GPR120 agonists.

  13. Tumor P-Glycoprotein Correlates with Efficacy of PF-3758309 in in vitro and in vivo Models of Colorectal Cancer

    OpenAIRE

    Bradshaw-Pierce, Erica Lynn; Pitts, Todd M.; Tan, Aik-Choon; McPhillips, Kelly; West, Mark; Gustafson, Daniel L.; Halsey, Charles; Nguyen, Leslie; Lee, Nathan V.; Kan, Julie L. C.; Murray, Brion William; Eckhardt, S. Gail

    2013-01-01

    P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309), a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB1...

  14. Inhibitory Effects of Neochamaejasmin B on P-Glycoprotein in MDCK-hMDR1 Cells and Molecular Docking of NCB Binding in P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Lanying Pan

    2015-02-01

    Full Text Available Stellera chamaejasme L. (Thymelaeaceae is widely distributed in Mongolia, Tibet and the northern parts of China. Its roots are commonly used as “Langdu”, which is embodied in the Pharmacopoeia of the P.R. China (2010 as a toxic Traditional Chinese Medicine. It is claimed to have antivirus, antitumor and antibacterial properties in China and other Asian countries. Studies were carried out to characterize the inhibition of neochamaejasmin B (NCB on P-glycoprotein (P-gp, ABCB1, MDR1. Rhodamine-123 (R-123 transport and accumulation studies were performed in MDCK-hMDR1 cells. ABCB1 (MDR1 mRNA gene expression and P-gp protein expression were analyzed. Binding selectivity studies based on molecular docking were explored. R-123 transport and accumulation studies in MDCK-hMDR1 cells indicated that NCB inhibited the P-gp-mediated efflux in a concentration-dependent manner. RT-PCR and Western blot demonstrated that the P-gp expression was suppressed by NCB. To investigate the inhibition type of NCB on P-gp, Ki and Ki’ values were determined by double-reciprocal plots in R-123 accumulation studies. Since Ki was greater than Ki’, the inhibition of NCB on P-gp was likely a mixed type of competitive and non-competitive inhibition. The results were confirmed by molecular docking in our current work. The docking data indicated that NCB had higher affinity to P-gp than to Lig1 ((S-5,7-dihydroxy-2-(4-hydroxyphenylchroman-4-one.

  15. Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen.

    Science.gov (United States)

    Arias, Mauricio A; Loxley, Andrew; Eatmon, Christy; Van Roey, Griet; Fairhurst, David; Mitchnick, Mark; Dash, Philip; Cole, Tom; Wegmann, Frank; Sattentau, Quentin; Shattock, Robin

    2011-02-01

    Induction of humoral responses to HIV at mucosal compartments without inflammation is important for vaccine design. We developed charged wax nanoparticles that efficiently adsorb protein antigens and are internalized by DC in the absence of inflammation. HIV-gp140-adsorbed nanoparticles induced stronger in vitro T-cell proliferation responses than antigen alone. Such responses were greatly enhanced when antigen was co-adsorbed with TLR ligands. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG. Importantly, intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhanced serum and vaginal IgG and IgA responses. Our results show that HIV-gp140-carrying wax nanoparticles can induce strong cellular/humoral immune responses without inflammation and may be of potential use as effective mucosal adjuvants for HIV vaccine candidates. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

  17. Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

    Science.gov (United States)

    Yuan, Wei-Qi; Zhang, Rong-Rong; Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

    2016-05-24

    Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

  18. Speeding up IA mechanically-steered multistatic radar scheduling with GP-GPUs

    CSIR Research Space (South Africa)

    Focke, RW

    2016-07-01

    Full Text Available In this paper, the authors investigate speeding up the execution time of Interval Algebra (IA) mechanically-steered multistatic and multisite radar scheduling using a general-purpose graphical processing unit (GP-GPU). Multistatic/multisite radar...

