WorldWideScience

Sample records for e1 glycoprotein involvement

  1. Monoclonal antibodies directed to E1 glycoprotein of rubella virus

    International Nuclear Information System (INIS)

    Umino, Y.; Sato, A.; Katow, S.; Matsuno, T.; Sugiura, A.

    1985-01-01

    We have prepared four monoclonal antibodies to rubella virus E1 glycoprotein. Three nonoverlapping antigenic sites were delineated on E1 protein by competitive binding assays. Antibodies binding to one site were characterized by high hemagglutination inhibition (HI) titer but poor neutralizing activity. The addition of antiglobulin conferred neutralizing activity. Antibodies directed to two other antigenic sites had modest hemolysis inhibition but little or no HI and neutralizing activities. The addition of antiglobulin markedly augmented HI activity but had little effect on neutralizing activity. Epitopes defined by three antibodies were conserved among four rubella virus strains examined. (Author)

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  3. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    Directory of Open Access Journals (Sweden)

    Goldbach Rob W

    2011-07-01

    Full Text Available Abstract Background Chikungunya virus (CHIKV is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...

  5. Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles

    DEFF Research Database (Denmark)

    Russell, R S; Kawaguchi, K; Meunier, J-C

    2009-01-01

    Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrov...

  6. 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 ...

  7. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    International Nuclear Information System (INIS)

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-01-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed

  8. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jimin, E-mail: jimin.wang@yale.edu; Li, Yue; Modis, Yorgo, E-mail: yorgo.modis@yale.edu

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  9. CYP2E1 Metabolism of Styrene Involves Allostery

    Science.gov (United States)

    Hartman, Jessica H.; Boysen, Gunnar

    2012-01-01

    We are the first to report allosterism during styrene oxidation by recombinant CYP2E1 and human liver microsomes. At low styrene concentrations, oxidation is inefficient because of weak binding to CYP2E1 (Ks = 830 μM). A second styrene molecule then binds CYP2E1 with higher affinity (Kss = 110 μM) and significantly improves oxidation to achieve a kcat of 6.3 nmol · min−1 · nmol CYP2E1−1. The transition between these metabolic cycles coincides with reported styrene concentrations in blood from exposed workers; thus, this CYP2E1 mechanism may be relevant in vivo. Scaled modeling of the in vitro-positive allosteric mechanism for styrene metabolism to its in vivo clearance led to significant deviations from the traditional model based on Michaelis-Menten kinetics. Low styrene levels were notably much less toxic than generally assumed. We interrogated the allosteric mechanism using the CYP2E1-specific inhibitor and drug 4-methylpyrazole, which we have shown binds two CYP2E1 sites. From the current studies, styrene was a positive allosteric effector on 4-methylpyrazole binding, based on a 10-fold increase in 4-methylpyrazole binding affinity from Ki 0.51 to Ksi 0.043 μM. The inhibitor was a negative allosteric effector on styrene oxidation, because kcat decreased 6-fold to 0.98 nmol · min−1 · nmol CYP2E1−1. Consequently, mixtures of styrene and other molecules can induce allosteric effects on binding and metabolism by CYP2E1 and thus mitigate the efficiency of their metabolism and corresponding effects on human health. Taken together, our elucidation of mechanisms for these allosteric reactions provides a powerful tool for further investigating the complexities of CYP2E1 metabolism of drugs and pollutants. PMID:22807108

  10. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    Science.gov (United States)

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-01-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. PMID:24725935

  11. Involvement of CYP 2E1 enzyme in ovotoxicity caused by 4-vinylcyclohexene and its metabolites

    International Nuclear Information System (INIS)

    Rajapaksa, Kathila S.; Cannady, Ellen A.; Sipes, I. Glenn; Hoyer, Patricia B.

    2007-01-01

    4-Vinylcyclohexene (VCH) is bioactivated by hepatic CYP 2A and 2B to a monoepoxide (VCM) and subsequently to an ovotoxic diepoxide metabolite (VCD). Studies suggest that the ovary can directly bioactivate VCH via CYP 2E1. The current study was designed to evaluate the role of ovarian CYP 2E1 in VCM-induced ovotoxicity. Postnatal day 4 B6C3F 1 and CYP 2E1 wild-type (+/+) and null (-/-) mouse ovaries were cultured (15 days) with VCD (30 μM), 1,2-VCM (125-1000 μM), or vehicle. Twenty-eight days female CYP 2E1 +/+ and -/- mice were dosed daily (15 days; ip) with VCH, 1,2-VCM, VCD or vehicle. Following culture or in vivo dosing, ovaries were histologically evaluated. In culture, VCD decreased (p 1 and CYP 2E1 +/+ ovaries, but not in CYP 2E1 -/- ovaries in culture. 1,2-VCM did not affect primary follicles in any group of mouse ovaries. Conversely, following in vivo dosing, primordial and primary follicles were reduced (p < 0.05) by VCD and VCM in CYP2E1 +/+ and -/-, and by VCH in +/+ mice. The data demonstrate that, whereas in vitro ovarian bioactivation of VCM requires CYP 2E1 enzyme, in vivo CYP 2E1 plays a minimal role. Thus, the findings support that hepatic metabolism dominates the contribution made by the ovary in bioactivation of VCM to its ovotoxic metabolite, VCD. This study also demonstrates the use of a novel ovarian culture system to evaluate ovary-specific metabolism of xenobiotics

  12. Involvement of prostaglandins F/sub 2α/ and E1 with rabbit endometrium

    International Nuclear Information System (INIS)

    Orlicky, D.J.

    1985-01-01

    Several growth factors and hormones are thought to play a role in the growth control of endometrial cells. The authors have shown that prostaglandin F/sub 2→/ (PGF/sub 2α/) is a growth factor for primary cultures of rabbit endometrium cultured in chemically-defined serum-free medium and that prostaglandin E 1 (PGE 1 ) antagonizes the PGF/sub 2→/ induction of growth. Both [ 3 H]PGF/sub 2α/ and [ 3 H]PGE 1 bind in a time and temperature dependent, dissociable, saturable and specific manner. The binding of [ 3 H]PGF/sub 2α/ and [ 3 H]PGE 1 can be both down and up regulated and is enzyme sensitive. PGE 1 stimulates intracellular cAMP synthesis and accumulation in a time and concentration dependent manner. PGF/sub 2α/ probably exerts its effects through an amiloride-sensitive intermediate. Both PGF/sub 2α/ and PGE 1 are constitutively synthesized by these primary cultures, and they have shown this synthesis to be both drug and hormone sensitive. They hypothesize that it is the ratio, rather than the absolute quantities, of PGF/sub 2α/ and PGE 1 which is of more importance in the regulation of endometrial cell growth. Furthermore, they believe this regulation of endometrial growth plays a role in control of proliferation during the decidual response and that a derangement in the ratio of these prostaglandins may lead to either infertility or hyperplasia. The ability of these cultures to synthesize prostaglandins in a hormonally regulatable manner may be of importance in the study of dysmenorrhea and uterine cramping as caused by the myometrial contracting prostaglandin, PGF/sub 2α/

  13. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Jennifer Pasquier

    2015-06-01

    Full Text Available The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp. The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading, we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

  14. Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control.

    Science.gov (United States)

    Satoh, Tadashi; Toshimori, Takayasu; Noda, Masanori; Uchiyama, Susumu; Kato, Koichi

    2016-11-01

    The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. © 2016 The Protein Society.

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

    Directory of Open Access Journals (Sweden)

    Kélen Fabíola Arroteia

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

  16. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    Energy Technology Data Exchange (ETDEWEB)

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

  17. Registration of DGE-2, a durum wheat disomic alien substitution line 1E(1A) involving a diploid wheatgrass chromosome

    Science.gov (United States)

    The durum wheat (Triticum turgidum L., 2n = 2x = 28; AABB genomes) alien disomic substitution 1E(1A) line DGE-2 (PI 663216) was developed by the USDA–ARS, Cereal Crops Research Unit, Northern Crop Science Laboratory, Fargo, North Dakota and released in 2011. DGE-2 has 2n = 28 chromosomes, which are...

  18. Registration of DGE-3, a durum wheat disomic substitution line 1E(1B) involving a wheatgrass chromosome

    Science.gov (United States)

    Durum wheat (Triticum turgidum L., 2n = 4x = 28; AABB genomes) alien disomic substitution 1E(1B) line DGE-3 (PI 665473) was developed by the U.S. Department of Agriculture – Agricultural Research Service, Northern Crop Science Lab, Cereal Crops Research Unit, Fargo, ND and released in 2012. It was ...

  19. Impairment of Akt activity by CYP2E1 mediated oxidative stress is involved in chronic ethanol-induced fatty liver

    Directory of Open Access Journals (Sweden)

    Tao Zeng

    2018-04-01

    Full Text Available Protein kinase B (PKB/Akt plays important roles in the regulation of lipid homeostasis, and impairment of Akt activity has been demonstrated to be involved in the development of non-alcoholic fatty liver disease (NAFLD. Previous studies suggest that cytochrome P4502E1 (CYP2E1 plays causal roles in the pathogenesis of alcoholic fatty liver (AFL. We hypothesized that Akt activity might be impaired due to CYP2E1-induced oxidative stress in chronic ethanol-induced hepatic steatosis. In this study, we found that chronic ethanol-induced hepatic steatosis was accompanied with reduced phosphorylation of Akt at Thr308 in mice liver. Chronic ethanol exposure had no effects on the protein levels of phosphatidylinositol 3 kinase (PI3K and phosphatase and tensin homologue deleted on chromosome ten (PTEN, and led to a slight decrease of phosphoinositide-dependent protein kinase 1 (PDK-1 protein level. Ethanol exposure resulted in increased levels of malondialdehyde (MDA and 4-hydroxynonenal (4-HNE-Akt adducts, which was significantly inhibited by chlormethiazole (CMZ, an efficient CYP2E1 inhibitor. Interestingly, N-acetyl-L-cysteine (NAC significantly attenuated chronic ethanol-induced hepatic fat accumulation and the decline of Akt phosphorylation at Thr308. In the in vitro studies, Akt phosphorylation was suppressed in CYP2E1-expressing HepG2 (CYP2E1-HepG2 cells compared with the negative control HepG2 (NC-HepG2 cells, and 4-HNE treatment led to significant decrease of Akt phosphorylation at Thr308 in wild type HepG2 cells. Lastly, pharmacological activation of Akt by insulin-like growth factor-1 (IGF-1 significantly alleviated chronic ethanol-induced fatty liver in mice. Collectively, these results indicate that CYP2E1-induced oxidative stress may be responsible for ethanol-induced suppression of Akt phosphorylation and pharmacological modulation of Akt in liver may be an effective strategy for the treatment of ethanol-induced fatty liver. Keywords

  20. CYP2E1 Rsa Ι/Pst Ι polymorphism and lung cancer susceptibility: a meta-analysis involving 10,947 subjects.

    Science.gov (United States)

    Shen, Ze-Tian; Wu, Xin-Hu; Li, Bing; Shen, Jun-shu; Wang, Zhen; Li, Jing; Zhu, Xi-Xu

    2015-09-01

    Many studies have examined the association between the CYP2E1 Rsa Ι/Pst Ι (rs3813867) polymorphism gene polymorphisms and lung cancer risk in various populations, but their results have been inconsistent. The PubMed and CNKI database was searched for case-control studies published up to October 2013. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. In this meta-analysis, we assessed 23 published studies involving comprising 4727 lung cancer cases and 6220 controls of the association between CYP2E1 Rsa Ι/Pst Ι polymorphism and lung cancer risk. For the homozygote c2/c2 and c2 allele carriers (c1/c2 + c2/c2), the pooled ORs for all studies were 0.73(95% CI = 0.62-0.84; P = 0.005 for heterogeneity) and 0.84 (95% CI = 0.77-0.92; P = 0.001 for heterogeneity) when compared with the homozygous wild-type genotype (c1/c1). In the stratified analysis by ethnicity, the same significantly risks were found among Asians and mixed population for both the c2 allele carriers and homozygote c2/c2. However, no significant associations were found in Caucasian population all genetic models. This updated meta-analysis suggests that CYP2E1 Rsa Ι/Pst Ι c2 allele is a decreased risk factor for the developing lung cancer among Asians and mixed population. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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

    Directory of Open Access Journals (Sweden)

    Fun-In Wang

    2015-06-01

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

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

    Science.gov (United States)

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

    2015-06-29

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

  3. A computational approach identifies two regions of Hepatitis C Virus E1 protein as interacting domains involved in viral fusion process

    Directory of Open Access Journals (Sweden)

    El Sawaf Gamal

    2009-07-01

    Full Text Available Abstract Background The E1 protein of Hepatitis C Virus (HCV can be dissected into two distinct hydrophobic regions: a central domain containing an hypothetical fusion peptide (FP, and a C-terminal domain (CT comprising two segments, a pre-anchor and a trans-membrane (TM region. In the currently accepted model of the viral fusion process, the FP and the TM regions are considered to be closely juxtaposed in the post-fusion structure and their physical interaction cannot be excluded. In the present study, we took advantage of the natural sequence variability present among HCV strains to test, by purely sequence-based computational tools, the hypothesis that in this virus the fusion process involves the physical interaction of the FP and CT regions of E1. Results Two computational approaches were applied. The first one is based on the co-evolution paradigm of interacting peptides and consequently on the correlation between the distance matrices generated by the sequence alignment method applied to FP and CT primary structures, respectively. In spite of the relatively low random genetic drift between genotypes, co-evolution analysis of sequences from five HCV genotypes revealed a greater correlation between the FP and CT domains than respect to a control HCV sequence from Core protein, so giving a clear, albeit still inconclusive, support to the physical interaction hypothesis. The second approach relies upon a non-linear signal analysis method widely used in protein science called Recurrence Quantification Analysis (RQA. This method allows for a direct comparison of domains for the presence of common hydrophobicity patterns, on which the physical interaction is based upon. RQA greatly strengthened the reliability of the hypothesis by the scoring of a lot of cross-recurrences between FP and CT peptides hydrophobicity patterning largely outnumbering chance expectations and pointing to putative interaction sites. Intriguingly, mutations in the CT

  4. Cytochrome P450 and P-Glycoprotein-Mediated Interactions Involving African Herbs Indicated for Common Noncommunicable Diseases

    Directory of Open Access Journals (Sweden)

    Gregory Ondieki

    2017-01-01

    Full Text Available Herbal remedies are regularly used to complement conventional therapies in the treatment of various illnesses in Africa. This may be because they are relatively cheap and easily accessible and are believed by many to be safe, cause fewer side effects, and are less likely to cause dependency. On the contrary, many herbs have been shown to alter the pharmacokinetics of coadministered allopathic medicines and can either synergize or antagonize therapeutic effects as well as altering the toxicity profiles of these drugs. Current disease burden data point towards epidemiological transitions characterised by increasing urbanization and changing lifestyles, risk factors for chronic diseases like hypertension, diabetes, and cancer which often present as multimorbidities. As a result, we highlight African herb-drug interactions (HDIs modulated via cytochrome P450 enzyme family (CYP and P-glycoprotein (P-gp and the consequences thereof in relation to antihypertensive, antidiabetic, and anticancer drugs. CYPs are enzymes which account for to up to 70% of drug metabolism while P-gp is an efflux pump that extrudes drug substrates out of cells. Consequently, regulation of the relative activity of both CYP and P-gp by African herbs influences the effective drug concentration at the site of action and modifies therapeutic outcomes.

  5. Cytochrome P450-2E1 is involved in aging-related kidney damage in mice through increased nitroxidative stress.

    Science.gov (United States)

    Abdelmegeed, Mohamed A; Choi, Youngshim; Ha, Seung-Kwoon; Song, Byoung-Joon

    2017-11-01

    The aim of this study was to investigate the role of cytochrome P450-2E1 (CYP2E1) in aging-dependent kidney damage since it is poorly understood. Young (7 weeks) and aged female (16-17 months old) wild-type (WT) and Cyp2e1-null mice were used. Kidney histology showed that aged WT mice exhibited typical signs of kidney aging such as cell vacuolation, inflammatory cell infiltration, cellular apoptosis, glomerulonephropathy, and fibrosis, along with significantly elevated levels of renal TNF-α and serum creatinine than all other groups. Furthermore, the highest levels of renal hydrogen peroxide, protein carbonylation and nitration were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of iNOS and mitochondrial nitroxidative stress through altered amounts and activities of the mitochondrial complex proteins and significantly reduced levels of the antioxidant glutathione (GSH). In contrast, the aged Cyp2e1-null mice exhibited significantly higher antioxidant capacity with elevated heme oxygenase-1 and catalase activities compared to all other groups, while maintaining normal GSH levels with significantly less mitochondrial nitroxidative stress compared to the aged WT mice. Thus, CYP2E1 is important in causing aging-related kidney damage most likely through increasing nitroxidative stress and that CYP2E1 could be a potential target in preventing aging-related kidney diseases. Published by Elsevier Ltd.

  6. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties.

    Directory of Open Access Journals (Sweden)

    Elodie Beaumont

    Full Text Available Various strategies involving the use of hepatitis C virus (HCV E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.

  7. The E1 proteins

    International Nuclear Information System (INIS)

    Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

    2013-01-01

    E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. - Highlights: • The papillomavirus E1 helicase orchestrates replication of the viral DNA genome. • E1 assembles into a double-hexamer at the viral origin with the help of E2. • E1 interacts with cellular DNA replication factors. • E1 unwinds DNA using a spiral escalator mechanism. • Nuclear accumulation of E1 is regulated by post-translational modifications

  8. 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.

  9. Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

    International Nuclear Information System (INIS)

    Petit, Chad M.; Melancon, Jeffrey M.; Chouljenko, Vladimir N.; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G.

    2005-01-01

    The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion

  10. Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ib alpha and alpha(IIb)beta(3)

    NARCIS (Netherlands)

    De Haas, C. J. C.; Weeterings, C.; Vughs, M. M.; De Groot, P. G.; Van Strijp, J. A.; Lisman, T.

    2009-01-01

    Objectives: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Methods and Results: When purified

  11. THE E1 PROTEINS

    OpenAIRE

    Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

    2013-01-01

    E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA usi...

  12. Characterization of a 105-kDa plasma membrane associated glycoprotein that is involved in West Nile virus binding and infection

    International Nuclear Information System (INIS)

    Chu, J.J.H.; Ng, M.L.

    2003-01-01

    This study attempts to isolate and characterize West Nile virus-binding molecules on the plasma membrane of Vero and murine neuroblastoma cells that is responsible for virus entry. Pretreatment of Vero cells with proteases, glycosidases (endoglycosidase H, α-mannosidase), and sodium periodate strongly inhibited West Nile virus infection, whereas treatments with phospholipases and heparinases had no effect. The virus overlay protein blot detected a 105-kDa molecule on the plasma membrane extract of Vero and murine neuroblastoma cells that bind to WN virus. Treatment of the 105-kDa molecules with β-mercaptoethanol resulted in the virus binding to a series of lower molecular weight bands ranging from 30 to 40 kDa. The disruption of disulfide-linked subunits did not affect virus binding. N-linked sugars with mannose residues on the 105-kDa membrane proteins were found to be important in virus binding. Specific antibodies against the 105-kDa glycoprotein were highly effective in blocking virus entry. These results strongly supported the possibility that the 105-kDa protease-sensitive glycoprotein with complex N-linked sugars could be the putative receptor for WN virus

  13. Glycoprotein on cell surfaces

    International Nuclear Information System (INIS)

    Muramatsu, T.

    1975-01-01

    There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

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

    Directory of Open Access Journals (Sweden)

    Kamel El Omari

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  16. Neutralization of feline immunodeficiency virus by polyclonal cat antibody: Simultaneous involvement of hypervariable regions 4 and 5 of the surface glycoprotein.

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees); W. Huisman (Willem); J.A. Karlas (Jos); G.F. Rimmelzwaan (Guus); M.L. Bosch (Marnix); A.D.M.E. Osterhaus (Albert)

    1995-01-01

    textabstractSites involved in antibody-mediated neutralization of feline immunodeficiency virus were mapped by reciprocal exchange of envelope fragments or amino acids between molecular clones of feline immunodeficiency virus with different susceptibilities to neutralization by a polyclonal cat

  17. Glycoprotein biosynthesis by human normal platelets

    International Nuclear Information System (INIS)

    Rodriguez, P.; Bello, O.; Apitz-Castro, R.

    1987-01-01

    Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation

  18. HIV-1 envelope glycoprotein

    Science.gov (United States)

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

    2017-08-22

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

  19. Glycoprotein and proteoglycan techniques

    International Nuclear Information System (INIS)

    Beeley, J.G.

    1985-01-01

    The aim of this book is to describe techniques which can be used to answer some of the basic questions about glycosylated proteins. Methods are discussed for isolation, compositional analysis, and for determination of the primary structure of carbohydrate units and the nature of protein-carbohydrate linkages of glycoproteins and proteoglycans. High resolution NMR is considered, as well as radioactive labelling techniques. (Auth.)

  20. Polydatin Protects Rat Liver against Ethanol-Induced Injury: Involvement of CYP2E1/ROS/Nrf2 and TLR4/NF-κB p65 Pathway

    Directory of Open Access Journals (Sweden)

    Qiong-Hui Huang

    2017-01-01

    Full Text Available Excessive alcohol consumption leads to serious liver injury, associating with oxidative stress and inflammatory response. Previous study has demonstrated that polydatin (PD exerted antioxidant and anti-inflammatory effects and attenuated ethanol-induced liver damage, but the research remained insufficient. Hence, this experiment aimed to evaluate the hepatoprotective effect and potential mechanisms of PD on ethanol-induced hepatotoxicity. Our results showed that PD pretreatment dramatically decreased the levels of alanine aminotransferase (ALT, aspartate aminotransferase (AST, alkaline phosphatase (ALP, and lactate dehydrogenase (LDH in the serum, suppressed the malonaldehyde (MDA and triglyceride (TG content and the production of reactive oxygen species (ROS, and enhanced the activities of superoxide dismutase (SOD, glutathione peroxidase (GSH-Px, catalase (CAT, andalcohol dehydrogenase (ADH, and aldehyde dehydrogenase (ALDH, paralleled by an improvement of histopathology alterations. The protective effect of PD against oxidative stress was probably associated with downregulation of cytochrome P450 2E1 (CYP2E1 and upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2 and its target gene haem oxygenase-1 (HO-1. Moreover, PD inhibited the release of proinflammatory cytokines (TNF-α, IL-1β, and IL-6 via downregulating toll-like receptor 4 (TLR4 and nuclear factor kappa B (NF-κB p65. To conclude, PD pretreatment protects against ethanol-induced liver injury via suppressing oxidative stress and inflammation.

  1. Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs.

    Science.gov (United States)

    Hook, Lauren M; Cairns, Tina M; Awasthi, Sita; Brooks, Benjamin D; Ditto, Noah T; Eisenberg, Roselyn J; Cohen, Gary H; Friedman, Harvey M

    2018-05-01

    Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to

  2. Involvement of P-glycoprotein and multidrug resistance associated protein 1 in the transport of tanshinone IIB, a primary active diterpenoid quinone from the roots of Salvia miltiorrhiza, across the blood-brain barrier.

    Science.gov (United States)

    Zhou, Zhi-Wei; Chen, Xiao; Liang, Jun; Yu, Xi-Yong; Wen, Jing-Yuan; Zhou, Shu-Feng

    2007-08-01

    Tanshinone IIB (TSB) is a major constituent of Salvia miltiorrhiza, which is widely used in treatment of cardiovascular and central nervous system (CNS) diseases such as coronary heart disease and stroke. This study aimed to investigate the role of various drug transporters in the brain penetration of TSB using several in vitro and in vivo mouse and rat models. The uptake and efflux of TSB in rat primary microvascular endothelial cells (RBMVECs) were ATP-dependent and significantly altered in the presence of a P-glycoprotein (P-gp) or multidrug resistance associated protein (Mrp1/2) inhibitor. A polarized transport of TSB was found in RBMVEC monolayers with facilitated efflux from the abluminal to luminal side. Addition of a P-gp inhibitor (e.g. verapamil) in both abluminal and luminal sides attenuated the polarized transport. In an in situ rat brain perfusion model, TSB crossed the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier at a greater rate than that for sucrose, and the brain penetration was increased in the presence of a P-gp or Mrp1/2 inhibitor. The brain levels of TSB were only about 30% of that in the plasma and it could be increased to up to 72% of plasma levels when verapamil, quinidine, or probenecid was co-administered in rats. The entry of TSB to CNS increased by 67-97% in rats subjected to middle cerebral artery occlusion or treatment with the neurotoxin, quinolinic acid, compared to normal rats. Furthermore, The brain levels of TSB in mdr1a(-/-) and mrp1(-/-) mice were 28- to 2.6-fold higher than those in the wild-type mice. TSB has limited brain penetration through the BBB due to the contribution of P-gp and to a lesser extent of Mrp1 in rodents. Further studies are needed to confirm whether these corresponding transporters in humans are involved in limiting the penetration of TSB across the BBB and the clinical relevance.

  3. Multiple efflux pumps are involved in the transepithelial transport of colchicine: combined effect of p-glycoprotein and multidrug resistance-associated protein 2 leads to decreased intestinal absorption throughout the entire small intestine.

    Science.gov (United States)

    Dahan, Arik; Sabit, Hairat; Amidon, Gordon L

    2009-10-01

    The purpose of this study was to thoroughly characterize the efflux transporters involved in the intestinal permeability of the oral microtubule polymerization inhibitor colchicine and to evaluate the role of these transporters in limiting its oral absorption. The effects of P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on colchicine bidirectional permeability were studied across Caco-2 cell monolayers, inhibiting one versus multiple transporters simultaneously. Colchicine permeability was then investigated in different regions of the rat small intestine by in situ single-pass perfusion. Correlation with the P-gp/MRP2 expression level throughout different intestinal segments was investigated by immunoblotting. P-gp inhibitors [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), verapamil, and quinidine], and MRP2 inhibitors [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), indomethacin, and p-aminohippuric acid (p-AH)] significantly increased apical (AP)-basolateral (BL) and decreased BL-AP Caco-2 transport in a concentration-dependent manner. No effect was obtained by the BCRP inhibitors fumitremorgin C (FTC) and pantoprazole. P-gp/MRP2 inhibitors combinations greatly reduced colchicine mucosal secretion, including complete abolishment of efflux (GF120918/MK571). Colchicine displayed low (versus metoprolol) and constant permeability along the rat small-intestine. GF120918 significantly increased colchicine permeability in the ileum with no effect in the jejunum, whereas MK571 augmented jejunal permeability without changing the ileal transport. The GF120918/MK571 combination caused an effect similar to that of MK571 alone in the jejunum and to that of GF120918 alone in the ileum. P-gp expression followed a gradient increasing from

  4. Fluid shear stress suppresses TNF-α-induced apoptosis in MC3T3-E1 cells: Involvement of ERK5-AKT-FoxO3a-Bim/FasL signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Bin, Geng; Bo, Zhang; Jing, Wang; Jin, Jiang; Xiaoyi, Tan; Cong, Chen; Liping, An; Jinglin, Ma; Cuifang, Wang; Yonggang, Chen [The Second Hospital of Lanzhou University, #82 Cuiyingmen, Lanzhou, 730000 Gansu (China); Orthopaedics Key Laboratory of Gansu Province, Lanzhou, 730000 Gansu (China); Yayi, Xia, E-mail: xiayayildey@163.com [The Second Hospital of Lanzhou University, #82 Cuiyingmen, Lanzhou, 730000 Gansu (China); Orthopaedics Key Laboratory of Gansu Province, Lanzhou, 730000 Gansu (China)

    2016-05-01

    TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis. - Highlights: • Fluid shear stress inhibits osteoblast apoptosis induced by TNF-α. • Inhibition of ERK5 activity by transfection of ERK5 siRNA blocks FSS-mediated anti-apoptotic effect in osteoblast. • Activated ERK5-AKT-FoxO3a-Bim/FasL signaling pathways by FSS is required to protect osteoblast from apoptosis.

  5. Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth

    International Nuclear Information System (INIS)

    Quackenbush, E.; Clabby, M.; Gottesdiener, K.M.; Barbosa, J.; Jones, N.H.; Strominger, J.L.; Speck, S.; Leiden, J.M.

    1987-01-01

    Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a λgt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggest that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution

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

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

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

  7. The hydroxyapatite-binding regions of a rat salivary glycoprotein.

    Science.gov (United States)

    Embery, G; Green, D R

    1989-09-01

    The regions of a salivary sulphated glycoprotein which are involved in its attachment to hydroxyapatite (Biogel HTP) have been characterised. The sulphated glycoprotein, a 35S-labelled preparation from mixed palatal and buccal minor gland secretions of the rat was bound onto hydroxyapatite and the resultant glycoprotein-hydroxyapatite complex was sequentially digested with pronase E and alpha-L-fucosidase, a treatment which released 86.8% +/- 1.7% of the radioactivity of the initially bound glycoprotein. The fragments which remained attached to the hydroxyapatite after enzymic digestion were fractionated on Sephadex G-25 and analysed for carbohydrate and amino acid components. A range of amino acids were detected which could reflect both glycosylated and non-glycosylated-binding regions. Sialic acid, although considered to be involved in the attachment process was not detected in any of the fragments remaining after enzymic digestion, a finding which provides indirect evidence that the enzymically liberated products do not subsequently re-attach to the hydroxyapatite surface. The notable feature of the fractions with average Mr estimated at 1000 or less is the high proportion of N-acetylhexosamine and N-acetylgalactosamine. It is apparent that the hexosamine residues, which normally bear the ester sulphate moieties of sulphated glycoproteins, play an important role in the attachment of sulphated glycoproteins to hydroxyapatite.

  8. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    NARCIS (Netherlands)

    Metz, S.W.H.; Geertsema, C.; Martina, Byron E.; Andrade, Paulina; Heldens, J.; Oers, van M.M.; Goldbach, R.W.; Vlak, J.M.; Pijlman, G.P.

    2011-01-01

    Background - Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of

  9. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    NARCIS (Netherlands)

    S.W. Metz (Stefan); C. Geertsema (Corinne); B.E.E. Martina (Byron); P. Andrade (Paulina); J.G.M. Heldens; M.M. van Oers (Monique); J.M. Vlak (Just); G.P. Pijlman (Gorben)

    2011-01-01

    textabstractBackground: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the

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

    Science.gov (United States)

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

    2018-03-01

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

  11. Salivary Mucin 19 Glycoproteins

    Science.gov (United States)

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

    2015-01-01

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

  12. The glycoprotein of measles virus

    International Nuclear Information System (INIS)

    Anttonen, O.; Jokinen, M.; Salmi, A.; Vainionpaeae, R.; Gahmberg, C.G.

    1980-01-01

    Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3 H after treatment with galactose oxidase/NaB 3 H 4 or with [ 3 H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79000. After labelling with periodate/NaB 3 H 4 , which would result in specific labelling of sialic acid residues, the 79000-mol.wt. glycoprotein was very weakly labelled. This suggested that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage. (author)

  13. Arterial portography using prostaglandin E1

    International Nuclear Information System (INIS)

    Seo, Heung Suk; Kim, Hyung Sik; Lee, Seung Chul; Lee, Seung Ro; Hahm, Chang Kok; Kim, Jung Jin; Cho, Suk Shin

    1987-01-01

    A total of 110 arterial portographies via superior mesenteric artery were performed on 100 patients at Hanyang University Hospital in the past 2 years. There were 20 control portographies and 90 portographies using prostaglandin E 1 Twenty μg prostaglandin E 1 was injected for 30 seconds in the superior mesenteric artery 30 seconds before injection of contrast media. Both control and prostaglandin E 1 portograms were evaluated for quality of opacification and side effects of prostaglandin E 1 were recorded. The results were as follows; 1.The appearance time and optimal opacification time of the portal vein system were obtained approximately 6 seconds earlier in the prostaglandin E 1 portograms than in the control portograms. 2.The incidence of opacification of the intrahepatic portal veins was greater in the prostaglandin E 1 portograms than in the control portograms. 3.The main portal vein and intrahepatic portal veins were more clearly opacified in the prostaglandin... portograms than in the control portograms. 4.The prostaglandin E 1 portograms provided clearer and more detailed opacification of the portal vein system than the control portograms in the same patients. 5.There was a minimal decrease in blood pressure with a concomitant small rise in heart rate and mild abdominal pain following the prostaglandin E 1 injection. The authors found arterial portography using prostaglandin E 1 simple, safe and useful for clear and detailed visualization of the portal vein system

  14. Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins

    OpenAIRE

    Buonocore, Linda; Blight, Keril J.; Rice, Charles M.; Rose, John K.

    2002-01-01

    We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein...

  15. Recent Progress in Electrochemical Biosensors for Glycoproteins

    Directory of Open Access Journals (Sweden)

    Uichi Akiba

    2016-12-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

  17. Secretion of hepatitis C virus envelope glycoproteins depends on assembly of apolipoprotein B positive lipoproteins.

    Directory of Open Access Journals (Sweden)

    Vinca Icard

    Full Text Available The density of circulating hepatitis C virus (HCV particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB positive and triglyceride rich lipoproteins (TRL likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1-E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.

  18. Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

    Science.gov (United States)

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

    2015-01-01

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

  19. The Implementation of E1 Clock Recovery

    Directory of Open Access Journals (Sweden)

    Wang Ziyu

    2016-01-01

    Full Text Available Clock transform and recovery is of significant importance in microwave TDM service, and it is always extracted from the E1 line data stream in most cases. However, intrinsically uncertain delay and jitter caused by packet transmission of E1 data information, may lead to the indexes of the data recovery clock exceed the clock performance template. Through analysis of the E1 clock indexes and measuring methods, this paper proposes a new clock recovery method. The method employs two buffers, the first RAM is used as a buffer to deduct excess information, and the second FIFO is used as a buffer to recovery the clock and data. The first buffer has a feedback from the second one, and is able to actively respond to changes in the data link and requests from the second one. The test results validate the effectiveness of the method, and the corresponding scheme is also valuable for the other communication systems.

  20. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

    OpenAIRE

    Kowalewski-Nimmerfall, Elisabeth; Sch?hs, Philipp; Maresch, Daniel; Rendic, Dubravko; Kr?mer, Helmut; Mach, Lukas

    2014-01-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be subst...

  1. Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

    Science.gov (United States)

    Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

    2008-01-01

    Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

  2. 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.

  3. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-26

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

  4. Features of the giant E1 resonance

    International Nuclear Information System (INIS)

    Bergere, R.

    1976-01-01

    Since most of the available experimental data concerning the giant dipole E1 resonance (GDR) have been obtained with real photons, the characteristics of real photon sources are reviewed with an attempt to connect the experimental particularities of each of them to the specific parameters of the GDR which it is best suited to reach. Some systematic properties gathered from experimental data of GDR (average energy, splitting and broadening of the GDR) are compared with the predictions of the static and dynamic collective models of the nuclei. The position in energy and the fine structure of the GDR are more closely connected to shell model predictions as nuclei get lighter, the various experimental integrated cross sections being also more easily understood by comparisons with microscopic models. Most of the reported data refer to the doorway state through which GDR is excited, however the competition between the decay channels for GDR states is also emphasized

  5. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

    Science.gov (United States)

    Song, Ehwang; Mechref, Yehia

    2015-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers.

  6. Nipah virus infection and glycoprotein targeting in endothelial cells

    Directory of Open Access Journals (Sweden)

    Maisner Andrea

    2010-11-01

    Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

  7. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    Directory of Open Access Journals (Sweden)

    Natascha Krömmelbein

    2016-02-01

    Full Text Available The human cytomegalovirus (HCMV replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production.

  8. 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

  9. Glycoprotein Enrichment Analytical Techniques: Advantages and Disadvantages.

    Science.gov (United States)

    Zhu, R; Zacharias, L; Wooding, K M; Peng, W; Mechref, Y

    2017-01-01

    Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were developed in recent years. Enrichment methods were used to improve the sensitivity of detection, while HPLC and mass spectrometry methods were developed to facilitate the separation of glycopeptides/proteins and enhance detection, respectively. Fragmentation techniques applied in modern mass spectrometers allow the structural interpretation of glycopeptides/proteins, while automated software tools started replacing manual processing to improve the reliability and throughput of the analysis. In this chapter, the current methodologies of glycoprotein analysis were discussed. Multiple analytical techniques are compared, and advantages and disadvantages of each technique are highlighted. © 2017 Elsevier Inc. All rights reserved.

  10. Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates

    DEFF Research Database (Denmark)

    Abraham, E H; Shrivastav, B; Salikhova, A Y

    2001-01-01

    P-glycoprotein is involved with the removal of drugs, most of them cations, from the plasma membrane and cytoplasm. Pgp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central question of this study is whether drug and ATP transport associated...

  11. Human CYP2E1 mediates the formation of glycidamide from acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Settels, Eva; Appel, Klaus E. [Federal Institute for Risk Assessment, Center for Experimental Toxicology, Berlin (Germany); Bernauer, Ulrike; Gundert-Remy, Ursula [Federal Institute for Risk Assessment, Department of Safety of Substances and Preparations, Berlin (Germany); Palavinskas, Richard; Klaffke, Horst S. [Federal Institute for Risk Assessment, Center for Analytical Chemistry, Berlin (Germany)

    2008-10-15

    Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes trademark, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes trademark, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes trademark; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species. (orig.)

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

    DEFF Research Database (Denmark)

    Yang, Zhang; Wang, Shengjun; Halim, Adnan

    2015-01-01

    Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferas...

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

    Science.gov (United States)

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

    2017-11-29

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

  14. Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction

    Directory of Open Access Journals (Sweden)

    Arthur I. Cederbaum

    2014-01-01

    Full Text Available The cytochrome P450 mixed function oxidase enzymes are the major catalysts involved in drug metabolism. There are many forms of P450. CYP2E1 metabolizes many toxicologically important compounds including ethanol and is active in generating reactive oxygen species. Since several of the contributions in the common theme series “Role of CYP2E1 and Oxidative/Nitrosative Stress in the Hepatotoxic Actions of Alcohol” discuss CYP2E1, this methodology review describes assays on how CYP2E1 catalytic activity and its induction by ethanol and other inducers can be measured using substrate probes such as the oxidation of para-nitrophenol to para-nitrocatechol and the oxidation of ethanol to acetaldehyde. Approaches to validate that a particular reaction e.g. oxidation of a drug or toxin is catalyzed by CYP2E1 or that induction of that reaction is due to induction of CYP2E1 are important and specific examples using inhibitors of CYP2E1, anti-CYP2E1 IgG or CYP2E1 knockout and knockin mice will be discussed.