  19. Immunizing Patients With Metastatic Melanoma Using Recombinant Adenoviruses Encoding MART-1 or gp100 Melanoma Antigens

    Science.gov (United States)

    Rosenberg, Steven A.; Zhai, Yifan; Yang, James C.; Schwartzentruber, Douglas J.; Hwu, Patrick; Marincola, Francesco M.; Topalian, Suzanne L.; Restifo, Nicholas P.; Seipp, Claudia A.; Einhorn, Jan H.; Roberts, Bruce; White, Donald E.

    2008-01-01

    Background: The characterization of the genes encoding melanoma-associated antigens MART-1 or gp100, recognized by T cells, has opened new possibilities for the development of immunization strategies for patients with metastatic melanoma. With the use of recombinant adenoviruses expressing either MART-1 or gp100 to immunize patients with metastatic melanoma, we evaluated the safety, immunologic, and potential therapeutic aspects of these immunizations. Methods: In phase I studies, 54 patients received escalating doses (between 107 and 1011 plaque-forming units) of recombinant adenovirus encoding either MART-1 or gp100 melanoma antigen administered either alone or followed by the administration of interleukin 2 (IL-2). The immunologic impact of these immunizations on the development of cellular and antibody reactivity was assayed. Results: Recombinant adenoviruses expressing MART-1 or gp100 were safely administered. One of 16 patients with metastatic melanoma receiving the recombinant adenovirus MART-1 alone experienced a complete response. Other patients achieved objective responses, but they had received IL-2 along with an adenovirus, and their responses could be attributed to the cytokine. Immunologic assays showed no consistent immunization to the MART-1 or gp100 transgenes expressed by the recombinant adenoviruses. High levels of neutralizing antibody were found in the pretreatment sera of the patients. Conclusions: High doses of recombinant adenoviruses could be safely administered to cancer patients. High levels of neutralizing antibody present in patients' sera prior to treatment may have impaired the ability of these viruses to immunize patients against melanoma antigens. PMID:9862627

  20. Basket Option Pricing Using GP-GPU Hardware Acceleration

    KAUST Repository

    Douglas, Craig C.

    2010-08-01

    We introduce a basket option pricing problem arisen in financial mathematics. We discretized the problem based on the alternating direction implicit (ADI) method and parallel cyclic reduction is applied to solve the set of tridiagonal matrices generated by the ADI method. To reduce the computational time of the problem, a general purpose graphics processing units (GP-GPU) environment is considered. Numerical results confirm the convergence and efficiency of the proposed method. © 2010 IEEE.

  1. Multiple Linear Regression Analysis Indicates Association of P-Glycoprotein Substrate or Inhibitor Character with Bitterness Intensity, Measured with a Sensor.

    Science.gov (United States)

    Yano, Kentaro; Mita, Suzune; Morimoto, Kaori; Haraguchi, Tamami; Arakawa, Hiroshi; Yoshida, Miyako; Yamashita, Fumiyoshi; Uchida, Takahiro; Ogihara, Takuo

    2015-09-01

    P-glycoprotein (P-gp) regulates absorption of many drugs in the gastrointestinal tract and their accumulation in tumor tissues, but the basis of substrate recognition by P-gp remains unclear. Bitter-tasting phenylthiocarbamide, which stimulates taste receptor 2 member 38 (T2R38), increases P-gp activity and is a substrate of P-gp. This led us to hypothesize that bitterness intensity might be a predictor of P-gp-inhibitor/substrate status. Here, we measured the bitterness intensity of a panel of P-gp substrates and nonsubstrates with various taste sensors, and used multiple linear regression analysis to examine the relationship between P-gp-inhibitor/substrate status and various physical properties, including intensity of bitter taste measured with the taste sensor. We calculated the first principal component analysis score (PC1) as the representative value of bitterness, as all taste sensor's outputs shared significant correlation. The P-gp substrates showed remarkably greater mean bitterness intensity than non-P-gp substrates. We found that Km value of P-gp substrates were correlated with molecular weight, log P, and PC1 value, and the coefficient of determination (R(2) ) of the linear regression equation was 0.63. This relationship might be useful as an aid to predict P-gp substrate status at an early stage of drug discovery. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

  2. Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug- Drug Interaction Risk Assessment Decision Criterias

    NARCIS (Netherlands)

    Bentz, J.; O'Connor, M.P.; Bednarczyk, D.; Coleman, J.; Lee, C.; Palm, J.; Pak, Y.A.; Perloff, E.S.; Reyner, E.; Balimane, P.; Brännström, M.; Chu, X.; Funk, C.; Guo, A.; Hanna, I.; Herédi-Szabó, K.; Hillgren, K.; Li, L.; Hollnack-Pusch, E.; Jamei, M.; Lin, X.; Mason, A.K.; Neuhoff, S.; Patel, A.; Podila, L.; Plise, E.; Rajaraman, G.; Salphati, L.; Sands, E.; Taub, M.E.; Taur, J.-S.; Weitz, D.; Wortelboer, H.M.; Xia, C.Q.; Xiao, G.; Yabut, J.; Yamagata, T.; Zhang, L.; Ellens, H.

    2013-01-01

    A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC 50 determinations. Each laboratory followed its in-house protocol to determine in vitro

  3. Inhibitory Effects of Daiokanzoto (Da-Huang-Gan-Cao-Tang on P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Yuka Watanabe

    2012-01-01

    Full Text Available We have studied the effects of various Kampo medicines on P-glycoprotein (P-gp, a drug transporter, in vitro. The present study focused on Daiokanzoto (Da-Huang-Gan-Cao-Tang, which shows the most potent inhibitory effects on P-gp among the 50 Kampo medicines studied, and investigated the P-gp inhibitory effects of Daiokanzoto herbal ingredients (rhubarb and licorice root and their components by an ATPase assay using human P-gp membrane. Both rhubarb and licorice root significantly inhibited ATPase activity, and the effects of rhubarb were more potent than those of licorice root. The content of rhubarb in Daiokanzoto is double that in licorice root, and the inhibition patterns of Daiokanzoto and rhubarb involve both competitive and noncompetitive inhibition, suggesting that the inhibitory effects of Daiokanzoto are mainly due to rhubarb. Concerning the components of rhubarb, concentration-dependent inhibitory effects were observed for (−-catechin gallate, (−-epicatechin gallate, and (−-epigallocatechin gallate. In conclusion, rhubarb may cause changes in the drug dispositions of P-gp substrates through the inhibition of P-gp. It appears that attention should be given to the interactions between these drugs and Kampo medicines containing rhubarb as an herbal ingredient.

  4. The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis

    Directory of Open Access Journals (Sweden)

    PAMELA ARAYA

    2001-01-01

    Full Text Available Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions. Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases. The gpNu1 subunit was obtained from E.coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein. Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively. The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes. Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends. The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments. This information is key to design new plasmids to overproduce these domains for further structural analysis.

  5. Small-angle scattering from GP zones in Al–Cu alloy

    Indian Academy of Sciences (India)

    ... and smallangle scattering experiments were carried on the powdered samples as a function of time during artificial aging. Small-angle scattering data were analysed, and evidence has been obtained for the occurrence of spinodal decomposition as the mechanism responsible in the early stages of formation of GP zones.

  6. Multiple protein-domain conservation architecture as a non ...

    African Journals Online (AJOL)

    Using two-sets of surface viral glycoproteins of human immunodeficiency virus type I, HIV-1 (gp120) and Ebola virus, EBOV (gp1,2 preprotein) (selected because their CD-architecture has widely been studied, their sequences are available in public databases, and the same are well annotated), the MPDCAs among three ...

  7. A study comparing the efficacy of antimicrobial agents versus enzyme (P-gp) inducers in the treatment of 2,4,6 trinitrobenzenesulfonic acid-induced colitis in rats.