  15. The pestivirus Erns glycoprotein interacts with E2 in both infected cells and mature virions

    International Nuclear Information System (INIS)

    Lazar, Catalin; Zitzmann, Nicole; Dwek, Raymond A.; Branza-Nichita, Norica

    2003-01-01

    E rns is a pestivirus envelope glycoprotein indispensable for virus attachment and infection of target cells. Unlike the other two envelope proteins E1 and E2, E rns lacks a transmembrane domain and a vast quantity is secreted into the medium of infected cells. The protein is also present in fractions of pure pestivirus virions, raising the important and intriguing question regarding the mechanism of its attachment to the pestivirus envelope. In this study a direct interaction between E rns and E2 glycoproteins was demonstrated in both pestivirus-infected cells and mature virions. By co- and sequential immunoprecipitation we showed that an E rns -E2 heterodimer is assembled very early after translation of the viral polyprotein and before its processing is completed. Our results suggest that E rns is attached to the pestivirus envelope via a direct interaction with E2 and explain the role of E rns in the initial virus-target cell interaction

  16. E1AF degradation by a ubiquitin-proteasome pathway

    International Nuclear Information System (INIS)

    Takahashi, Akiko; Higashino, Fumihiro; Aoyagi, Mariko; Yoshida, Koichi; Itoh, Miyuki; Kobayashi, Masanobu; Totsuka, Yasunori; Kohgo, Takao; Shindoh, Masanobu

    2005-01-01

    E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway, which has some effect on E1AF function

  17. Isolation of glycoproteins from brown algae

    DEFF Research Database (Denmark)

    2015-01-01

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

  18. 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

  19. Platelet Glycoprotein Ib-IX and Malignancy

    Science.gov (United States)

    2010-09-01

    provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

  20. Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

    International Nuclear Information System (INIS)

    Schaehs, Philipp; Weidinger, Petra; Probst, Olivia C.; Svoboda, Barbara; Stadlmann, Johannes; Beug, Hartmut; Waerner, Thomas; Mach, Lukas

    2008-01-01

    Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes

  1. Identification of a mouse synaptic glycoprotein gene in cultured neurons.

    Science.gov (United States)

    Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

    2005-10-01

    Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Backovic, Marija [Northwestern Univ., Evanston, IL (United States); Longnecker, Richard [Northwestern Univ., Chicago, IL (United States); Jardetzky, Theodore S [Northwestern Univ., Evanston, IL (United States)

    2009-03-16

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

  3. IgA antibodies against β2 glycoprotein I in hemodialysis patients are an independent risk factor for mortality.

    Science.gov (United States)

    Serrano, Antonio; García, Florencio; Serrano, Manuel; Ramírez, Elisa; Alfaro, F Javier; Lora, David; de la Cámara, Agustín Gómez; Paz-Artal, Estela; Praga, Manuel; Morales, Jose M

    2012-06-01

    Cardiovascular complications are the most important cause of death in patients on dialysis with end-stage renal disease. Antibodies reacting with β-glycoprotein I seem to play a pathogenic role in antiphospholipid syndrome and stroke and are involved in the origin of atherosclerosis. Here we evaluated the presence of anticardiolipin and anti-β-glycoprotein I antibodies together with other vascular risk factors and their relationship with mortality and cardiovascular morbidity in a cohort of 124 hemodialysis patients prospectively followed for 2 years. Of these, 41 patients were significantly positive for IgA anti-β-glycoprotein I, and the remaining had normal values. At 24 months, overall and cardiovascular mortality and thrombotic events were all significantly higher in patients with high anti-β-glycoprotein I antibodies. Multivariate analysis using Cox regression modeling found that age, hypoalbuminemia, use of dialysis catheters, and IgA β-glycoprotein I antibodies were independent risk factors for death. Thus, IgA antibodies to β-glycoprotein I are detrimental to the clinical outcome of hemodialysis patients.

  4. A Diverse Panel of Hepatitis C Virus Glycoproteins for Use in Vaccine Research Reveals Extremes of Monoclonal Antibody Neutralization Resistance.

    Science.gov (United States)

    Urbanowicz, Richard A; McClure, C Patrick; Brown, Richard J P; Tsoleridis, Theocharis; Persson, Mats A A; Krey, Thomas; Irving, William L; Ball, Jonathan K; Tarr, Alexander W

    2015-12-23

    Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies. Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral therapies. A safe and

  5. Interaction of E2 glycoprotein with heparan sulfate is crucial for cellular infection of Sindbis virus.

    Directory of Open Access Journals (Sweden)

    Wuyang Zhu

    Full Text Available Cell culture-adapted strains of Sindbis virus (SINV initially attach to cells by the ability to interact with heparan sulfate (HS through selective mutation for positively charged amino acid (aa scattered in E2 glycoprotein (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72: 7357-7366, 1998. Here we have further confirmed that interaction of E2 protein with HS is crucial for cellular infection of SINV based on the reverse genetic system of XJ-160 virus, a Sindbis-like virus (SINLV. Both SINV YN87448 and SINLV XJ-160 displayed similar infectivity on BHK-21, Vero, or C6/36 cells, but XJ-160 failed to infect mouse embryonic fibroblast (MEF cells. The molecular mechanisms underlying the selective infectivity of XJ-160 were approached by substituting the E1, E2, or both genes of XJ-160 with that of YN87448, and the chimeric virus was denominated as XJ-160/E1, XJ-160/E2, or XJ-160/E1E2, respectively. In contrast to the parental XJ-160, all chimeric viruses became infectious to wild-type MEF cells (MEF-wt. While MEF-Ext(-/- cells, producing shortened HS chains, were resistant not only to XJ-160, but also to YN87448 as well as the chimeric viruses, indicating that the inability of XJ-160 to infect MEF-wt cells likely due to its incompetent discrimination of cellular HS. Treatment with heparin or HS-degrading enzyme resulted in a substantial decrease in plaque formation by YN87448, XJ-160/E2, and XJ-160/E1E2, but had marginal effect on XJ-160 and XJ-160/E1, suggesting that E2 glycoprotein from YN87448 plays a more important role than does E1 in mediating cellular HS-related cell infection. In addition, the peptide containing 145-150 aa from E2 gene of YN87448 specifically bound to heparin, while the corresponding peptide from the E2 gene of XJ-160 essentially showed no binding to heparin. As a new dataset, these results clearly confirm an essential role of E2 glycoprotein, especially the domain of 145-150 aa, in SINV cellular infection

  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. Labelled antibody techniques in glycoprotein estimation

    International Nuclear Information System (INIS)

    Hazra, D.K.; Ekins, R.P.; Edwards, R.; Williams, E.S.

    1977-01-01

    The problems in the radioimmunoassay of the glycoprotein hormones (pituitary LH, FSH and TSH and human chlorionic gonadotrophin HGG) are reviewed viz: limited specificity and sensitivity in the clinical context, interpretation of disparity between bioassay and radioimmunoassay, and interlaboratory variability. The advantages and limitations of the labelled antibody techniques - classical immonoradiometric methods and 2-site or 125 I-anti-IgG indirect labelling modifications are reviewed in general, and their theoretical potential in glycoprotein assays examined in the light of previous work. Preliminary experiments in the development of coated tube 2-site assay for glycoproteins using 125 I anti-IgG labelling are described, including conditions for maximizing solid phase extraction of the antigen, iodination of anti-IgG, and assay conditions such as effects of temperature of incubation with antigen 'hormonefree serum', heterologous serum and detergent washing. Experiments with extraction and antigen-specific antisera raised in the same or different species are described as exemplified by LH and TSH assay systems, the latter apparently promising greater sensitivity than radioimmunoassay. Proposed experimental and mathematical optimisation and validation of the method as an assay system is outlined, and the areas for further work delineated. (orig.) [de

  8. C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

    International Nuclear Information System (INIS)

    Tzeng, W.-P.; Frey, Teryl K.

    2006-01-01

    Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles

  9. CYP 2E1 mutant mice are resistant to DDC-induced enhancement of MPTP toxicity.

    Science.gov (United States)

    Viaggi, C; Vaglini, F; Pardini, C; Sgadò, P; Caramelli, A; Corsini, G U

    2007-01-01

    In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, we showed that CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. Furthermore we showed that CYP 2E1 is present in the brain and in the basal ganglia of mice (Vaglini et al., 2004). However, because DAS and PIC are not selective CYP 2E1 inhibitors and in order to provide direct evidence for CYP 2E1 involvement in the enhancement of MPTP toxicity, CYP 2E1 knockout mice (GONZ) and wild type animals (SVI) of the same genetic background were treated with MPTP or the combined DDC + MPTP treatment. In CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity, although enhancement of MPTP toxicity was regularly present in the SVI control animals. The immunohistochemical study confirms our results and suggests that CYP 2E1 may have a detoxifying role.

  10. Dimers of beta 2-glycoprotein I mimic the in vitro effects of beta 2-glycoprotein I-anti-beta 2-glycoprotein I antibody complexes

    NARCIS (Netherlands)

    Lutters, B. C.; Meijers, J. C.; Derksen, R. H.; Arnout, J.; de Groot, P. G.

    2001-01-01

    Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a

  11. The nuclear receptor NR2E1/TLX controls senescence

    Science.gov (United States)

    Krusche, Benjamin; Pemberton, Helen; Alonso, Marta M.; Chandler, Hollie; Brookes, Sharon; Parrinello, Simona; Peters, Gordon; Gil, Jesús

    2014-01-01

    The nuclear receptor NR2E1 (also known as TLX or tailless) controls the self-renewal of neural stem cells (NSCs) and has been implied as an oncogene which initiates brain tumours including glioblastomas. Despite NR2E1 regulating targets like p21CIP1 or PTEN we still lack a full explanation for its role in NSC self-renewal and tumorigenesis. We know that Polycomb repressive complexes (PRC) also control stem cell self-renewal and tumorigenesis, but so far, no formal connection has been established between NR2E1 and PRCs. In a screen for transcription factors regulating the expression of the Polycomb protein CBX7, we identified NR2E1 as one of its more prominent regulators. NR2E1 binds at the CBX7 promoter, inducing its expression. Notably CBX7 represses NR2E1 as part of a regulatory loop. Ectopic NR2E1 expression inhibits cellular senescence, extending cellular lifespan in fibroblasts via CBX7-mediated regulation of p16INK4a and direct repression of p21CIP1. In addition NR2E1 expression also counteracts oncogene-induced senescence (OIS). The importance of NR2E1 to restrain senescence is highlighted through the process of knocking down its expression, which causes premature senescence in human fibroblasts and epithelial cells. We also confirmed that NR2E1 regulates CBX7 and restrains senescence in NSCs. Finally, we observed that the expression of NR2E1 directly correlates with that of CBX7 in human glioblastoma multiforme. Overall we identified control of senescence and regulation of Polycomb action as two possible mechanisms that can join those so far invoked to explain the role of NR2E1 in control of NSC self-renewal and cancer. PMID:25328137

  12. The nuclear receptor NR2E1/TLX controls senescence.

    Science.gov (United States)

    O'Loghlen, Ana; Martin, Nadine; Krusche, Benjamin; Pemberton, Helen; Alonso, Marta M; Chandler, Hollie; Brookes, Sharon; Parrinello, Simona; Peters, Gordon; Gil, Jesús

    2015-07-30

    The nuclear receptor NR2E1 (also known as TLX or tailless) controls the self-renewal of neural stem cells (NSCs) and has been implied as an oncogene which initiates brain tumors including glioblastomas. Despite NR2E1 regulating targets like p21(CIP1) or PTEN we still lack a full explanation for its role in NSC self-renewal and tumorigenesis. We know that polycomb repressive complexes also control stem cell self-renewal and tumorigenesis, but so far, no formal connection has been established between NR2E1 and PRCs. In a screen for transcription factors regulating the expression of the polycomb protein CBX7, we identified NR2E1 as one of its more prominent regulators. NR2E1 binds at the CBX7 promoter, inducing its expression. Notably CBX7 represses NR2E1 as part of a regulatory loop. Ectopic NR2E1 expression inhibits cellular senescence, extending cellular lifespan in fibroblasts via CBX7-mediated regulation of p16(INK4a) and direct repression of p21(CIP1). In addition NR2E1 expression also counteracts oncogene-induced senescence. The importance of NR2E1 to restrain senescence is highlighted through the process of knocking down its expression, which causes premature senescence in human fibroblasts and epithelial cells. We also confirmed that NR2E1 regulates CBX7 and restrains senescence in NSCs. Finally, we observed that the expression of NR2E1 directly correlates with that of CBX7 in human glioblastoma multiforme. Overall we identified control of senescence and regulation of polycomb action as two possible mechanisms that can join those so far invoked to explain the role of NR2E1 in control of NSC self-renewal and cancer.

  13. Cell wall O-glycoproteins and N-glycoproteins: biosynthesis and some functional aspects.

    Directory of Open Access Journals (Sweden)

    Eric eNguema-Ona

    2014-10-01

    Full Text Available Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensin, the O-glycan chains of arabinogalactan proteins are highly heterogeneous consisting mostly of (i a short oligo-arabinoside chain of three to four residues, and (ii a larger -1,3-linked galactan backbone with -1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core 1,2-xylose, core 1,3-fucose residues and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins only extensin and arabinogalactan proteins are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review.

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

    Science.gov (United States)

    Gardner, Thomas J.

    2016-01-01

    SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

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

    Directory of Open Access Journals (Sweden)

    Yannan Qin

    2014-11-01

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

  16. Isolation of allergenically active glycoprotein from Prosopis juliflora pollen.

    Science.gov (United States)

    Thakur, I S

    1989-03-01

    An allergenically active glycoprotein was homogeneously isolated from the aqueous extract of Prosopis juliflora pollen by ConA-Sepharose affinity chromatography. The molecular weight of this glycoprotein was 20,000 dalton, determined by gel filtration and SDS-PAGE. This fraction showed a total carbohydrate concentration of 25%. The purified glycoprotein revealed immunochemically most antigenic or allergenic and demonstrated homogeneous after reaction with P. juliflora pollen antiserum, characterized by gel diffusion, Immunoelectrophoresis and Radioallergosorbent test.

  17. 26 CFR 1.642(e)-1 - Depreciation and depletion.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Depreciation and depletion. 1.642(e)-1 Section 1... (CONTINUED) INCOME TAXES Estates, Trusts, and Beneficiaries § 1.642(e)-1 Depreciation and depletion. An estate or trust is allowed the deductions for depreciation and depletion, but only to the extent the...

  18. 26 CFR 31.3231(e)-1 - Compensation.

    Science.gov (United States)

    2010-04-01

    .... (a) Definition—(1) The term compensation has the same meaning as the term wages in section 3121(a... regarding the inclusion of fringe benefits in compensation, see § 31.3121(a)-1T. (6) Split-dollar life... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Compensation. 31.3231(e)-1 Section 31.3231(e)-1...

  19. Change of mitotic cycle and DNA repair in embryonic cells of rat, immortalized by E1 A oncogene and transformated by E1 A and c-Ha-Ras oncogenes under ionizing radiation action

    International Nuclear Information System (INIS)

    Kirillova, T.V.

    1997-01-01

    Comparison investigation into the repair of mitotic cycle and the reunion of DN single- and double-strand breaks in gamma-ray irradiated initial E1 A oncogene immortalized and E1 A and c-Ha-Ras oncogene transformed (mutant form) lines of rat embryonic fibroblasts was carried out. Possible involvement of Ras gene product in DNA repair speed governing and absence of tumor suppression function of p 53 protein in the embryonic and E1 A oncogene immortalized cells of rat fibroblast, as well as, presence of the mentioned function of p 53 protein in E1 A and c-Ha-Ras oncogene transformed cells were studied [ru

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    , and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human m......Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81......bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies...

  1. Synthesis of Structures Related to Antifreeze Glycoproteins

    OpenAIRE

    Fyrner, Timmy

    2005-01-01

    In this thesis, synthesis of structures related to antifreeze glycoproteins (AFGPs) are presented. Synthetic routes to a protected carbohydrate derivative, 2,3,4,6-tetra-O-benzyl-β-galactopyranosyl-(1→3)-2-deoxy-2-azido-4,6-di-O-benzyl-β-D-thio-1-galactopyranoside, and a tBu-Ala-Thr-Ala-Fmoc tripeptide, are described. These compounds are meant to be used in the assembly of AFGPs and analogues thereof. A Gal-GlcN disaccharide was synthesized via glycosylation between the donor, bromo-2-O-benzo...

  2. Podoplanin - a small glycoprotein with many faces

    OpenAIRE

    Ugorski, Maciej; Dziegiel, Piotr; Suchanski, Jaroslaw

    2016-01-01

    Podoplanin is a small membrane glycoprotein with a large number of O-glycoside chains and therefore it belongs to mucin-type proteins. It can be found on the surface of many types of normal cells originating from various germ layers. It is present primarily on the endothelium of lymphatic vessels, type I pneumocytes and glomerular podocytes. Increased levels of podoplanin or its neo-expression have been found in numerous types of human carcinomas, but it is especially common in squamous cell ...

  3. [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.

  4. Glycoprotein component of plant cell walls

    International Nuclear Information System (INIS)

    Cooper, J.B.; Chen, J.A.; Varner, J.E.

    1984-01-01

    The primary wall surrounding most dicotyledonous plant cells contains a hydroxyproline-rich glycoprotein (HRGP) component named extensin. A small group of glycopeptides solubilized from isolated cell walls by proteolysis contained a repeated pentapeptide glycosylated by tri- and tetraarabinosides linked to hydroxyproline and, by galactose, linked to serine. Recently, two complementary approaches to this problem have provided results which greatly increase the understanding of wall extensin. In this paper the authors describe what is known about the structure of soluble extensin secreted into the walls of the carrot root cells

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

    Science.gov (United States)

    Estelrich, J.; Pouplana, R.

    1988-01-01

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

  6. Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell

    Directory of Open Access Journals (Sweden)

    A.M. Al-Sulaiman

    2017-11-01

    Full Text Available Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses’ proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD and varicella zoster glycoprotein E (VZV gE. Recombinant plasmids were generated, transfected into insect cells (SF9, and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.

  7. Activation of the human beta interferon gene by the adenovirus type 12 E1B gene

    International Nuclear Information System (INIS)

    Shiroki, K.; Toth, M.

    1988-01-01

    The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogeneously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT

  8. Multifactorial comparative proteomic study of cytochrome P450 2E1 function in chronic alcohol administration.

    Directory of Open Access Journals (Sweden)

    Yuan Wang

    Full Text Available With the use of iTRAQ technique, a multifactorial comparative proteomic study can be performed. In this study, to obtain an overview of ethanol, CYP2E1 and gender effects on liver injury and gain more insight into the underlying molecular mechanism, mouse liver proteomes were quantitatively analyzed using iTRAQ under eight conditions including mice of different genders, wild type versus CYP2E1 knockout, and normal versus alcohol diet. A series of statistical and bioinformatic analyses were explored to simplify and clarify multifactorial comparative proteomic data. First, with the Principle Component analysis, six proteins, CYP2E1, FAM25, CA3, BHMT, HIBADH and ECHS1, involved in oxidation reduction, energy and lipid metabolism and amino acid metabolism, were identified as the most differentially expressed gene products across all of the experimental conditions of our chronic alcoholism model. Second, hierarchical clustering analysis showed CYP2E1 knockout played a primary role in the overall differential protein expression compared with ethanol and gender factors. Furthermore, pair-wise multiple comparisons have revealed that the only significant expression difference lied in wild-type and CYP2E1 knockout mice both treated with ethanol. Third, K-mean clustering analysis indicated that the CYP2E1 knockout had the reverse effect on ethanol induced oxidative stress and lipid oxidation. More importantly, IPA analysis of proteomic data inferred that the gene expressions of two upstream regulators, NRF2 and PPARα, regulated by chronic alcohol feeding and CYP2E1 knockout, are involved in ethanol induced oxidative stress and lipid oxidation. The present study provides an effectively comprehensive data analysis strategy to compare multiple biological factors, contributing to biochemical effects of alcohol on the liver. The mass spectrometry proteomics data have been deposited to the ProteomeXchange with data set identifier of PXD000635.

  9. 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

  10. (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

  11. Membrane glycoproteins of differentiating skeletal muscle cells

    International Nuclear Information System (INIS)

    Miller, K.R.; Remy, C.N.; Smith, P.B.