    Science.gov (United States)

    Toklu, H Z; Kabasakal, L; Imeryuz, N; Kan, B; Celikel, C; Cetinel, S; Orun, O; Yuksel, M; Dulger, G A

    2013-08-01

    The intestinal microflora is an important cofactor in the pathogenesis of intestinal inflammation; and the epithelial cell barrier function is critical in providing protection against the stimulation of mucosal immune system by the microflora. In the present study, therapeutic role of the antibacterial drugs rifampicin and ciprofloxacine were investigated in comparison to spironolactone, an enzyme inducer, in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis of the rats. Drugs were administered for 14 days following induction of colitis. All drug treatments ameliorated the clinical hallmarks of colitis as determined by body weight loss and assessment of diarrhea, colon length, and histology. Oxidative damage and neutrophil infiltration as well as nuclear factor κB (NF-κB) and tumor necrosis factor α (TNF-α) expressions that were increased during colitis, were decreased significantly. Rifampicin and ciprofloxacin were probably effective due to their antibacterial and immunomodulating properties. The multidrug resistence gene (MDR1) and its product p-glycoprotein (P-gp) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). In the present study, findings of the P-gp expression were inconclusive but regarding previous studies, it can be suggested that the beneficial effects of rifampicin and spironolactone may be partly due to their action as a P-gp ligand. Spironolactone has been reported to supress the transcription of proinflamatory cytokines that are considered to be of importance in immunoinflammatory diseases. It is also a powerful pregnane X receptor (PXR) inducer; thus, inhibition of the expression of NF-κB and TNF-α, and amelioration of inflammation by spironolactone suggest that this may have been through the activation of PXR. However, our findings regarding PXR expression were inconclusive. Activation of PXR by spironolactone probably also contributed to the induction of P-gp, resulting in extrusion of noxious substances

  8. Effect of Ebola virus proteins GP, NP and VP35 on VP40 VLP morphology

    Directory of Open Access Journals (Sweden)

    Harty Ronald N

    2006-05-01

    Full Text Available Abstract Recently we described a role for Ebola virus proteins, NP, GP, and VP35 in enhancement of VP40 VLP budding. To explore the possibility that VLP structure was altered by co-expression of EBOV proteins leading to the observed enhancement of VP40 VLP budding, we performed density gradient analysis as well as electron microscopy studies. Our data suggest that VP40 is the major determinant of VLP morphology, as co-expression of NP, GP and VP35 did not significantly change VLP density, length, and diameter. Ultra-structural changes were noted in the core of the VLPs when NP was co-expressed with VP40. Overall, these findings indicate that major changes in morphology of VP40 VLPs were likely not responsible for enhanced budding of VP40 VLPs in the presence of GP, NP and/or VP35.

  9. Observing the temperature dependent transition of the GP2 peptide using terahertz spectroscopy.

    Directory of Open Access Journals (Sweden)

    Yiwen Sun

    Full Text Available The GP2 peptide is derived from the Human Epidermal growth factor Receptor 2 (HER2/nue, a marker protein for breast cancer present in saliva. In this paper we study the temperature dependent behavior of hydrated GP2 at terahertz frequencies and find that the peptide undergoes a dynamic transition between 200 and 220 K. By fitting suitable molecular models to the frequency response we determine the molecular processes involved above and below the transition temperature (T(D. In particular, we show that below T(D the dynamic transition is dominated by a simple harmonic vibration with a slow and temperature dependent relaxation time constant and that above T(D, the dynamic behavior is governed by two oscillators, one of which has a fast and temperature independent relaxation time constant and the other of which is a heavily damped oscillator with a slow and temperature dependent time constant. Furthermore a red shifting of the characteristic frequency of the damped oscillator was observed, confirming the presence of a non-harmonic vibration potential. Our measurements and modeling of GP2 highlight the unique capabilities of THz spectroscopy for protein characterization.

  10. Genetic deletion of P-glycoprotein alters stress responsivity and increases depression-like behavior, social withdrawal and microglial activation in the hippocampus of female mice.