    1987-01-01

    The composition of N-linked glycoprotein oligosaccharides was studied in myoblasts and myotubes of the C2 muscle cell line. Oligosaccharides were radioactively labelled for 15 hr with [ 3 H] mannose and plasma membranes isolated. Ten glycopeptides were detected by SDS-PAGE and fluorography. The extent of labelling was 4-6 fold greater in myoblasts vs myotubes. A glycopeptide of Mr > 100,000 was found exclusively in myoblast membranes. Lectin chromatography revealed that the proportion of tri-, tetranntenary, biantennary and high mannose chains was similar throughout differentiation. The high mannose chain fraction was devoid of hybrid chains. The major high mannose chain contained nine mannose residues. The higher level of glycopeptide labelling in myoblasts vs myotubes corresponded to a 5-fold greater rate of protein synthesis. Pulse-chase experiments were used to follow the synthesis of the Dol-oligosaccharides. Myoblasts and myotubes labelled equivalently the glucosylated tetradecasaccharide but myoblasts labelled the smaller intermediates 3-4 greater than myotubes. Myoblasts also exhibited a 2-3 fold higher Dol-P dependent glycosyl transferase activity for chain elongation and Dol-sugar synthesis. Together these results show that the degree of protein synthesis and level of Dol-P are contributing factors in the higher capacity of myoblasts to produce N-glycoproteins compared to myotubes

  12. Annotating Human P-Glycoprotein Bioassay Data.

    Science.gov (United States)

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

    2012-08-01

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

  13. Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

    Science.gov (United States)

    Asfor, A S; Wakeley, P R; Drew, T W; Paton, D J

    2014-08-30

    Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Humanizing recombinant glycoproteins from Chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Hansen, Anders Holmgaard; Amann, Thomas; Kol, Stefan

    With new tools for gene-editing like zinc-fingers, TALENS and CRISPR, it is now feasible totailor-make the N-Glycoforms for therapeutic glycoproteins that have previously been almost impossible. We here demonstrate a case of humanizing a recombinant human glycoprotein that in Wild type (WT) Chinese...

  15. Bioinformatics Analysis of Envelope Glycoprotein E epitopes of ...

    African Journals Online (AJOL)

    The E glycoprotein of dengue virus is responsible for the viral binding to the receptor. The crystal structure of envelope glycoprotein has already been determined. However, where the well-defined Bcell and T-cell epitopes are located is still a question. Because of the large variations among the four dengue genotypes, it is ...

  16. Ammonia transport in the kidney by Rhesus glycoproteins

    Science.gov (United States)

    Verlander, Jill W.

    2014-01-01

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

  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. CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Jie; Krausz, Kristopher W. [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Li, Feng; Ma, Xiaochao [Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, 4089 KLSIC, MS 1018, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States); Gonzalez, Frank J., E-mail: fjgonz@helix.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-15

    Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation. -- Highlights: ► Isoniazid metabolites were elevated only in wild-type mice. ► Isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice. ► Isoniazid elevated serum cholesterol and triglycerides, and hepatic bile acids. ► Bile acid transporters were significantly decreased in isoniazid-treated mice.

  19. CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid

    International Nuclear Information System (INIS)

    Cheng, Jie; Krausz, Kristopher W.; Li, Feng; Ma, Xiaochao; Gonzalez, Frank J.

    2013-01-01

    Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation. -- Highlights: ► Isoniazid metabolites were elevated only in wild-type mice. ► Isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice. ► Isoniazid elevated serum cholesterol and triglycerides, and hepatic bile acids. ► Bile acid transporters were significantly decreased in isoniazid-treated mice.

  20. An improved radioimmunoassay for urinary Tamm-Horsfall glycoprotein

    International Nuclear Information System (INIS)

    Dawnay, A.B. St. J.; Thornley, C.; Cattell, W.R.

    1982-01-01

    A rapid specific radioimmunoassay has been used to measure Tamm-Horsfall glycoprotein (TH glycoprotein) in urine, and the method described. The apparent concentration increased with increasing dilution of urine in water, reaching a plateau at 1 in 20. This increase was greater the higher the osmolality and TH glycoprotein concentration and the lower the pH of the original sample. The apparent concentration of TH glycoprotein in neat or diluted urine was not affected by freezing or by storage at 4 0 C or room temperature for at least 2 days. A physiological range for the urinary excretion rate was established as 22-56 mg/24h, (considerably higher than the amount present in serum) based on samples from 29 individuals with normal renal function, as defined by their creatinine clearance. There was no significant correlation between serum concentrations of TH glycoprotein and its urinary excretion rate, nor between urinary excretion rate and creatinine clearance. (author)

  1. Solubilization of glycoproteins of envelope viruses by detergents

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  2. Genetic polymorphism analysis of cytochrome P4502E1 (CYP2E1) in a Chinese Tibetan population

    Science.gov (United States)

    Wang, Li; Ren, Guoxia; Li, Jingjie; Zhu, Linhao; Niu, Fanglin; Yan, Mengdan; Li, Jing; Yuan, Dongya; Jin, Tianbo

    2017-01-01

    Abstract Cytochrome P4502E1 (CYP2E1) gene genetic polymorphisms vary markedly in frequency among different ethnic and racial groups. We studied the genotype distributions and allele frequencies of 3 CYP2E1 polymorphisms: CYP2E1∗1A, CYP2E1∗7A, and CYP2E1∗7C by polymerase chain reaction technique in a sample of 100 healthy subjects representing Tibetan population. The frequencies of CYP2E1∗1A, ∗7A, and ∗7C alleles were 0.705, 0.125, and 0.170, respectively. Compared with other populations, we found that the allele frequencies of the variants −352A>G (rs2070672) and −333A>T (rs2070673) in this Tibetan population have significant differences compared with European-American, African-American, Japanese, Korean, and other different geographic areas in Chinese Han population. Furthermore, the results of protein prediction revealed that the variant 6397G>A (rs61710826) could influence the protein structure and function. These findings in this study would be valuable for pharmacogenetics for drug therapy and drug discovery. However, further studies in larger samples are warranted to confirm our results. PMID:29381998

  3. 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...

  4. Raman optical activity of proteins and glycoproteins

    International Nuclear Information System (INIS)

    Smyth, E.

    2000-03-01

    Raman optical activity (ROA), measured in this project as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarised incident laser light, offers the potential to provide more information about the structure of biological molecules in aqueous solution than conventional spectroscopic techniques. Chapter one contains a general discussion of the relative merits of different spectroscopic techniques for structure determination of biomolecules, as well as a brief introduction to ROA. In Chapter two a theoretical analysis of ROA is developed, which extends the discussion in chapter one. The spectrometer setup and sample preparation is then discussed in chapter three. Instrument and sample conditions are monitored to ensure that the best results are obtained. As with any experimental project problems occur, which may result in a degradation of the spectra obtained. The cause of these problems was explored and remedied whenever possible. Chapter four introduces a brief account of protein, glycoprotein and carbohydrate structure and function, with a particular emphasis on the structure of proteins. In the remaining chapters experimental ROA results on proteins and glycoproteins, with some carbohydrate samples, from a wide range of sources are examined. For example, in chapter five some β-sheet proteins are examined. Structural features in these proteins are examined in the extended amide III region of their ROA spectra, revealing that ROA is sensitive to the rigidity or flexibility inherent in proteins. Chapter six concentrates on a group of proteins (usually glycoproteins) known as the serine proteinase inhibitors (serpins). Medically, the serpins are one of the most important groups of proteins of current interest, with wide-ranging implications in conditions such as Down's syndrome, Alzheimer's disease, and emphysema with associated cirrhosis of the liver. With favourable samples and conditions ROA may offer the

  5. Characterization of papillomavirus E1 helicase mutants defective for interaction with the SUMO-conjugating enzyme Ubc9

    International Nuclear Information System (INIS)

    Fradet-Turcotte, Amelie; Brault, Karine; Titolo, Steve; Howley, Peter M.; Archambault, Jacques

    2009-01-01

    The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and binding to DNA and ATP.

  6. The inactivation of human CYP2E1 by phenethyl isothiocyanate, a naturally occurring chemopreventive agent, and its oxidative bioactivation.

    Science.gov (United States)

    Yoshigae, Yasushi; Sridar, Chitra; Kent, Ute M; Hollenberg, Paul F

    2013-04-01

    Phenethylisothiocyanate (PEITC), a naturally occurring isothiocyanate and potent cancer chemopreventive agent, works by multiple mechanisms, including the inhibition of cytochrome P450 (P450) enzymes, such as CYP2E1, that are involved in the bioactivation of carcinogens. PEITC has been reported to be a mechanism-based inactivator of some P450s. We describe here the possible mechanism for the inactivation of human CYP2E1 by PEITC, as well as the putative intermediate that might be involved in the bioactivation of PEITC. PEITC inactivated recombinant CYP2E1 with a partition ratio of 12, and the inactivation was not inhibited in the presence of glutathione (GSH) and not fully recovered by dialysis. The inactivation of CYP2E1 by PEITC is due to both heme destruction and protein modification, with the latter being the major pathway for inactivation. GSH-adducts of phenethyl isocyanate (PIC) and phenethylamine were detected during the metabolism by CYP2E1, indicating formation of PIC as a reactive intermediate following P450-catalyzed desulfurization of PEITC. Surprisingly, PIC bound covalently to CYP2E1 to form protein adducts but did not inactivate the enzyme. Liquid chromatography mass spectroscopy analysis of the inactivated CYP2E1 apo-protein suggests that a reactive sulfur atom generated during desulfurization of PEITC is involved in the inactivation of CYP2E1. Our data suggest that the metabolism of PEITC by CYP2E1 that results in the inactivation of CYP2E1 may occur by a mechanism similar to that observed with other sulfur-containing compounds, such as parathion. Digestion of the inactivated enzyme and analysis by SEQUEST showed that Cys 268 may be the residue modified by PIC.

  7. P-glycoprotein in autoimmune rheumatic diseases.

    Science.gov (United States)

    García-Carrasco, M; Mendoza-Pinto, C; Macias Díaz, S; Vera-Recabarren, M; Vázquez de Lara, L; Méndez Martínez, S; Soto-Santillán, P; González-Ramírez, R; Ruiz-Arguelles, A

    2015-07-01

    P-glycoprotein (Pgp) is a transmembrane protein of 170 kD encoded by the multidrug resistance 1 (MDR-1) gene, localized on chromosome 7. More than 50 polymorphisms of the MDR-1 gene have been described; a subset of these has been shown to play a pathophysiological role in the development of inflammatory bowel disease, femoral head osteonecrosis induced by steroids, lung cancer and renal epithelial tumors. Polymorphisms that have a protective effect on the development of conditions such as Parkinson disease have also been identified. P-glycoprotein belongs to the adenosine triphosphate binding cassette transporter superfamily and its structure comprises a chain of approximately 1280 aminoacid residues with an N-C terminal structure, arranged as 2 homologous halves, each of which has 6 transmembrane segments, with a total of 12 segments with 2 cytoplasmic nucleotide binding domains. Many cytokines like interleukin 2 and tumor necrosis factor alpha increase Pgp expression and activity. Pgp functions as an efflux pump for a variety of toxins in order to protect particular organs and tissues as the central nervous system. Pgp transports a variety of substrates including glucocorticoids while other drugs such as tacrolimus and cyclosporine A act as modulators of this protein. The most widely used method to measure Pgp activity is flow cytometry using naturally fluorescent substrates such as anthracyclines or rhodamine 123. The study of drug resistance and its association to Pgp began with the study of resistance to chemotherapy in the treatment of cancer and antiretroviral therapy for human immunodeficiency virus; however, the role of Pgp in the treatment of systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis has been a focus of study lately and has emerged as an important mechanism by which treatment failure occurs. The present review analyzes the role of Pgp in these autoimmune diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Oncolytic Replication of E1b-Deleted Adenoviruses

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    2015-11-01

    Full Text Available Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption.

  9. Regulation of glycoprotein synthesis in yeast by mating pheromones

    International Nuclear Information System (INIS)

    Tanner, W.

    1984-01-01

    In Saccharomyces cerevisiae, glycosylated proteins amount to less than 2% of the cell protein. Two intensively studied examples of yeast glycoproteins are the external cell wall - associated invertase and the vacuolar carboxypeptidase Y. Recently, it was shown that the mating pheromone, alpha factor, specifically and strongly inhibits the synthesis of N-glycosylated proteins in haploid a cells, whereas O-glycosylated proteins are not affected. In this paper, the pathways of glycoprotein biosynthesis are summarized briefly, and evidence is presented that mating pheomones have a regulatory function in glycoprotein synthesis

  10. 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...

  11. Acetaldehyde and parkinsonism: role of CYP450 2E1

    Directory of Open Access Journals (Sweden)

    Francesca eVaglini

    2013-06-01

    Full Text Available The present review update the relationship between acetaldehyde and parkinsonism with a specific focus on the role of P450 system and CYP 2E1 isozyme particularly.We have indicated that acetaldehyde is able to enhance the parkinsonism induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin able to damage the nigrostriatal dopaminergic pathway. Similarly diethyldithiocarbamate, the main metabolite of disulfiram, a drug widely used to control alcoholism, diallylsulfide and phenylisothiocyanate also markedly enhance the toxin-related parkinsonism. All these compounds are substrate/inhibitors of CYP450 2E1 isozyme. The presence of CYP 2E1 has been detected in the dopamine neurons of rodent Substantia Nigra, but a precise function of the enzyme has not been elucidated yet. By treating CYP 2E1 knockout mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the Substantia Nigra induced lesion was significantly reduced when compared with the lesion observed in wild-type animals. Several in vivo and in vitro studies led to the conclusion that CYP 2E1 may enhance the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice by increasing free radical production inside the dopaminergic neurons. Acetaldehyde is a good substrate for CYP 2E1 enzyme as the other substrate-inhibitors and by this way may facilitate the susceptibility of dopaminergic neurons to toxic events. The literature suggests that ethanol and/or disulfiram may be responsible for toxic parkinsonism in human and it indicates that basal ganglia are the major targets of disulfiram toxicity. A very recent study reports that there are a decreased methylation of the CYP 2E1 gene and increased expression of CYP 2E1 mRNA in Parkinson’s Disease patient brains. This study suggests that epigenetic variants of this cytochrome contribute to the susceptibility, thus confirming multiples lines of evidence which indicate a link between environmental toxins and

  12. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    International Nuclear Information System (INIS)

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-01-01

    E2, along with E rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818 CPIGWTGVIEC 828 indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion

  13. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Sainz, I.J. [Plum Island Animal Disease Center, ARS, USDA (United States); Largo, E. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Gladue, D.P.; Fletcher, P. [Plum Island Animal Disease Center, ARS, USDA (United States); O’Donnell, V. [Plum Island Animal Disease Center, ARS, USDA (United States); Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Holinka, L.G. [Plum Island Animal Disease Center, ARS, USDA (United States); Carey, L.B. [Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona (Spain); Lu, X. [Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Nieva, J.L. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Borca, M.V., E-mail: manuel.borca@ars.usda.gov [Plum Island Animal Disease Center, ARS, USDA (United States)

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  14. Carbohydrates of influenza virus. I. Glycopeptides derived from viral glycoproteins after labeling with radioactive sugars

    International Nuclear Information System (INIS)

    Schwarz, R.T.; Schmidt, M.F.G.; Anwer, U.; Klenk, H.D.

    1977-01-01

    The carbohydrate moiety of the influenza glycoproteins NA, HA 1 , and HA 2 were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA 1 , and HA 2 . The side chain of type II contains a high amount of mannose and is found only on NA and HA 2 . The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken ambryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains

  15. Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

    Science.gov (United States)

    Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

    2016-07-01

    Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection.

  16. Multi-Laboratory Validation of Estrone (E1) ELISA Methods

    Science.gov (United States)

    This project is a round-robin evaluation of commercially available Enzyme-Linked Immunosorbent Assay (ELISA) technology to quantitatively or qualitatively measure the hormone estrone (E1) in combined animal feeding operation (CAFO) receiving streams. ELISA is meant to be a simpl...

  17. Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode

    OpenAIRE

    Haag, Lars; Garoff, Henrik; Xing, Li; Hammar, Lena; Kan, Sin-Tau; Cheng, R.Holland

    2002-01-01

    In the icosahedral (T = 4) Semliki Forest virus, the envelope protomers, i.e. E1–E2 heterodimers, make one-to-one interactions with capsid proteins below the viral lipid bilayer, transverse the membrane and form an external glycoprotein shell with projections. The shell is organized by protomer domains interacting as hexamers and pentamers around shell openings at icosahedral 2- and 5-fold axes, respectively, and the projections by other domains associating as trimers at 3- and quasi 3-fold a...

  18. Bioinformatics Analysis of Envelope Glycoprotein E epitopes of ...

    African Journals Online (AJOL)

    User

    2011-05-02

    May 2, 2011 ... 1National Centre of Excellence in Molecular Biology, University of the Punjab Lahore, Pakistan. 2Department of ..... E glycoprotein and its interaction with antibody with the method of molecular dynamics and molecular model ...

  19. Extra-oviductal expression of oviductal glycoprotein 1 in mouse ...

    Indian Academy of Sciences (India)

    2017-01-11

    Jan 11, 2017 ... oestrogen-dependent protein, oviduct-specific glycoprotein. 1 (Ovgp1) also ... the tissues collected were testis, epididymis, prostate gland and seminal vesicle. ... The antigenic sites were unmasked by incuba- tion of sections ...

  20. The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors

    Science.gov (United States)

    Zvejniece, L; Vavers, E; Svalbe, B; Vilskersts, R; Domracheva, I; Vorona, M; Veinberg, G; Misane, I; Stonans, I; Kalvinsh, I; Dambrova, M

    2014-01-01

    Background and Purpose Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. Experimental Approach E1R was tested for sigma receptor binding activity in a [3H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca2+ concentration ([Ca2+]i) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. Key Results Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca2+]i increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. Conclusion and Implications E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases. PMID:24490863

  1. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-02-01

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

  2. Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36

    Directory of Open Access Journals (Sweden)

    Roger S. Holmes

    2012-09-01

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

  3. Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.

    Directory of Open Access Journals (Sweden)

    Thomas A Bowden

    Full Text Available The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1980-01-01

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

  5. P-glycoprotein targeted nanoscale drug carriers

    KAUST Repository

    Li, Wengang

    2013-02-01

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

  6. Analysis of the selective advantage conferred by a C-E1 fusion protein synthesized by rubella virus DI RNAs

    International Nuclear Information System (INIS)

    Claus, Claudia; Tzeng, W.-P.; Liebert, Uwe Gerd; Frey, Teryl K.

    2007-01-01

    During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and

  7. Therapeutic Strategies Against Cyclin E1 Amplified Ovarian Cancers

    Science.gov (United States)

    2017-10-01

    13-14 ( References ) 1. INTRODUCTION: Approximately 20% of high grade serous ovarian cancers harbor Cyclin E1 (CCNE1) amplification and are associated... Harvard Medical School and was named Director of Translational Research in the Gynecologic Oncology Program at Dana-Farber Cancer Institute. How...on HDAC6 activity. Nat Cell Biol 19:962-973. PMID: 28737768. PMC5541905. Books or other non-periodical, one-time publications. “Nothing to Report

  8. Statistical decay of the E1 giant resonance

    International Nuclear Information System (INIS)

    Teruya, N.; Dias, H.; Wolynec, E.

    1987-10-01

    Available esperimental data on neutron decay spectra from the E1 giant resonances in 208 Pb and 209 Bi are compared with the predicted spectra for statistical decay. The calculations are performed using the Hauser-Feshbach formalism with the experimental levels of the residual nuclei. The particle-vibrator model is used to assign spins and parities to experimental levels when those are unknown and also to predict the levels where there is not enough experimental information. (author) [pt

  9. Project FOOTPRINT: Substation modeling and simulations for E1 pulses

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Scott D. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Larson, D. J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Kirkendall, B. A. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2017-11-06

    This report includes a presentation with an: Introduction to CW coupling; Introduction to single-pulse coupling; Description of E1 waveforms; Structures in a substation yard --articulated (as part of the substation's defined electrical functionality)--unarticulated (not as part of the substation's defined electrical functionality); Coupling --electrical coupling (capacitive coupling) --magnetic coupling (inductive coupling); Connectivity to long-line transmission lines; Control infrastructure; Summary; and References.

  10. Effect of dietary fat on hepatic liver X receptor expression in P-glycoprotein deficient mice: implications for cholesterol metabolism

    Directory of Open Access Journals (Sweden)

    Lee Stephen D

    2008-06-01

    Full Text Available Abstract Pgp (P-glycoprotein, MDR1, ABCB1 is an energy-dependent drug efflux pump that is a member of the ATP-binding cassette (ABC family of proteins. Preliminary studies have reported that nonspecific inhibitors of Pgp affect synthesis and esterification of cholesterol, putatively by blocking trafficking of cholesterol from the plasma membrane to the endoplasmic reticulum, and that relative increases in Pgp within a given cell type are associated with increased accumulation of cholesterol. Several key efflux proteins involved in the cholesterol metabolic pathway are transcriptionally regulated by the nuclear hormone liver X receptor (LXR. Therefore, to examine the interplay between P-glycoprotein and the cholesterol metabolic pathway, we utilized a high fat, normal cholesterol diet to upregulate LXRα without affecting dietary cholesterol. Our research has demonstrated that mice lacking in P-glycoprotein do not exhibit alterations in hepatic total cholesterol storage, circulating plasma total cholesterol levels, or total cholesterol concentration in the bile when compared to control animals on either a normal (25% calories from dietary fat or high fat (45% calories from dietary fat diet. However, p-glycoprotein deficient mice (Mdr1a-/-/1b-/- exhibit increased hepatic LXRα protein expression and an elevation in fecal cholesterol concentration when compared to controls.

  11. Mammalian cytochrome CYP2E1 triggered differential gene regulation in response to trichloroethylene (TCE) in a transgenic poplar.

    Science.gov (United States)

    Kang, Jun Won; Wilkerson, Hui-Wen; Farin, Federico M; Bammler, Theo K; Beyer, Richard P; Strand, Stuart E; Doty, Sharon L

    2010-08-01

    Trichloroethylene (TCE) is an important environmental contaminant of soil, groundwater, and air. Studies of the metabolism of TCE by poplar trees suggest that cytochrome P450 enzymes are involved. Using poplar genome microarrays, we report a number of putative genes that are differentially expressed in response to TCE. In a previous study, transgenic hybrid poplar plants expressing mammalian cytochrome P450 2E1 (CYP2E1) had increased metabolism of TCE. In the vector control plants for this construct, 24 h following TCE exposure, 517 genes were upregulated and 650 genes were downregulated over 2-fold when compared with the non-exposed vector control plants. However, in the transgenic CYP2E1 plant, line 78, 1,601 genes were upregulated and 1,705 genes were downregulated over 2-fold when compared with the non-exposed transgenic CYP2E1 plant. It appeared that the CYP2E1 transgenic hybrid poplar plants overexpressing mammalian CYP2E1 showed a larger number of differentially expressed transcripts, suggesting a metabolic pathway for TCE to metabolites had been initiated by activity of CYP2E1 on TCE. These results suggest that either the over-expression of the CYP2E1 gene or the abundance of TCE metabolites from CYP450 2E1 activity triggered a strong genetic response to TCE. Particularly, cytochrome p450s, glutathione S-transferases, glucosyltransferases, and ABC transporters in the CYP2E1 transgenic hybrid poplar plants were highly expressed compared with in vector controls.

  12. Induction of brain CYP2E1 by chronic ethanol treatment and related oxidative stress in hippocampus, cerebellum, and brainstem

    International Nuclear Information System (INIS)

    Zhong, Yanjun; Dong, Guicheng; Luo, Haiguang; Cao, Jie; Wang, Chang; Wu, Jianyuan; Feng, Yu-Qi; Yue, Jiang

    2012-01-01

    Ethanol is one of the most commonly abused substances, and oxidative stress is an important causative factor in ethanol-induced neurotoxicity. Cytochrome P450 2E1 (CYP2E1) is involved in ethanol metabolism in the brain. This study investigates the role of brain CYP2E1 in the susceptibility of certain brain regions to ethanol neurotoxicity. Male Wistar rats were intragastrically treated with ethanol (3.0 g/kg, 30 days). CYP2E1 protein, mRNA expression, and catalytic activity in various brain regions were respectively assessed by immunoblotting, quantitative quantum dot immunohistochemistry, real-time RT-PCR, and LC–MS. The generation of reactive oxygen species (ROS) was analyzed using a laser confocal scanning microscope. The hippocampus, cerebellum, and brainstem were selectively damaged after ethanol treatment, indicated by both lactate dehydrogenase (LDH) activity and histopathological analysis. Ethanol markedly increased the levels of CYP2E1 protein, mRNA expression, and activity in the hippocampus and cerebellum. CYP2E1 protein and activity were significantly increased by ethanol in the brainstem, with no change in mRNA expression. ROS levels induced by ethanol paralleled the enhanced CYP2E1 proteins in the hippocampus, granular layer and white matter of cerebellum as well as brainstem. Brain CYP2E1 activity was positively correlated with the damage to the hippocampus, cerebellum, and brainstem. These results suggest that the selective sensitivity of brain regions to ethanol neurodegeneration may be attributed to the regional and cellular-specific induction of CYP2E1 by ethanol. The inhibition of CYP2E1 levels may attenuate ethanol-induced oxidative stress via ROS generation.

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

    Science.gov (United States)

    Banzato, Alessandra; Pengo, Vittorio

    2014-06-01

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

  14. Novel CYP2E1 haplotype identified in a South African cohort

    Directory of Open Access Journals (Sweden)

    Laura J. Heathfield

    2014-09-01

    Full Text Available Alcohol abuse accounts for approximately 2.5 million deaths annually and is the third highest risk factor for disease and disability. Alcohol is metabolised by polymorphic enzymes and the status of an individual with respect to alcohol metabolising enzymes may have forensic relevance in post-mortems. Baseline frequencies of gene variants involved in alcohol metabolism need to be established to aid the identification of suitable population-specific polymorphisms to genotype during molecular autopsies. The principal alcohol metabolising enzymes include alcohol dehydrogenase (ADH, aldehyde dehydrogenase (ALDH and cytochrome P450 2E1 (CYP2E1. Six single nucleotide polymorphisms (SNPs – rs1229984G>A and rs2066702C>T in ADH1B, rs671G>A in ALDH2, and rs3813867G>C, rs2031920C>T and rs6413432T>A in CYP2E1 – were genotyped in 150 individuals from four South African populations: Xhosa, Zulu, South African white and South African coloured. Allele frequencies for each SNP in the four population groups were 0–10% for rs1229984A, 2–12% for rs2066702T, 0–2% for rs671A, 1–4% for rs3813867C, 0–1% for rs2031920T and 3–15% for rs6413432A. Haplotype analysis revealed a novel combination of three SNPs in CYP2E1 whose effects on alcohol metabolism need further investigation. Establishment of baseline frequencies adds to our knowledge of genetic variation in alcohol metabolising enzymes and additional research is required to determine the functional significance of this novel CYP2E1 haplotype.

  15. Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

    Science.gov (United States)

    Jallet, Corinne; Jacob, Yves; Bahloul, Chokri; Drings, Astrid; Desmezieres, Emmanuel; Tordo, Noël; Perrin, Pierre

    1999-01-01

    The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6). PMID:9847325

  16. Sites of disruption within E1 and E2 genes of HPV16 and association with cervical dysplasia.

    Science.gov (United States)

    Tsakogiannis, D; Gortsilas, P; Kyriakopoulou, Z; Ruether, I G A; Dimitriou, T G; Orfanoudakis, G; Markoulatos, P

    2015-11-01

    Integration of HPV16 DNA into the host chromosome usually disrupts the E1 and/or E2 genes. The present study investigated the disruption of E1, E2 genes in a total of eighty four HPV16-positive precancerous and cervical cancer specimens derived from Greek women (seventeen paraffin-embedded cervical biopsies and sixty seven Thin Prep samples). Complete E2 and E1 genes were amplified using three and nine overlapping primer sets respectively, in order to define the sites of disruption. Extensive mapping analysis revealed that disruption/deletion events within E2 gene occurred in high grade and cervical cancer samples (x(2) test, P disruption was documented among low grade cervical intraepithelial neoplasias. In addition, disruptions within the E1 gene occur both in high and low grade cervical intraepithelial neoplasia. This leads to the assumption that in low grade cervical intraepithelial neoplasias only E1 gene disruption was involved (Fisher's exact test, P disruption of E1 gene was located between nucleotides 1059 and 1323, while the most prevalent deleted region of the E2 gene was located between nucleotides 3172 and 3649 (E2 hinge region). Therefore, it is proposed that each population has its own profile of frequencies and sites of disruptions and extensive mapping analysis of E1 and E2 genes is mandatory in order to determine suitable markers for HPV16 DNA integration analysis in distinct populations. © 2015 Wiley Periodicals, Inc.

  17. The chang’E-1 topographic atlas of the Moon

    CERN Document Server

    Li, Chunlai; Mu, Lingli; Ren, Xin; Zuo, Wei

    2016-01-01

    This atlas is based on the lunar global Digital Elevation Models (DEM) of Chang'E-1 (CE-1), and presents CCD stereo image data with digital photogrammetry. The spatial resolution of the DEM in this atlas is 500m, with horizontal accuracy of 192m and vertical accuracy of 120m. Color-shaded relief maps with contour lines are used to show the lunar topographical characteristics. The topographical data gathered by CE-1 can provide fundamental information for the study of lunar topographical, morphological and geological structures, as well as for lunar evolution research.

  18. Alternative promoter usage of the membrane glycoprotein CD36

    Directory of Open Access Journals (Sweden)

    Whatling Carl

    2006-03-01

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

  19. 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.

  20. 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

  1. Synthetic glycopeptides and glycoproteins with applications in biological research

    Directory of Open Access Journals (Sweden)

    Ulrika Westerlind

    2012-05-01

    Full Text Available Over the past few years, synthetic methods for the preparation of complex glycopeptides have been drastically improved. The need for homogenous glycopeptides and glycoproteins with defined chemical structures to study diverse biological phenomena further enhances the development of methodologies. Selected recent advances in synthesis and applications, in which glycopeptides or glycoproteins serve as tools for biological studies, are reviewed. The importance of specific antibodies directed to the glycan part, as well as the peptide backbone has been realized during the development of synthetic glycopeptide-based anti-tumor vaccines. The fine-tuning of native chemical ligation (NCL, expressed protein ligation (EPL, and chemoenzymatic glycosylation techniques have all together enabled the synthesis of functional glycoproteins. The synthesis of structurally defined, complex glycopeptides or glyco-clusters presented on natural peptide backbones, or mimics thereof, offer further possibilities to study protein-binding events.

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development......The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus...... blot studies using chromosomal or restricted DNA, the major surface glycoproteins are the products of a multicopy family of genes. The predicted protein has an M(r) of approximately 123,000, is relatively rich in cysteine residues (5.5%) that are very strongly conserved, and contains a well conserved...

  3. Intracellular localization of hydroxyproline-rich glycoprotein biosynthesis

    International Nuclear Information System (INIS)

    Robinson, D.G.; Andreae, M.; Glas, A.R.; Sauer, A.

    1984-01-01

    The structural proteins of plant cell walls are glycoproteins characterized by O-glucosidic linkages to hydroxyproline or serine. Proline, not hydroxyproline, is the translatable amino acid in hydroxyproline-rich glycoproteins (HRGP). Hydroxylation and arabinosylation of proline are sequential, post-translational events. Because of this, there is no a priori reason for expecting HRGP synthesis to follow the well-established route for secretory and plasma membrane (PM) glycoproteins, i.e., from endoplasmic reticulum (ER) via the Golgi apparatus (GA) to the PM. In this paper, two plausible alternatives for HRGO secretion are examined. Because a feature of the majority of dicotyledons is overlapping GA and PM regions in sucrose density gradients, the authors have used two monocotyledonous systems to determine the distribution of HRGP and enzyme activity

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

    International Nuclear Information System (INIS)

    Tropea, J.E.; Molyneux, R.J.; Kaushal, G.P.; Pan, Y.T.; Mitchell, M.; Elbein, A.D.

    1989-01-01

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

  5. Alteration of a second putative fusion peptide of structural glycoprotein E2 of Classical Swine Fever Virus alters virus replication and virulence in swine

    Science.gov (United States)

    E2, the major envelope glycoprotein of Classical Swine Fever Virus (CSFV), is involved in several critical virus functions including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on Wimley-White interfacial hydrophobicity dis...

  6. 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

  7. A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

    Directory of Open Access Journals (Sweden)

    Broder Christopher C

    2010-11-01

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

  8. Preparation of 131I-asialo-α1-acid glycoprotein

    International Nuclear Information System (INIS)

    Rijk, P.P. van

    1975-01-01

    α 1 -Acid glycoprotein (orosomucoid) was prepared from a byproduct of the ethanol plasma fractionation by means of ion-exchange procedures. Immunoelectrophoresis suggested a high degree of purity; the purified protein contained 13.5% sialic acid and 17.8% hexose. The α 1 -acid glycoprotein was modified by removal of sialic acid with neurominidase (E.C. 3.2.1.18) followed by iodination with 131 I. The purpose of the preparation, its potential use as a pharmacon for liver-function studies in nuclear medicine, is the subject of further study

  9. Intestinal mucus and juice glycoproteins have a liquid crystalline structure

    International Nuclear Information System (INIS)

    Denisova, E.A.; Lazarev, P.I.; Vazina, A.A.; Zheleznaya, L.A.

    1985-01-01

    X-ray diffraction patterns have been obtained from the following components of canine gastrointestinal tract: (1) native small intestine mucus layer; (2) the precipitate of the flocks formed in the duodenal juice with decreasing pH; (3) concentrated solutions of glycoproteins isolated from the duodenal juice. The X-ray patterns consist of a large number of sharp reflections of spacings between about 100 and 4 A. Some reflections are common for all components studied. All the patterns are interpreted as arising from the glycoprotein molecules ordered into a liquid crystalline structure. (author)

  10. Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation.

    Science.gov (United States)

    Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W Andy

    2016-11-30

    Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

  11. Synthesis of 11-(Piperazin-1-yl-5H-dibenzo[b,e] [1,4]diazepine on Kilo Scale

    Directory of Open Access Journals (Sweden)

    Rahul S. Kalhapure

    2011-01-01

    Full Text Available A synthesis of 11-(piperazin-1yl-5 H-dibenzo[b,e][1,4]diazepine on kilo scale without any chromatographic purification step is reported. Key steps involved are Ullmann condensation, catalytic hydrogenation, and catalyzed cyclization.

  12. Monoclonal antibody to an external epitope of the human mdr1 P-glycoprotein

    NARCIS (Netherlands)

    Arceci, R. J.; Stieglitz, K.; Bras, J.; Schinkel, A.; Baas, F.; Croop, J.

    1993-01-01

    A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents. P-glycoprotein, encoded in humans by the mdr1 gene, functions as an energy-dependent efflux pump to exclude these

  13. Genomic clone encoding the α chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules

    International Nuclear Information System (INIS)

    Cosgrove, L.J.; Sandrin, M.S.; Rajasekariah, P.; McKenzie, I.F.C.

    1986-01-01

    LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa β subunit but are distinguished by their α chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. The authors report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in λ phase Charon 4A and subsequent rescue of the λ phage. Transfection with the purified recombinant λ DNA yielded a transfectant that expressed the three human α chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine β chain

  14. Rat macrophages: membrane glycoproteins in differentiation and function

    NARCIS (Netherlands)

    van den Berg, T. K.; Döpp, E. A.; Dijkstra, C. D.

    2001-01-01

    Macrophages (mphi) play a crucial role in the immune system. The rat offers unique advantages for studying the biology of mphi. Firstly, monoclonal antibodies (mAb) against many rat mphi surface glycoproteins have become available. These have not only demonstrated a considerable heterogeneity among

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus this a...

  16. Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins

    NARCIS (Netherlands)

    Baranowski, E.; Keil, G.; Lyaku, J.; Rijsewijk, F.A.M.; Oirschot, van J.T.; Pastoret, P.P.; Thiry, E.

    1996-01-01

    This paper focuses on the structure and functions of bovine herpesvirus 1 minor glycoproteins gH, gE, gG and gp42. It reviews the progress which has been made in their identification and characterization, in the study of their temporal expression and processing in infected cells, and finally in the

  17. Separation and identification of carp pituitary proteins and glycoproteins

    Czech Academy of Sciences Publication Activity Database

    Ryšlavá, H.; Janatová, M.; Čalounová, G.; Selicharová, Irena; Barthová, J.; Barth, Tomislav

    2005-01-01

    Roč. 50, č. 9 (2005), 430-437 ISSN 1212-1819 R&D Projects: GA MZe(CZ) QF3028 Institutional research plan: CEZ:AV0Z4055905 Keywords : carp hormones * glycoproteins * oligosaccharide chains Subject RIV: CE - Biochemistry Impact factor: 0.254, year: 2005

  18. Glycoprotein of the wall of sycamore tissue-culture cells.

    Science.gov (United States)

    Heath, M F; Northcote, D H

    1971-12-01

    1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the alpha-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the alpha-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.

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

    Directory of Open Access Journals (Sweden)

    David Clark

    2012-01-01

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

  20. Glycoprotein expression by adenomatous polyps of the colon

    Science.gov (United States)

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

    2008-03-01

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

  1. 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

  2. Glycoprotein Ibα clustering in platelet storage and function

    NARCIS (Netherlands)

    Gitz, E.

    2013-01-01

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

  3. Glycoproteins and sialyl transferase of human B lymphoblastoid cell lines

    International Nuclear Information System (INIS)

    Lui, S.W.L.; Ng, M.H.

    1980-01-01

    We used two radiolabeling methods to study glycoproteins on the surface of lymphoblastoid cells. One of the methods affects tritiation of residues which are oxidized with galactose oxidase and the other causes tritiation of neuraminic acid residues. This approach was shown to allow a better resolution of cell surface glycoproteins than if either method were used alone. Glycoproteins of B 1 - 19 cells which harbor the Epstein-Barr virus genomes were compared with those of its parental cell line, BJAB, which does not harbor the viral genomes. These studies did not reveal a unique viral protein. A 28,000 mol. wt. glycoprotein was found to be the most prominent neuraminic acidlabeled product of B 1 - 19 cells and also of the two other cell lines, Raji and Ly38, which harbor the EBV genomes. A similar molecular weight species from BJAB cells identified by galactose oxidase labeling might be deficient in neuraminic acid residues as it was poorly labeled by the periodate oxidation method. The neuraminic acid content and level of sialyl transferase of BJAB cells were found to be lower than those of the other cell lines studied. (auth.)

  4. Direct chemical modification and voltammetric detection of glycans in glycoproteins

    Czech Academy of Sciences Publication Activity Database

    Trefulka, Mojmír; Paleček, Emil

    2014-01-01

    Roč. 48, NOV2014 (2014), s. 52-55 ISSN 1388-2481 R&D Projects: GA ČR(CZ) GAP301/11/2055 Institutional support: RVO:68081707 Keywords : Glycoproteins * Chemical modification * Os(VI)L complexes Subject RIV: BO - Biophysics Impact factor: 4.847, year: 2014

  5. MDR1 P-glycoprotein transports endogenous opioid peptides

    NARCIS (Netherlands)

    Oude Elferink, R. P.; Zadina, J.