    Science.gov (United States)

    Brzozowska, Natalia I; Smith, Kristie L; Zhou, Cilla; Waters, Peter M; Cavalcante, Ligia Menezes; Abelev, Sarah V; Kuligowski, Michael; Clarke, David J; Todd, Stephanie M; Arnold, Jonathon C

    2017-10-01

    P-glycoprotein (P-gp) is an ABC transporter expressed at the blood brain barrier and regulates the brain uptake of various xenobiotics and endogenous mediators including glucocorticoid hormones which are critically important to the stress response. Moreover, P-gp is expressed on microglia, the brain's immune cells, which are activated by stressors and have an emerging role in psychiatric disorders. We therefore hypothesised that germline P-gp deletion in mice might alter the behavioral and microglial response to stressors. Female P-gp knockout mice displayed an unusual, frantic anxiety response to intraperitoneal injection stress in the light-dark test. They also tended to display reduced conditioned fear responses compared to wild-type (WT) mice in a paradigm where a single electric foot-shock stressor was paired to a context. Foot-shock stress reduced social interaction and decreased microglia cell density in the amygdala which was not varied by P-gp genotype. Independently of stressor exposure, female P-gp deficient mice displayed increased depression-like behavior, idiosyncratic darting behavior, age-related social withdrawal and hyperactivity, facilitated sensorimotor gating and altered startle reactivity. In addition, P-gp deletion increased microglia cell density in the CA3 region of the hippocampus, and the microglial cells exhibited a reactive, hypo-ramified morphology. Further, female P-gp KO mice displayed increased glucocorticoid receptor (GR) expression in the hippocampus. In conclusion, this research shows that germline P-gp deletion affected various behaviors of relevance to psychiatric conditions, and that altered microglial cell activity and enhanced GR expression in the hippocampus may play a role in mediating these behaviors. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Identification of the Interaction between P-Glycoprotein and Anxa2 in Multidrug-resistant Human Breast Cancer Cells

    International Nuclear Information System (INIS)

    Zhang, Hai-chang; Zhang, Fei; Wu, Bing; Han, Jing-hua; Ji, Wei; Zhou, Yan; Niu, Rui-fang

    2012-01-01

    To explore the interaction of Anxa2 with P-Glycoprotein (P-gp) in the migration and invasion of the multidrug-resistant (MDR) human breast cancer cell line MCF-7/ADR. A pair of short hairpin RNA (shRNA) targeting P-gp was transfected into MCF-7/ADR cells, and monoclonal cell strains were screened. The expression of P-gp was detected by Western blot. Transwell chambers were used to observe the cell migration capacity and invasion ability. The interaction between P-gp and Anxa2 was examined by immunoprecipitation and immunofluorescence confocal microscopy analyses. P-gp expression was significantly knocked down, and there were notable decreasing trends in the migration and invasion capability of MDR breast cancer cells (P<0.05). There was a close interaction between Anxa2 and P-gp. MCF-7/ADR is an MDR human breast cancer cell line with high migration and invasion abilities. The knockdown of P-gp notably impaired the migration and invasion abilities of the tumor cells. The interaction of Anxa2 with P-pg may play an important role in the enhanced invasiveness of MDR human breast cancer cells

  12. Glycoprotein Ibalpha signalling in platelet apoptosis and clearance

    NARCIS (Netherlands)

    van der Wal, E.

    2010-01-01

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

  13. In vivo evaluation of [{sup 123}I]-4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(4-iodobenzyl)piperidine, an iodinated SPECT tracer for imaging the P-gp transporter

    Energy Technology Data Exchange (ETDEWEB)

    De Bruyne, Sylvie; Wyffels, Leonie [Laboratory for Radiopharmacy, Ghent University, 9000 Ghent (Belgium); Boos, Terrence L. [Chemical Biology Research Branch, National Institute on Drug Abuse and National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD (United States); Staelens, Steven; Deleye, Steven [IBITECH-Medisip, Ghent University-IBBT, 9000 Ghent (Belgium); Rice, Kenner C. [Chemical Biology Research Branch, National Institute on Drug Abuse and National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD (United States); De Vos, Filip [Laboratory for Radiopharmacy, Ghent University, 9000 Ghent (Belgium)], E-mail: filipx.devos@ugent.be

    2010-05-15

    Introduction: P-glycoprotein (P-gp) is an energy-dependent transporter that contributes to the efflux of a wide range of xenobiotics at the blood-brain barrier playing a role in drug-resistance or therapy failure. In this study, we evaluated [{sup 123}I]-4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(4-iodobenzyl)piperidine ([{sup 123}I]-FMIP) as a novel single photon emission computed tomography (SPECT) tracer for imaging P-gp at the brain in vivo. Methods: The tissue distribution and brain uptake as well as the metabolic profile of [{sup 123}I]-FMIP in wild-type and mdr1a (-/-) mice after pretreatment with physiological saline or cyclosporin A (CsA) (50 mg/kg) was investigated. The influence of increasing doses CsA on brain uptake of [{sup 123}I]-FMIP was explored. {mu}SPECT images of mice brain after injection of 11.1 MBq [{sup 123}I]-FMIP were obtained for different treatment strategies thereby using the Milabs U-SPECT-II. Results: Modulation of P-gp with CsA (50 mg/kg) as well as mdr1a gene depletion resulted in significant increase in cerebral uptake of [{sup 123}I]-FMIP with only minor effect on blood activity. [{sup 123}I]-FMIP is relative stable in vivo with >80% intact [{sup 123}I]-FMIP in brain at 60 min p.i. in the different treatment regiments. A dose-dependent sigmoidal increase in brain uptake of [{sup 123}I]-FMIP with increasing doses of CsA was observed. In vivo region of interest-based SPECT measurements correlated well with the observations of the biodistribution studies. Conclusions: These findings indicate that [{sup 123}I]-FMIP can be applied to assess the efficacy of newly developed P-gp modulators. It is also suggested that [{sup 123}I]-FMIP is a promising SPECT tracer for imaging P-gp at the blood-brain barrier.

  14. Council tax valuation bands and contacts with a GP out-of-hours service.

    Science.gov (United States)

    Beale, Norman; Taylor, Gordon; Gwynne, Mark; Peart, Carole

    2006-04-01

    UK GPs are no longer responsible for the organisation of out-of-hours care for their patients, but resources remains capitation-based. This cross-sectional study tests whether council tax valuation bands can predict the demand for such services. All out-of-hours contacts made by patients in North Wiltshire over 4 months were classified by council tax band; frequencies compared with official population statistics. Council tax band predicts out-of-hours GP workload irrespective of age and sex: the more modest the home, the higher the GP contact rate. It may prove more difficult to sustain out-of-hours services in deprived parts of the UK.

  15. B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

    Directory of Open Access Journals (Sweden)

    Mattias N E Forsell

    2008-10-01

    Full Text Available The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3. Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4 rabbits with envelope glycoprotein (Env trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

  16. B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

    Science.gov (United States)

    Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

    2008-10-03

    The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

  17. Assessment of reliability of Greulich and Pyle (gp) method for ...

    African Journals Online (AJOL)

    Background: Greulich and Pyle standards are the most widely used age estimation standards all over the world. The applicability of the Greulich and Pyle standards to populations which differ from their reference population is often questioned. This study aimed to assess the reliability of Greulich and Pyle (GP) method for ...

  18. Acute Effects of Viral Exposure on P-Glycoprotein Function in the Mouse Fetal Blood-Brain Barrier

    Directory of Open Access Journals (Sweden)

    Enrrico Bloise

    2017-02-01

    Full Text Available Background/Aims: Viral infection during pregnancy is known to affect the fetal brain. The toll-like receptor (TLR-3 is a pattern recognition receptor activated by viruses known to elicit adverse fetal neurological outcomes. The P-glycoprotein (P-gp efflux transporter protects the developing fetus by limiting the transfer of substrates across both the placenta and the fetal blood-brain barrier (BBB. As such, inhibition of P-gp at these blood-barrier sites may result in increased exposure of the developing fetus to environmental toxins and xenobiotics present in the maternal circulation. We hypothesized that viral exposure during pregnancy would impair P-gp function in the placenta and in the developing BBB. Here we investigated whether the TLR-3 ligand, polyinosinic:polycytidylic acid (PolyI:C, increased accumulation of one P-gp substrate in the fetus and in the developing fetal brain. Methods: Pregnant C57BL/6 mice (GD15.5 were injected (i.p. with PolyI:C (5 mg/kg or 10 mg/kg or vehicle (saline. [3H]digoxin (P-gp substrate was injected (i.v. 3 or 23h post-treatment and animals were euthanized 1h later. Maternal plasma, ‘fetal-units’ (fetal membranes, amniotic fluid and whole fetus, and fetal brains were collected. Results: PolyI:C exposure (4h significantly elevated maternal plasma IL-6 (P<0.001 and increased [3H]digoxin accumulation in the fetal brain (P<0.05. In contrast, 24h after PolyI:C exposure, no effect on IL-6 or fetal brain accumulation of P-gp substrate was observed. Conclusion: Viral infection modeled by PolyI:C causes acute increases in fetal brain accumulation of P-gp substrates and by doing so, may increase fetal brain exposure to xenobiotics and environmental toxins present in the maternal circulation.