    2001-01-01

    MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore

  6. Cereal n-glycoproteins enrichment by lectin affinity monolithic chromatography

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Bobálová, Janette; Laštovičková, Markéta

    2016-01-01

    Roč. 44, č. 2 (2016), s. 286-297 ISSN 0133-3720 R&D Projects: GA ČR(CZ) GPP503/12/P395 Institutional support: RVO:68081715 Keywords : barley * wheat * glycoprotein * mass spectrometry * lectin chromatography Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.496, year: 2016

  7. Molecular cloning of S1 glycoprotein gene of infectious bronchitis ...

    African Journals Online (AJOL)

    In vitro protein expression is an important method of obtaining large amounts of viral proteins to investigate their biological properties. The S1 glycoprotein of infectious bronchitis virus, due to its effective immune-dominant role is an appropriate candidate for production of recombinant vaccine against infectious bronchitis ...

  8. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Morrison, A.I.; Keeble, S.; Watt, F.M.

    1988-01-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [ 14 C]galactose- or [ 14 C]mannose- and [ 14 C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification

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

    International Nuclear Information System (INIS)

    Korecka, Lucie; Jezova, Jana; Bilkova, Zuzana; Benes, Milan; Horak, Daniel; Hradcova, Olga; Slovakova, Marcela; Viovy, Jean-Louis

    2005-01-01

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

  10. E1-forbidden transition rates in ions of astrophysical interest

    International Nuclear Information System (INIS)

    Träbert, E

    2014-01-01

    Transition rates in atomic systems may appear to be of little importance in steady-state plasmas that are observed at great distances from Earth. However, some of the transition rates compete with collision rates, and in these cases certain line intensity ratios are affected and can serve as remote indicators of density. In the low-density environments of stellar coronae and planetary nebulae, the transition rates of interest are mostly spin-forbidden E1 decays, higher-multipole order transitions (M1, E2, M2, M3), and hyperfine-induced transitions. On Earth, measurements of the long upper level lifetimes of these atomic systems require the use of ion traps. A fair number of test cases with lifetimes in the range from nanoseconds to many seconds have been treated successfully, and the evolution of calculations along with the experimental progress is notable. A new generation of cold ion traps is expected to extend the atomic lifetime measurements on multiply charged ions into the range of many minutes. (paper)

  11. E1 and M1 strength functions at low energy

    Science.gov (United States)

    Schwengner, Ronald; Massarczyk, Ralph; Bemmerer, Daniel; Beyer, Roland; Junghans, Arnd R.; Kögler, Toni; Rusev, Gencho; Tonchev, Anton P.; Tornow, Werner; Wagner, Andreas

    2017-09-01

    We report photon-scattering experiments using bremsstrahlung at the γELBE facility of Helmholtz-Zentrum Dresden-Rossendorf and using quasi-monoenergetic, polarized γ beams at the HIγS facility of the Triangle Universities Nuclear Laboratory in Durham. To deduce the photoabsorption cross sections at high excitation energy and high level density, unresolved strength in the quasicontinuum of nuclear states has been taken into account. In the analysis of the spectra measured by using bremsstrahlung at γELBE, we perform simulations of statistical γ-ray cascades using the code γDEX to estimate intensities of inelastic transitions to low-lying excited states. Simulated average branching ratios are compared with model-independent branching ratios obtained from spectra measured by using monoenergetic γ beams at HIγS. E1 strength in the energy region of the pygmy dipole resonance is discussed in nuclei around mass 90 and in xenon isotopes. M1 strength in the region of the spin-flip resonance is also considered for xenon isotopes. The dipole strength function of 74Ge deduced from γELBE experiments is compared with the one obtained from experiments at the Oslo Cyclotron Laboratory. The low-energy upbend seen in the Oslo data is interpreted as M1 strength on the basis of shell-model calculations.

  12. E1 and M1 strength functions at low energy

    Directory of Open Access Journals (Sweden)

    Schwengner Ronald

    2017-01-01

    Full Text Available We report photon-scattering experiments using bremsstrahlung at the γELBE facility of Helmholtz-Zentrum Dresden-Rossendorf and using quasi-monoenergetic, polarized γ beams at the HIγS facility of the Triangle Universities Nuclear Laboratory in Durham. To deduce the photoabsorption cross sections at high excitation energy and high level density, unresolved strength in the quasicontinuum of nuclear states has been taken into account. In the analysis of the spectra measured by using bremsstrahlung at γELBE, we perform simulations of statistical γ-ray cascades using the code γDEX to estimate intensities of inelastic transitions to low-lying excited states. Simulated average branching ratios are compared with model-independent branching ratios obtained from spectra measured by using monoenergetic γ beams at HIγS. E1 strength in the energy region of the pygmy dipole resonance is discussed in nuclei around mass 90 and in xenon isotopes. M1 strength in the region of the spin-flip resonance is also considered for xenon isotopes. The dipole strength function of 74Ge deduced from γELBE experiments is compared with the one obtained from experiments at the Oslo Cyclotron Laboratory. The low-energy upbend seen in the Oslo data is interpreted as M1 strength on the basis of shell-model calculations.

  13. The E1 beta-subunit of pyruvate dehydrogenase is surface-expressed in Lactobacillus plantarum and binds fibronectin.

    Science.gov (United States)

    Vastano, Valeria; Salzillo, Marzia; Siciliano, Rosa A; Muscariello, Lidia; Sacco, Margherita; Marasco, Rosangela

    2014-01-01

    Lactobacillus plantarum is among the species with a probiotic activity. Adhesion of probiotic bacteria to host tissues is an important principle for strain selection, because it represents a crucial step in the colonization process of either pathogens or commensals. Most bacterial adhesins are proteins, and a major target for them is fibronectin, an extracellular matrix glycoprotein. In this study we demonstrate that PDHB, a component of the pyruvate dehydrogenase complex, is a factor contributing to fibronectin-binding in L. plantarum LM3. By means of fibronectin overlay immunoblotting assay, we identified a L. plantarum LM3 surface protein with apparent molecular mass of 35 kDa. Mass spectrometric analysis shows that this protein is the pyruvate dehydrogenase E1 beta-subunit (PDHB). The corresponding pdhB gene is located in a 4-gene cluster encoding pyruvate dehydrogenase. In LM3-B1, carrying a null mutation in pdhB, the 35 kDa adhesin was not anymore detectable by immunoblotting assay. Nevertheless, the pdhB null mutation did not abolish pdhA, pdhC, and pdhD transcription in LM3-B1. By adhesion assays, we show that LM3-B1 cells bind to immobilized fibronectin less efficiently than wild type cells. Moreover, we show that pdhB expression is negatively regulated by the CcpA protein and is induced by bile. Copyright © 2013. Published by Elsevier GmbH.

  14. Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development.

    Science.gov (United States)

    Kowalewski-Nimmerfall, Elisabeth; Schähs, Philipp; Maresch, Daniel; Rendic, Dubravko; Krämer, Helmut; Mach, Lukas

    2014-12-01

    Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of >95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development. Copyright © 2014. Published by Elsevier B.V.

  15. Statins Suppress Ebola Virus Infectivity by Interfering with Glycoprotein Processing.

    Science.gov (United States)

    Shrivastava-Ranjan, Punya; Flint, Mike; Bergeron, Éric; McElroy, Anita K; Chatterjee, Payel; Albariño, César G; Nichol, Stuart T; Spiropoulou, Christina F

    2018-05-01

    Ebola virus (EBOV) infection is a major public health concern due to high fatality rates and limited effective treatments. Statins, widely used cholesterol-lowering drugs, have pleiotropic mechanisms of action and were suggested as potential adjunct therapy for Ebola virus disease (EVD) during the 2013-2016 outbreak in West Africa. Here, we evaluated the antiviral effects of statin (lovastatin) on EBOV infection in vitro Statin treatment decreased infectious EBOV production in primary human monocyte-derived macrophages and in the hepatic cell line Huh7. Statin treatment did not interfere with viral entry, but the viral particles released from treated cells showed reduced infectivity due to inhibition of viral glycoprotein processing, as evidenced by decreased ratios of the mature glycoprotein form to precursor form. Statin-induced inhibition of infectious virus production and glycoprotein processing was reversed by exogenous mevalonate, the rate-limiting product of the cholesterol biosynthesis pathway, but not by low-density lipoprotein. Finally, statin-treated cells produced EBOV particles devoid of the surface glycoproteins required for virus infectivity. Our findings demonstrate that statin treatment inhibits EBOV infection and suggest that the efficacy of statin treatment should be evaluated in appropriate animal models of EVD. IMPORTANCE Treatments targeting Ebola virus disease (EVD) are experimental, expensive, and scarce. Statins are inexpensive generic drugs that have been used for many years for the treatment of hypercholesterolemia and have a favorable safety profile. Here, we show the antiviral effects of statins on infectious Ebola virus (EBOV) production. Our study reveals a novel molecular mechanism in which statin regulates EBOV particle infectivity by preventing glycoprotein processing and incorporation into virus particles. Additionally, statins have anti-inflammatory and immunomodulatory effects. Since inflammation and dysregulation of the immune

  16. Development of glycoprotein capture-based label-free method for the high-throughput screening of differential glycoproteins in hepatocellular carcinoma.

    Science.gov (United States)

    Chen, Rui; Tan, Yexiong; Wang, Min; Wang, Fangjun; Yao, Zhenzhen; Dong, Liwei; Ye, Mingliang; Wang, Hongyang; Zou, Hanfa

    2011-07-01

    A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.

  17. Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells

    NARCIS (Netherlands)

    Westra, DF; Glazenburg, KL; Harmsen, MC; Tiran, A; Scheffer, AJ; Welling, GW; The, TH; WellingWester, S

    In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell

  18. 26 CFR 53.4941(e)-1 - Definitions.

    Science.gov (United States)

    2010-04-01

    ... value of the use of property shall be the higher of the rate (that is, fair rental value per period in... that the private foundation receives new stock worth only $95,000 in exchange for the stock which it... that the estate receives less than fair market value for the property exchanged, the amount involved is...

  19. Activation of the classical pathway of complement by tobacco glycoprotein (TGP).

    Science.gov (United States)

    Koethe, S M; Nelson, K E; Becker, C G

    1995-07-15

    Tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein isolated from tobacco leaves, activates the classical complement pathway through a mechanism that appears to involve direct interaction with C1q. A binding site on C1q for TGP can be localized by competitive inhibition with DNA to a region located in the junction between the collagen-like and globular regions of the molecule. A protein with activity similar to TGP has also been isolated from cigarette smoke condensate (TGP-S); it shares a binding site on C1q with TGP and has similar functional activity, with the exception that complement activation does not proceed to formation of a C3 cleaving enzyme. The ability of TGP and TGP-S to activate complement can be partially duplicated using polyphenols associated with tobacco leaf and smoke, i.e., chlorogenic acid and rutin. These polyphenols also compete with TGP for a binding site on immobilized C1q, suggesting that the polyphenol portion of TGP is critical for activation of complement. These results provide an additional mechanism for complement activation by cigarette products that, in vivo, could result in a localized complement depletion, generation of biologically active complement cleavage products, and initiation of an inflammatory response.

  20. Solution structure, copper binding and backbone dynamics of recombinant Ber e 1-the major allergen from Brazil nut.

    Directory of Open Access Journals (Sweden)

    Louise Rundqvist

    Full Text Available BACKGROUND: The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. METHODOLOGY/PRINCIPAL FINDINGS: The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using (15N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu(2+ in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. CONCLUSIONS/SIGNIFICANCE: The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu(2+ ions. This Cu(2+ binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu(2+ can in turn affect the interaction that is essential to provoke an allergenic response.

  1. Biosynthesis of ascites sialoglycoprotein-1, the major O-linked glycoprotein of 13762 rat mammary adenocarcinoma ascites cells

    International Nuclear Information System (INIS)

    Spielman, J.

    1987-01-01

    The present studies were undertaken to determine the timing of the major events in biosynthesis, and to characterize the contributions of chain initiation and elongation in maturation of the glycoprotein. Initiation of the earliest O-linked chains was detected by analysis of conversion of 3 H-thr to 3 H 2-aminobutyrate following mild alkaline borohydride elimination of O-linked sugars from peanut lectin-precipitated ASGP-1. Initiation was detected within 5 min of translation; amino sugar analysis of GlcNH 2 -labeled, trypsinized cells also showed that GalNAc was added as late as 5 min prior to arrival of ASGP-1 at the cell surface. Thus initiation occurs throughout biosynthesis. Maturation of the glycoprotein from a lightly-glycosylated immature form to the heavily-glycosylated mature from involved both continued initiation of new chains and chain elongation, and occurred with a half-time of about 30 min. Analysis of labeled ASGP-1 released from the cell surface by trypsinization showed that although some newly-synthesized ASGP-1 reached the cell surface within 70-80 min of protein synthesis, the half-time for appearance of mature glycoprotein was in excess of 4 hr, indicating that most molecules reside in an intracellular compartment(s) for a considerable time

  2. The role of reactive oxygen species (ROS and cytochrome P-450 2E1 in the generation of carcinogenic etheno-DNA adducts

    Directory of Open Access Journals (Sweden)

    Kirsten Linhart

    2014-01-01

    Full Text Available Exocyclic etheno-DNA adducts are mutagenic and carcinogenic and are formed by the reaction of lipidperoxidation (LPO products such as 4-hydoxynonenal or malondialdehyde with DNA bases. LPO products are generated either via inflammation driven oxidative stress or via the induction of cytochrome P-450 2E1 (CYP2E1. In the liver CYP2E1 is induced by various compounds including free fatty acids, acetone and ethanol. Increased levels of CYP2E1 and thus, oxidative stress are observed in the liver of patients with non-alcoholic steatohepatitis (NASH as well as in the chronic alcoholic. In addition, chronic ethanol ingestion also increases CYP2E1 in the mucosa of the oesophagus and colon. In all these tissues CYP2E1 correlates significantly with the levels of carcinogenic etheno-DNA adducts. In contrast, in patients with non-alcoholic steatohepatitis (NASH hepatic etheno-DNA adducts do not correlate with CYP2E1 indicating that in NASH etheno-DNA adducts formation is predominately driven by inflammation rather than by CYP2E1 induction. Since etheno-DNA adducts are strong mutagens producing various types of base pair substitution mutations as well as other types of genetic damage, it is strongly believed that they are involved in ethanol mediated carcinogenesis primarily driven by the induction of CYP2E1.

  3. Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant

    International Nuclear Information System (INIS)

    Whitt, M.A.; Chong, L.; Rose, J.K.

    1989-01-01

    The authors have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, they determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, they were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. The results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding

  4. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design

    Directory of Open Access Journals (Sweden)

    Alexander W. Tarr

    2015-07-01

    Full Text Available In the 26 years since the discovery of Hepatitis C virus (HCV a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design.

  5. A Novel Method for Detection of Glycoproteins on Sodium Dodecyl Sulphate Polyacrylamide Gel Using Radio-Iodinated Tyrosine

    DEFF Research Database (Denmark)

    Nalla, Amarnadh; Draz, Hossam M.; Dole, Anita

    2009-01-01

    The aim of this study is to develop a novel method for detection of glycoproteins on polyacrylamide gel. In this method, radio-iodinated-tyrosine (125I-tyrosine) was conjugated to glycoprotein by schiff's base mechanism on the sodium dodecyl sulfate- polyacrylamide gel. Ovalbumin and Concanavalin...... of glycoproteins using 125I-tyrosine selectively detected ovalbumin. Present results showed that MPD enhanced glycoprotein detection method can be used as a sensitive tool for the detection of glycoproteins on polyacrylamide gel...

  6. A recombinant E1-deleted porcine adenovirus-3 as an expression vector

    International Nuclear Information System (INIS)

    Zakhartchouk, Alexander; Zhou Yan; Tikoo, Suresh Kumar

    2003-01-01

    Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B large coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B large of PAV-3 and also complemented PAV214 (E1A+E1B small deleted). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B large coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B small + E1B large ) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B large was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines

  7. Determinants of foamy virus envelope glycoprotein mediated resistance to superinfection

    International Nuclear Information System (INIS)

    Berg, Angelika; Pietschmann, Thomas; Rethwilm, Axel; Lindemann, Dirk

    2003-01-01

    Little is known about the nature of foamy virus (FV) receptor molecules on target cells and their interaction with the viral glycoproteins. Similar to other viruses, cellular expression of the FV Env protein is sufficient to induce resistance to exogenous FV, a phenomenon called superinfection resistance (SIR). In this study we define determinants of the FV Env protein essential for mediating SIR. FV Env requires the extracellular domains of the SU and the TM subunits as well as membrane anchorage, efficient cell surface transport, and most probably correct subunit processing. This is in contrast to murine leukemia virus where secreted proteins comprising the receptor-binding domain in SU are sufficient to induce SIR. Furthermore, we demonstrate that cellular expression of the prototype FV envelope proteins induces SIR against pseudotypes with glycoproteins of other FV species, including of simian, feline, bovine, and equine origin. This implies that all of them use the same receptor molecules for viral entry

  8. TROPHOBLASTIC β1 – GLYCOPROTEIN SYNTHESIS IN SEROPOSITIVE PREGNANT WOMEN

    Directory of Open Access Journals (Sweden)

    R. N. Bogdanovich

    2005-01-01

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

  9. Comparison of glycoprotein expression between ovarian and colon adenocarcinomas

    DEFF Research Database (Denmark)

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

    1999-01-01

    , carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both...... primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use...... in distinguishing between these 2 entities. CONCLUSION: A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot...

  10. Antigiardial activity of glycoproteins and glycopeptides from Ziziphus honey.

    Science.gov (United States)

    Mohammed, Seif Eldin A; Kabashi, Ahmed S; Koko, Waleed S; Azim, M Kamran

    2015-01-01

    Natural honey contains an array of glycoproteins, proteoglycans and glycopeptides. Size-exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular masses in the range from 10 to >200 kDa. The fractionated proteins exhibited in vitro activities against Giardia lamblia with IC50 values ≤ 25 μg/mL. Results indicated that honey proteins were more active as antiprotozoal agents than metronidazole. This study indicated the potential of honey proteins and peptides as novel antigiardial agents.

  11. A Simplified Model of Glycoprotein Production within Cell Culture

    OpenAIRE

    Lambert, A. B.; Smith, F. T.; Velayudhan, A.

    2017-01-01

    Complex biological products, such as those used to treat various forms of cancer, are typically produced by mammalian cells in bioreactors. The most important class of such biological medicines is proteins. These typically bind to sugars (glycans) in a process known as glycosylation, creating glycoproteins, which are more stable and effective medicines. The glycans are large polymers that are formed by a long sequence of enzyme catalysed reactions. This sequence is not always completed, thus ...

  12. Tumor specific glycoproteins and method for detecting tumorigenic cancers

    International Nuclear Information System (INIS)

    Davidson, E.A.; Bolmer, S.D.

    1981-01-01

    The detection of tumour specific glycoproteins (TSGP) in human sera often indicates the presence of a malignant tumour in a patient. The distinguishing characteristics of TSGP isolated from the blood sera of cancer patients are described in detail together with methods of TSGP isolation and purification. Details are also given of radioimmunoassay techniques capable of detecting very low levels of serum TSGP with high specificity. (U.K.)

  13. Mucus glycoprotein secretion by tracheal explants: effects of pollutants

    International Nuclear Information System (INIS)

    Last, J.A.; Kaizu, T.

    1980-01-01

    Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might by the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone. After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose. Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with [3H] glucosamine, does not contain radioactivity when Na2 35SO4 is used as the precursor. Acidic fractions I to IV are all labeled with either 3H-glucosamine or Na2 35SO4 as precursor. Acidic fraction II contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns of wheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques

  14. Structure, dynamics, and interaction of Mycobacterium tuberculosis (Mtb DprE1 and DprE2 examined by molecular modeling, simulation, and electrostatic studies.