  19. Mechanistic kinetic modeling generates system-independent P-glycoprotein mediated transport elementary rate constants for inhibition and, in combination with 3D SIM microscopy, elucidates the importance of microvilli morphology on P-glycoprotein mediated efflux activity.

    Science.gov (United States)

    Ellens, Harma; Meng, Zhou; Le Marchand, Sylvain J; Bentz, Joe

    2018-06-01

    In vitro transporter kinetics are typically analyzed by steady-state Michaelis-Menten approximations. However, no clear evidence exists that these approximations, applied to multiple transporters in biological membranes, yield system-independent mechanistic parameters needed for reliable in vivo hypothesis generation and testing. Areas covered: The classical mass action model has been developed for P-glycoprotein (P-gp) mediated transport across confluent polarized cell monolayers. Numerical integration of the mass action equations for transport using a stable global optimization program yields fitted elementary rate constants that are system-independent. The efflux active P-gp was defined by the rate at which P-gp delivers drugs to the apical chamber, since as much as 90% of drugs effluxed by P-gp partition back into nearby microvilli prior to reaching the apical chamber. The efflux active P-gp concentration was 10-fold smaller than the total expressed P-gp for Caco-2 cells, due to their microvilli membrane morphology. The mechanistic insights from this analysis are readily extrapolated to P-gp mediated transport in vivo. Expert opinion: In vitro system-independent elementary rate constants for transporters are essential for the generation and validation of robust mechanistic PBPK models. Our modeling approach and programs have broad application potential. They can be used for any drug transporter with minor adaptations.

  20. Nonsense mutation in the glycoprotein Ibα coding sequence associated with Bernard-Soulier syndrome

    International Nuclear Information System (INIS)

    Ware, J.; Russell, S.R.; Vicente, V.; Scharf, R.E.; Tomer, A.; McMillian, R.; Ruggeri, Z.M.

    1990-01-01

    Three distinct gene products, the α and β chains of glycoprotein (GP) Ib and GP IX, constitute the platelet membrane GP Ib-IX complex, a receptor for von Willebrand factor and thrombin involved in platelet adhesion and aggregation. Defective function of the GP Ib-IX complex is the hallmark of a rare congenital bleeding disorder of still undefined pathogenesis, the Bernard-Soulier syndrome. The authors have analyzed the molecular basis of the disease in one patient in whom immunoblotting of solubilized platelets demonstrated absence of normal GP Ibα but presence of a smaller immunoreactive species. The truncated polypeptide was also present, along with normal protein, in platelets from the patient's mother and two of his four children. Genetic characterization identified a nucleotide transition changing the Trp-343 codon (TGG) to a nonsense codon (TGA). Such a mutation explains the origin of the smaller GP Ibα, which by lacking half of the sequence on the carboxyl-terminal side, including the transmembrane domain, cannot be properly inserted in the platelet membrane. Both normal and mutant codons were found in the patient, suggesting that he is a compound heterozygote with a still unidentified defect in the other GP Ibα allele. Nonsense mutation and truncated GP Ibα polypeptide were found to cosegregate in four individuals through three generations and were associated with either Bernard-Soulier syndrome or carrier state phenotype. The molecular abnormality demonstrated in this family provides evidence that defective synthesis of GP Ibα alters the membrane expression of the GP Ib-IX complex and may be responsible for Bernard-Soulier syndrome