    Directory of Open Access Journals (Sweden)

    Isha Bhutani

    Full Text Available The enzymes decaprenylphosphoryl-β-D-ribose oxidase (DprE1 and decaprenylphosphoryl-β-D-ribose-2-epimerase (DprE2 catalyze epimerization of decaprenylphosporyl ribose (DPR todecaprenylphosporyl arabinose (DPA and are critical for the survival of Mtb. Crystal structures of DprE1 so far reported display significant disordered regions and no structural information is known for DprE2. We used homology modeling, protein threading, molecular docking and dynamics studies to investigate the structural and dynamic features of Mtb DprE1 and DprE2 and DprE1-DprE2 complex. A three-dimensional model for DprE2 was generated using the threading approach coupled with ab initio modeling. A 50 ns simulation of DprE1 and DprE2 revealed the overall stability of the structures. Principal Component Analysis (PCA demonstrated the convergence of sampling in both DprE1 and DprE2. In DprE1, residues in the 269-330 area showed considerable fluctuation in agreement with the regions of disorder observed in the reported crystal structures. In DprE2, large fluctuations were detected in residues 95-113, 146-157, and 197-226. The study combined docking and MD simulation studies to map and characterize the key residues involved in DprE1-DprE2 interaction. A 60 ns MD simulation for DprE1-DprE2 complex was also performed. Analysis of data revealed that the docked complex is stabilized by H-bonding, hydrophobic and ionic interactions. The key residues of DprE1 involved in DprE1-DprE2 interactions belong to the disordered region. We also examined the docked complex of DprE1-BTZ043 to investigate the binding pocket of DprE1 and its interactions with the inhibitor BTZ043. In summary, we hypothesize that DprE1-DprE2 interaction is crucial for the synthesis of DPA and DprE1-DprE2 complex may be a new therapeutic target amenable to pharmacological validation. The findings have important implications in tuberculosis (TB drug discovery and will facilitate drug development efforts against

  15. Extracellular Glycoproteins in Embryogenic Culture of Pumpkin (Cucurbita pepo L.

    Directory of Open Access Journals (Sweden)

    Hana Čipčić Paljetak

    2011-01-01

    Full Text Available The extracellular proteins in three distinctly induced embryogenic lines of pumpkin (Cucurbita pepo L. cultivated in four MS media modified regarding the nitrogen composition or auxin presence/absence have been analyzed. Extracellular glycoproteins containing α-D-mannose were specifically detected by the lectine concavalin A. During the cultivation of embryogenic tissue in the medium supplemented with reduced nitrogen, the embryos were mostly arrested at preglobular and globular developmental stages, which coincide with the absence of protein secretion. Secreted glycoproteins of 76, 68, 37 and 34 kDa were detected only if any of the three lines were cultivated in the medium that stimulates embryo development, irrespectively of the addition of 2,4-dichlorophenoxyacetic acid or tunicamycin. The glycoprotein of 64 kDa was detected in all lines cultivated in hormone-free MS medium with conventional nitrogen sources and it appears to be associated with embryo maturation. Tunicamycin treatment did not influence embryogenesis, although it specifically affected glycosylation of proteins in the investigated lines. Our results show that besides auxin, the source of nitrate is of great importance for proper protein glycosylation, excretion and developmental transition of pumpkin somatic embryos.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Beata Olejnik

    2015-04-01

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

  18. The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

    Science.gov (United States)

    Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

    2015-01-02

    Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-03-07

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

  20. Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection

    International Nuclear Information System (INIS)

    Tsvitov, Marianna; Frampton, Arthur R.; Shah, Waris A.; Wendell, Steven K.; Ozuer, Ali; Kapacee, Zoher; Goins, William F.; Cohen, Justus B.; Glorioso, Joseph C.

    2007-01-01

    Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection

  1. Retention and topology of the bovine viral diarrhea virus glycoprotein E2.

    Science.gov (United States)

    Radtke, Christina; Tews, Birke Andrea

    2017-10-01

    Pestiviruses are enveloped viruses that bud intracellularly. They have three envelope glycoproteins, E rns , E1, and E2. E2 is the receptor binding protein and the main target for neutralizing antibodies. Both E rns and E2 are retained intracellularly. Here, E2 of the bovine viral diarrhea virus (BVDV) strain CP7 was used to study the membrane topology and intracellular localization of the protein. E2 is localized in the ER and there was no difference between E2 expressed alone or in the context of the viral polyprotein. The mature E2 protein was found to possess a single span transmembrane anchor. For the mapping of a retention signal CD72-E2 fusion proteins, as well as E2 alone were analysed. This confirmed the importance of the transmembrane domain and arginine 355 for intracellular retention, but also revealed a modulating effect on retention through the cytoplasmic tail of the E2 protein, especially through glutamine 370. Mutants with a strong impact on retention were tested in the viral context and we were able to rescue BVDV with certain mutations that in E2 alone impaired intracellular retention and lead to export of E2 to the cells surface.

  2. Tumor Suppression and Sensitization to Taxol Induced Apoptosis of E1A In Breast Cancer Cells

    National Research Council Canada - National Science Library

    Liao, Yong

    2004-01-01

    .... Because a phase I E1A gene therapy protocol for human breast and ovarian cancers was completed and a phase II clinical trial is undergoing, we also plan to develop an alternative E1A mutant construct...

  3. Genotype and allelic frequencies of CYP2E1*5B polymorphism in the southwest population of Iran

    Directory of Open Access Journals (Sweden)

    Fatemeh Zanganeh

    2014-10-01

    Full Text Available Background: Cytochrome P450 2E1 (CYP2E1 is a main enzyme which plays a major role in activating and detoxifying many xenobiotics, carcinogens and drugs. Available studies suggest that CYP2E1 single nucleotide polymorphisms (SNPs are involved in the risk of developing certain cancers after exposure to carcinogens. The purpose of the present study was to assess genotype and allele frequencies of polymorphic CYP2E1*5B in the Iranian population. Material and Methods: This study was performed on 200 healthy individuals (female: 100, male: 100 in medical laboratories of Ahvaz during 2011. The CYP2E1 *5B (rs3813867 G-1293C assessment was carried out using PCR-RFLP method. The data were analyzed with ĸ2 and hardy-Weinberg Equation statistically methods. Results: The frequency of *1A/*1A (c1/c1, *1A/*5B (c1/c2 and *5B/*5B (c2/c2 genotypes was computed 97, 3 and 0 percent, respectively. The frequency of *1A (c1 and *5B (c2 alleles was computed 98.5 and 1.5 percent, respectively. No statistically significant difference was between two genders (p>0.05. Conclusion: The genotype distribution and allele frequencies of CYP2E1*5B polymorphism were similar to Turkish and some of the European populations. However, there are significant interethnic differences when the Iranian population is compared with the Eastern Asian, American and some of the European populations. The allelic distribution of this polymorphism did not vary with gender.

  4. Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

    Science.gov (United States)

    Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

    2006-01-01

    Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

  5. Isolation and characterization of two antigenic glycoproteins from the pollen of Prosopis juliflora.

    Science.gov (United States)

    Thakur, I S

    1991-03-01

    Two antigenically active glycoprotein fractions were isolated from crude extract of the pollen of Prosopis juliflora using DEAE-cellulose ion exchange chromatography. The glycoproteins gave single band on polyacrylamide gel electrophoresis. The molecular weight of these two glycoprotein was 20,000 and 10,000 as determined by gel filtration on Sephadex G-75. With the help of crossed immunoelectrophoresis and gel diffusion crude extract exhibited twelve and three precipitating antigens suggesting its heterogeneous nature; and the purified glycoprotein fractions however formed single precipitin band on gel diffusion test and immunoelectrophoresis. As tested by ELISA the polyclonal antisera raised in rabbit showed strong binding affinity with glycoprotein of MW 20,000. These result indicates that the two glycoprotein fractions are not antigenically identical.

  6. Three-dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation

    OpenAIRE

    Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W. Andy

    2016-01-01

    Glycoproteins have vast structural diversity which plays an important role in many biological processes and have great potential as disease biomarkers. Here we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase GlycoProtein Array (polyGPA), to specifically capture and profile glycoproteomes, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture pre-oxidized glycan...

  7. Development of a radioimmunoassay for 'Tamm-Horsfall-like' glycoprotein in serum and cerebrospinal fluid

    International Nuclear Information System (INIS)

    Hartmann, L.; Bringuier, A.-F.; Schuller, E.

    1983-01-01

    Affinity chromatography purification was combined with a radioimmunoassay for 'Tamm-Horsfall-like' glycoprotein. This enabled serum comcentrations to be established and to demonstrate its presence in cerebrospinal fluid for the first time. This assay method used in different circumstances suggests a multifocal synthesis. Nevertheless, urinary Tamm-Horsfall glycoprotein so far must be distinguished from the serum or cerebrospinal fluid Tamm-Horsfall-like glycoprotein. (Auth.)

  8. Infection with E1B-mutant adenovirus stabilizes p53 but blocks p53 acetylation and activity through E1A

    DEFF Research Database (Denmark)

    Savelyeva, I.; Dobbelstein, M.

    2011-01-01

    to the suppression of p21 transcription. Depending on the E1A conserved region 3, E1B-defective adenovirus impaired the ability of the transcription factor Sp1 to bind the p21 promoter. Moreover, the amino terminal region of E1A, binding the acetyl transferases p300 and CREB-binding protein, blocked p53 K382...... accumulation of p53, without obvious defects in p53 localization, phosphorylation, conformation and oligomerization. Nonetheless, p53 completely failed to induce its target genes in this scenario, for example, p21/CDKN1A, Mdm2 and PUMA. Two regions of the E1A gene products independently contributed...... acetylation in infected cells. Mutating either of these E1A regions, in addition to E1B, partially restored p21 mRNA levels. Our findings argue that adenovirus attenuates p53-mediated p21 induction, through at least two E1B-independent mechanisms. Other virus species and cancer cells may employ analogous...

  9. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production

    International Nuclear Information System (INIS)

    Jarvis, Donald L.

    2003-01-01

    The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains

  10. CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro

    Directory of Open Access Journals (Sweden)

    A. Rossini

    2006-02-01

    Full Text Available Cytochrome P450 (CYP is a superfamily of enzymes involved in the metabolism of endogenous compounds and xenobiotics. CYP2A6 catalyzes the oxidation of nicotine and the activation of carcinogens such as aflatoxin B1 and nitrosamines. CYP2E1 metabolizes ethanol and other low-molecular weight compounds and can also activate nitrosamines. The CYP2A6 and CYP2E1 genes are polymorphic, altering their catalytic activities and susceptibility to cancer and other diseases. A number of polymorphisms described are ethnic-dependent. In the present study, we determined the genotype and allele frequencies of the main CYP2A6 and CYP2E1 polymorphisms in a group of 289 volunteers recruited at the Central Laboratory of Hospital Universitário Pedro Ernesto. They had been residing in the city of Rio de Janeiro for at least 6 months and were divided into two groups according to skin color (white and non-white. The alleles were determined by allele specific PCR (CYP2A6 or by PCR-RFLP (CYP2E1. The frequencies of the CYP2A6*1B and CYP2A6*2 alleles were 0.29 and 0.02 for white individuals and 0.24 and 0.01 for non-white individuals, respectively. The CYP2A6*5 allele was not found in the population studied. Regarding the CYP2E1*5B allele, we found a frequency of 0.07 in white individuals, which was statistically different (P < 0.05 from that present in non-white individuals (0.03. CYP2E1*6 allele frequency was the same (0.08 in both groups. The frequencies of CYP2A6*1B, CYP2A6*2 and CYP2E1*6 alleles in Brazilians are similar to those found in Caucasians and African-Americans, but the frequency of the CYP2E1*5B allele is higher in Brazilians.

  11. CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro

    Directory of Open Access Journals (Sweden)

    Rossini A.

    2006-01-01

    Full Text Available Cytochrome P450 (CYP is a superfamily of enzymes involved in the metabolism of endogenous compounds and xenobiotics. CYP2A6 catalyzes the oxidation of nicotine and the activation of carcinogens such as aflatoxin B1 and nitrosamines. CYP2E1 metabolizes ethanol and other low-molecular weight compounds and can also activate nitrosamines. The CYP2A6 and CYP2E1 genes are polymorphic, altering their catalytic activities and susceptibility to cancer and other diseases. A number of polymorphisms described are ethnic-dependent. In the present study, we determined the genotype and allele frequencies of the main CYP2A6 and CYP2E1 polymorphisms in a group of 289 volunteers recruited at the Central Laboratory of Hospital Universitário Pedro Ernesto. They had been residing in the city of Rio de Janeiro for at least 6 months and were divided into two groups according to skin color (white and non-white. The alleles were determined by allele specific PCR (CYP2A6 or by PCR-RFLP (CYP2E1. The frequencies of the CYP2A6*1B and CYP2A6*2 alleles were 0.29 and 0.02 for white individuals and 0.24 and 0.01 for non-white individuals, respectively. The CYP2A6*5 allele was not found in the population studied. Regarding the CYP2E1*5B allele, we found a frequency of 0.07 in white individuals, which was statistically different (P < 0.05 from that present in non-white individuals (0.03. CYP2E1*6 allele frequency was the same (0.08 in both groups. The frequencies of CYP2A6*1B, CYP2A6*2 and CYP2E1*6 alleles in Brazilians are similar to those found in Caucasians and African-Americans, but the frequency of the CYP2E1*5B allele is higher in Brazilians.

  12. Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

    Directory of Open Access Journals (Sweden)

    A.T. Da Poian

    2005-06-01

    Full Text Available Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

  13. High-performance liquid chromatography of human glycoprotein hormones.

    Science.gov (United States)

    Chlenov, M A; Kandyba, E I; Nagornaya, L V; Orlova, I L; Volgin, Y V

    1993-02-12

    The chromatographic behavior of the glycoprotein hormones from human pituitary glands and of placental origin [thyroid-stimulating hormone, luteinizing hormone and chorionic gonadotropin (CG)] was studied. It was shown that hydrophobic interaction chromatography on a microparticulate packing and anion-exchange HPLC can be applied for the purification of these hormones. Reversed-phase HPLC on wide-pore C4-bonded silica at neutral pH can be applied for the determination of the above hormones and for the isolation of pure CG and its subunits.

  14. Cooperative role of calnexin and TigA in Aspergillus oryzae glycoprotein folding.

    Science.gov (United States)

    Wang, Ning; Seko, Akira; Takeda, Yoichi; Kikuma, Takashi; Ito, Yukishige

    2015-10-01

    Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G1M9GN2-proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae, which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G1M9GN2-ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G1M9GN2-RNase was chemoenzymatically synthesized from G1M9GN-oxazoline and GN-RNase. Denatured G1M9GN2-RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae, TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Role of CYP2E1-mediated metabolism in the acute and vestibular toxicities of nineteen nitriles in the mouse.

    Science.gov (United States)

    Saldaña-Ruíz, Sandra; Soler-Martín, Carla; Llorens, Jordi

    2012-01-25

    Allylnitrile, cis-crotononitrile, and 3,3'-iminodipropionitrile are known to cause vestibular toxicity in rodents, and evidence is available indicating that cis-2-pentenenitrile shares this effect. We evaluated nineteen nitriles for vestibular toxicity in wild type (129S1) and CYP2E1-null mice, including all the above, several neurotoxic nitriles, and structurally similar nitriles. A new acute toxicity test protocol was developed to facilitate evaluation of the vestibular toxicity by a specific behavioral test battery at doses up to sub-lethal levels while using a limited number of animals. A mean number of 8.5±0.3 animals per nitrile, strain and sex was necessary to obtain evidence of vestibular toxicity and optionally an estimation of the lethal dose. For several but not all nitriles, lethal doses significantly increased in CYP2E1-null mice. The protocol revealed the vestibular toxicity of five nitriles, including previously identified ototoxic compounds and one nitrile (trans-crotononitrile) known to have a different profile of neurotoxic effects in the rat. In all five cases, both sexes were affected and no decrease in susceptibility was apparent in CYP2E1-null mice respect to 129S1 mice. Fourteen nitriles caused no vestibular toxicity, including six nitriles tested in CYP2E1-null mice at doses significantly larger than the maximal doses that can be tested in wild type animals. We conclude that only a subset of low molecular weight nitriles is toxic to the vestibular system, that species-dependent differences exist in this vestibular toxicity, and that CYP2E1-mediated metabolism is not involved in this effect of nitriles although it has a role in the acute lethality of some of these compounds. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA Is a Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Isabelle Bleiziffer

    2017-01-01

    Full Text Available Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA strains by SDS-PAGE and subsequent periodic acid-Schiff's staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa and two (>300 and ∼175 kDa glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCCmec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and

  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. The cell surface expressed nucleolin is a glycoprotein that triggers calcium entry into mammalian cells

    International Nuclear Information System (INIS)

    Losfeld, Marie-Estelle; Khoury, Diala El; Mariot, Pascal; Carpentier, Mathieu; Krust, Bernard; Briand, Jean-Paul; Mazurier, Joel; Hovanessian, Ara G.; Legrand, Dominique

    2009-01-01

    Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. It has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Several studies have demonstrated that surface nucleolin serves as a receptor for various extracellular ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the presence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon metabolic labeling of cells with [ 3 H]glucosamine. Besides its well-demonstrated role in the internalization of specific ligands, here we show that ligand binding to surface nucleolin could also induce Ca 2+ entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopeptide, which binds specifically surface nucleolin, triggers rapid and intense membrane Ca 2+ fluxes in various types of cells. The use of several drugs then indicated that Store-Operated Ca 2+ Entry (SOCE)-like channels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to surface nucleolin could be involved in the activation of signaling pathways by promoting Ca 2+ entry into cells

  19. Parental involvement

    Directory of Open Access Journals (Sweden)

    Ezra S Simon

    2005-01-01

    Full Text Available Parent-Teacher Associations and other community groups can play a significant role in helping to establish and run refugee schools; their involvement can also help refugee adults adjust to their changed circumstances.

  20. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing, E-mail: allenylq@hotmail.com; Liao, Er-Yuan, E-mail: eyliao@21cn.com

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  1. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    International Nuclear Information System (INIS)

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing; Liao, Er-Yuan

    2013-01-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  2. Gamma-radiolysis of some glycoproteins in dilute aqueous solutions

    Energy Technology Data Exchange (ETDEWEB)

    Nagrani, S

    1981-01-01

    A study has been made of the radiation-induced damage of some glycoproteins in dilute aqueous solutions. By use of specific radical scavengers, the roles of the individual free radicals, formed by ..gamma..-radiolysis, in causing damage has been assessed. The most effective radical in causing damage to human and porcine glycopolypeptide is the OH radical. The structure of the different blood group glycopolypeptides determines the sensitivity towards the free radical attack. The glycopolypeptide shows depolymerization and a characteristic absorption at approximately 270 nm due to the formation of additional products on irradiation. Chemical changes of the irradiated glycopolypeptide solutions revealed significant damage to the oligosaccharide chain and the polypeptide core of the glycopolypeptide. The radiation-induced inactivation of another glycoprotein, external yeast invertase, due to different radical species at pH 7.0 decreases in the following order: ea-barq > OH radical > (SCN) radical/sub 2//sup -/ > Br radical/sub 2//sup -/. The structure of this enzyme, accounts for the mechanism of enzyme inactivation and the relative damage of carbohydrate and amino acid residues. The irradiated enzyme solutions show significant changes in their electrophoretic behaviour on cellogel electrophoresis due to the formation of radiolysis products, which also show characteristic absorption maxima at approximately 275 nm. (author).

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

    Directory of Open Access Journals (Sweden)

    Aurélie A Albertini

    2012-02-01

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

  4. The Lyssavirus glycoprotein: A key to cross-immunity.

    Science.gov (United States)

    Buthelezi, Sindisiwe G; Dirr, Heini W; Chakauya, Ereck; Chikwamba, Rachel; Martens, Lennart; Tsekoa, Tsepo L; Stoychev, Stoyan H; Vandermarliere, Elien

    2016-11-01

    Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. The Lyssavirus glycoprotein: A key to cross-immunity

    International Nuclear Information System (INIS)

    Buthelezi, Sindisiwe G.; Dirr, Heini W.; Chakauya, Ereck; Chikwamba, Rachel; Martens, Lennart; Tsekoa, Tsepo L.; Stoychev, Stoyan H.; Vandermarliere, Elien

    2016-01-01

    Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. -- Highlights: •The current PEP has raised safety and availability concerns. •Cocktails of mAbs have been proposed as alternative treatment. •Amino acid conservation amongst Lyssavirus G proteins was studied. •Possible cross-neutralizing B-cell epitopes were proposed.

  6. The Lyssavirus glycoprotein: A key to cross-immunity

    Energy Technology Data Exchange (ETDEWEB)

    Buthelezi, Sindisiwe G. [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg (South Africa); Dirr, Heini W. [Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg (South Africa); Chakauya, Ereck; Chikwamba, Rachel [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Martens, Lennart [Unit for Computational Omics and Systems Biology, Medical Biotechnology Center, VIB, Ghent (Belgium); Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent (Belgium); Tsekoa, Tsepo L. [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Stoychev, Stoyan H., E-mail: Sstoychev@csir.co.za [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Vandermarliere, Elien [Unit for Computational Omics and Systems Biology, Medical Biotechnology Center, VIB, Ghent (Belgium); Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent (Belgium); Bioinformatics Institute Gent, Ghent University, Ghent (Belgium)

    2016-11-15

    Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. -- Highlights: •The current PEP has raised safety and availability concerns. •Cocktails of mAbs have been proposed as alternative treatment. •Amino acid conservation amongst Lyssavirus G proteins was studied. •Possible cross-neutralizing B-cell epitopes were proposed.

  7. 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

  8. 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

  9. Filamentous fungi as production organisms for glycoproteins of bio-medical interest

    NARCIS (Netherlands)

    Maras, M.; Die, I. van; Contreras, R.; Hondel, C.A.M.J.J. van den

    1999-01-01

    Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20-40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but

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

    International Nuclear Information System (INIS)

    Ahn, Hae-Young.

    1988-01-01

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

  11. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    Science.gov (United States)

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-05

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  12. Shell effects on the E1 moments of Ra-Th nuclei

    International Nuclear Information System (INIS)

    Leander, G.A.

    1984-01-01

    Large systematic shell effects on intrinsic E1 moments are found, which should modulate any E1 moment induced by β 3 deformation. The calculated shell effects can explain an emerging trend for E1 data in Ra-Th nuclei, if and only if the gross β 3 -induced polarization of finite nuclear matter goes in the same direction as the lightning rod effect. 16 references

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

    Science.gov (United States)

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

    2016-10-02

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

  14. Improved radioimmunoassay for urinary Tamm-Horsfall glycoprotein. Investigation and resolution of factors affecting its quantification

    Energy Technology Data Exchange (ETDEWEB)

    Dawney, A B.St.J.; Thornley, C; Cattell, W R [Saint Bartholomew' s Hospital, London (UK)

    1982-09-15

    A rapid specific radioimmunoassay has been used to measure Tamm-Horsfall glycoprotein (TH glycoprotein) in urine, and the method described. The apparent concentration increased with increasing dilution of urine in water, reaching a plateau at 1 in 20. This increase was greater the higher the osmolality and TH glycoprotein concentration and the lower the pH of the original sample. The apparent concentration of TH glycoprotein in neat or diluted urine was not affected by freezing or by storage at 4/sup 0/C or room temperature for at least 2 days. A physiological range for the urinary excretion rate was established as 22-56 mg/24h, (considerably higher than the amount present in serum) based on samples from 29 individuals with normal renal function, as defined by their creatinine clearance. There was no significant correlation between serum concentrations of TH glycoprotein and its urinary excretion rate, nor between urinary excretion rate and creatinine clearance.

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

    DEFF Research Database (Denmark)

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

    1993-01-01

    We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had...... in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate...... the complexity of glycoconjugate synthesis in mouse vagina and reveal the distinct cycle-specific patterns of individual glycoprotein expression. These cyclic glycoproteins could serve as vaginal biochemical markers for the specific phases of the estrous cycle....

  16. Purification and characterization of the glycoprotein allergen from Prosopis juliflora pollen.

    Science.gov (United States)

    Thakur, I S

    1991-02-01

    Highly active glycoprotein allergens have been isolated from pollen of Prosopis juliflora by a combination of Sephadex G-100 gel filtration and Sodium dodecyl sulphate-Poly-acrylamide gel electrophoresis. The glycoprotein fraction was homogeneous, and had molecular weight 20,000. The purified glycoprotein allergen contained 20% carbohydrate, mainly arabinose and galactose. Enzymatic digestion of glycoprotein with protease released glycopeptides of molecular weight ranging from less than 1,000 to more than 5,000 on Sephadex G-25 gel filtration. Antigenicity or allergenicity testing of these glycopeptides by immunodiffusion, immunoelectrophoresis, and radioallergosorbent test indicated complete loss of allergenic activity after digestion with protease whereas incubation with beta-D-galactosidase and periodate oxidation had little affect on the allergenic activity of the glycoprotein fraction. But incubation with alpha-D-glucosidase did not affect the allergenic activity significantly. All these tests indicated that protein played significant role in allergenicity of P. juliflora pollen.

  17. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    Energy Technology Data Exchange (ETDEWEB)

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R. [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Yousef, Ahmed F. [Department of Chemical and Environmental Engineering, Masdar Institute, Abu Dhabi (United Arab Emirates); Zhang, Zhiying [College of Animal Science and Technologies, Northwest A and F University, Yangling, Shaanxi 712100 (China); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Department of Oncology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada)

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  18. The effect of lycopene on cytochrome P450 isoenzymes and P-glycoprotein by using human liver microsomes and Caco-2 cell monolayer model.

    Science.gov (United States)

    Kong, Lingti; Song, Chunli; Ye, Linhu; Xu, Jian; Guo, Daohua; Shi, Qingping

    2018-01-11

    Lycopene is widely used as a dietary supplement. However, the effects of lycopene on cytochrome P450 (CYP) enzymes or P-glycoprotein (P-gp) are not comprehensive. The present study was performed to investigate the effects of lycopene on the CYP enzymes and P-gp activity. A cocktail method was used to evaluate the activities of CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Caco-2 cell monolayer model was carried out to assay lycopene on P-gp activity. The results indicated that lycopene had a moderate inhibitory effect on CYP2E1, with IC50 value of 43.65 μM, whereas no inhibitory effects on CYP3A4, CYP2C19, CYP2D6 and CYP2E1, with IC50 values all over 100 μM. In addition, lycopene showed almost no inhibitory effect on rhodamine-123 efflux and uptake (p > .05), indicated no effects on P-gp activity. In conclusion, there should be required attention when lycopene are coadministered with other drugs that are metabolised by CYP2E1.

  19. Determination of P-Glycoprotein Expression by Flow Cytometry in Hematological Malignancies

    Directory of Open Access Journals (Sweden)

    Berkay Saraymen

    2016-03-01

    Full Text Available Objective: Determination the expression of P-glycoprotein is especially problematic for normal tissues because immuno­logical methods are limited in terms of sensitivity. We aimed to determine the expression of P-glycoprotein and CD34 by flow cytometry, and to evaluate the level of expression of P-glycoprotein and CD34 with unresponsive to treatment in pa­tients diagnosed with hematologic malignancy. Methods: Our study included fifty patients diagnosed with acute myeloblastic leukemia and acute lymphoblastic leuke­mia, and twenty healthy controls who were admitted to Erci­yes University Hematology-Oncology Hospital. The suspend­ed cells from bone marrow samples of patients and the pe­ripheral blood samples of healthy people were marked with P-glycoprotein phycoerythrin and CD34 FITC or PerCP Cy 5.5; and then surface expression was measured by means of flow cytometry. Results: In 6 of 30 acute myeloblastic leukemia patients P-glycoprotein and CD34 expression, in 6 of 20 acute lympho­blastic leukemia patients P-glycoprotein, in 5 of them CD34 expression were determined. A significant relation between P-glycoprotein and CD34 expressions in acute myeloblas­tic leukemia and acute lymphoblastic leukemia bone marrow samples was reported. Conclusion: Our data indicate that flow cytometry is more reliable, precise and faster than molecular methods for mea­suring P-glycoprotein expression and suggests the pos­sibility of a significant relationship between P-glycoprotein and CD34 expressions in acute myeloblastic leukemia and acute lymphoblastic leukemia bone marrow samples. The blast cells expressing CD34 on their surface along with P-glycoprotein simultaneously show that multi drug resistance 1 gene is mostly active in immature cells.

  20. Human platelet glycoprotein IX: An adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures

    International Nuclear Information System (INIS)

    Hickey, M.J.; Williams, S.A.; Roth, G.J.

    1989-01-01

    The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (M r , 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex. GPIb (M r , 165,000). Using antibody screening, the authors cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage λgt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A) + RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acids followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined

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

    Directory of Open Access Journals (Sweden)

    Sasnauskas Kęstutis

    2011-05-01

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

  2. Correlation between spermatogenesis disorders and rat testes CYP2E1 mRNA contents under experimental alcoholism or type I diabetes.

    Science.gov (United States)

    Shayakhmetova, Ganna M; Bondarenko, Larysa B; Matvienko, Anatoliy V; Kovalenko, Valentina M

    2014-09-01

    The aim of the study was to investigate the correlation between spermatogenesis disorders and CYP2E1 mRNA contents in testes of rats with experimental alcoholism or type I diabetes. Two pathological states characterized by CYP2E1 induction were simulated on Wistar male rats: experimental alcoholism and type I diabetes. As controls for each state, equal number of animals (of the same age and weight) were used. Morphological evaluation of rat testes was carried out. The spermatogenic epithelium state was estimated by four points system. CYP2E1 mRNA expression was rated by method of reverse transcriptase polymerase chain reaction. Pearson correlation coefficients were used for describing relationships between variables. The presence of alcoholism and diabetes-mediated quantitative and qualitative changes in male rat spermatogenic epithelium in comparison with norm has been demonstrated. The increased levels of testes CYP2E1 have been fixed simultaneously. CYP2E1 mRNA content negatively strongly correlated with spermatogenic index value (r=-0.99; Palcoholism. The strong correlation between CYP2E1 mRNA content and number of spermatogonia (r=0.99; P<0.001) and "windows" occurrence (r=0.96; P<0.001) has been fixed in diabetic rats testes. Present investigation has demonstrated that the testicular failure following chronic ethanol consumption and diabetes type I in male rats accompanied CYP2E1 mRNA over-expression in testes. The correlation between the levels of CYP2E1 mRNA in testes and spermatogenesis disorders allow supposing the involvement of CYP2E1 into the non-specific pathogenetic mechanisms of male infertility under above-mentioned pathologies. Copyright © 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  3. Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

    Science.gov (United States)

    Schmouth, Jean-François; Arenillas, David; Corso-Díaz, Ximena; Xie, Yuan-Yun; Bohacec, Slavita; Banks, Kathleen G; Bonaguro, Russell J; Wong, Siaw H; Jones, Steven J M; Marra, Marco A; Simpson, Elizabeth M; Wasserman, Wyeth W

    2015-07-24

    Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

  4. Monomer-dimer control of the ColE1 P(cer) promoter.

    Science.gov (United States)

    Chatwin, H M; Summers, D K

    2001-11-01

    XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P(cer), located centrally within cer, is also required for stable plasmid maintenance. P(cer) is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P(cer) in plasmid monomers. P(cer) is unusual in that the -35 and -10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P(cer) activation involves realignment of the -35 and -10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

  5. California serogroup GC (G1) glycoprotein is the principal determinant of pH-dependent cell fusion and entry

    International Nuclear Information System (INIS)

    Plassmeyer, Matthew L.; Soldan, Samantha S.; Stachelek, Karen M.; Martin-Garcia, Julio; Gonzalez-Scarano, Francisco

    2005-01-01

    Members of the California serogroup of orthobunyaviruses, particularly La Crosse (LAC) and Tahyna (TAH) viruses, are significant human pathogens in areas where their mosquito vectors are endemic. Previous studies using wild-type LAC and TAH181/57, a highly neurovirulent strain with low neuroinvasiveness (Janssen, R., Gonzalez-Scarano, F., Nathanson, N., 1984. Mechanisms of bunyavirus virulence. Comparative pathogenesis of a virulent strain of La Crosse and an avirulent strain of Tahyna virus. Lab. Invest. 50 (4), 447-455), have demonstrated that the neuroinvasive phenotype maps to the M segment, the segment that encodes the two viral glycoproteins GN (G2) and GC (G1), as well as a non-structural protein NSm. To further define the role of GN and GC in fusion and entry, we prepared a panel of recombinant M segment constructs using LAC, TAH181/57, and V22F, a monoclonal-resistant variant of LAC with deficient fusion function. These M segment constructs were then tested in two surrogate assays for virus entry: a cell-to-cell fusion assay based on T7-luciferase expression, and a pseudotype transduction assay based on the incorporation of the bunyavirus glycoproteins on an MLV backbone. Both assays demonstrated that GC is the principal determinant of virus fusion and cell entry, and furthermore that the region delineated by amino acids 860-1442, corresponding to the membrane proximal two-thirds of GC, is key to these processes. These results, coupled with structural modeling suggesting homologies between the carboxy region of GC and Sindbis virus E1, suggest that the LAC GC functions as a type II fusion protein

  6. Roles of the multifunctional glycoprotein, emmprin (basigin; CD147), in tumour progression.

    Science.gov (United States)

    Yan, Li; Zucker, Stanley; Toole, Bryan P

    2005-02-01

    Emmprin (basigin;CD147) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of malignant tumour cells. Emmprin is involved in numerous physiological and pathological systems and exhibits several molecular and cellular characteristics, but a major function of emmprin is stimulation of synthesis of several matrix metalloproteinases. In tumours, emmprin most likely stimulates matrix metalloproteinase production in stromal fibroblasts and endothelial cells as well as in tumour cells themselves by a mechanism involving homophilic interactions between emmprin molecules on apposing cells or on neighbouring cells after membrane vesicle shedding. Membrane-associated cofactors, including caveolin-1 and annexin II, regulate emmprin activity. Emmprin induces angiogenesis via stimulation of VEGF production, invasiveness via stimulation of matrix metalloproteinase production and multidrug resistance via hyaluronan-mediated up-regulation of ErbB2 signaling and cell survival pathway activities. Although the detailed mechanisms whereby it regulates these numerous phenomena are not yet known, it is clear that emmprin is a major mediator of malignant cell behavior.

  7. 26 CFR 31.3402(e)-1 - Included and excluded wages.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Included and excluded wages. 31.3402(e)-1... SOURCE Collection of Income Tax at Source § 31.3402(e)-1 Included and excluded wages. (a) If a portion of... not more than 31 consecutive days constitutes wages, and the remainder does not constitute wages, all...

  8. Expression of CYP2E1 in human nasopharynx and its metabolic effect in vitro.

    Science.gov (United States)

    Hou, De-Fu; Wang, Shui-Liang; He, Zhi-Min; Yang, Fang; Chen, Zhu-Chu

    2007-04-01

    It was evident that nitrosamines can act directly on target tissue and result in carcinogenesis. As has been shown, the carcinogenic activity of nitrosamines relied on its bioactivation by Cytochrome P450 2E1 (CYP2E1). In this study, we investigated the expression of CYP2E1 in Nasopharyngeal carcinoma (NPC) cells, embryonic nasopharyngeal epithelial tissue (ENET) specimens, and NPC biopsies by RT-PCR analysis. CYP2E1 was expressed in all NPC cell lines (6/6, including 7429) and ENET (6/6), and 80% of NPC biopsie (8/10). The fact that Human nasopharynx expresses CYP2E1 suggests that CYP2E1 may play an important role in the course of NPC by indirect carcinogens nitrosamines. To further evaluate the function of CYP2E1, the CYP2E1 was stably expressed in the cell line NIH 3T3/rtTA under a tetracycline-controlled transactivator. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by Doxycycline (Dox) When the catalytic activity of CYP2E1 was assayed, the result showed that the generation of 6-hydroxychlorzoxazone (6-OH-CZ) from chlorzoxazone (CZ) was dose- and time-dependent on Dox addition to the medium. In the presence of 1 microg/ml Dox, the CZ 6-hydroxylase activity of the cell line was found to be 0.986 +/- 0.034 nmol/10(6) cells/h. The metabolic activation of Tet/3T3/2E1-6 cells was also assayed by N,N'-dinitrosopiperazine (DNP) cytotoxicity, and the viability of Tet/3T3/2E1-6 cells treated with Dox was lower than that of untreated cells with a significant difference between them in 80 and 160 microg/ml DNP (P ( 0.05, t test. This cell line will be useful not only to assess the metabolic characteristics of CYP2E1, but also will be useful to investigate the role of CYP2E1 in metabolic activation of carcinogenic nitrosamines in vitro.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    Science.gov (United States)

    Liu, Bo

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-01-15

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

  13. The chaperone BAG6 captures dislocated glycoproteins in the cytosol.

    Directory of Open Access Journals (Sweden)

    Jasper H L Claessen

    Full Text Available Secretory and membrane (glycoproteins are subject to quality control in the endoplasmic reticulum (ER to ensure that only functional proteins reach their destination. Proteins deemed terminally misfolded and hence functionally defective may be dislocated to the cytosol, where the proteasome degrades them. What we know about this process stems mostly from overexpression of tagged misfolded proteins, or from situations where viruses have hijacked the quality control machinery to their advantage. We know of only very few endogenous substrates of ER quality control, most of which are degraded as part of a signaling pathway, such as Insig-1, but such examples do not necessarily represent terminally misfolded proteins. Here we show that endogenous dislocation clients are captured specifically in association with the cytosolic chaperone BAG6, or retrieved en masse via their glycan handle.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Dash R

    2017-03-01

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

  16. O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport.

    Directory of Open Access Journals (Sweden)

    Ravin Seepersaud

    2017-08-01

    Full Text Available The serine-rich repeat (SRR glycoproteins are a family of adhesins found in many Gram-positive bacteria. Expression of the SRR adhesins has been linked to virulence for a variety of infections, including streptococcal endocarditis. The SRR preproteins undergo intracellular glycosylation, followed by export via the accessory Sec (aSec system. This specialized transporter is comprised of SecA2, SecY2 and three to five accessory Sec proteins (Asps that are required for export. Although the post-translational modification and transport of the SRR adhesins have been viewed as distinct processes, we found that Asp2 of Streptococcus gordonii also has an important role in modifying the SRR adhesin GspB. Biochemical analysis and mass spectrometry indicate that Asp2 is an acetyltransferase that modifies N-acetylglucosamine (GlcNAc moieties on the SRR domains of GspB. Targeted mutations of the predicted Asp2 catalytic domain had no effect on transport, but abolished acetylation. Acetylated forms of GspB were only detected when the protein was exported via the aSec system, but not when transport was abolished by secA2 deletion. In addition, GspB variants rerouted to export via the canonical Sec pathway also lacked O-acetylation, demonstrating that this modification is specific to export via the aSec system. Streptococci expressing GspB lacking O-acetylated GlcNAc were significantly reduced in their ability bind to human platelets in vitro, an interaction that has been strongly linked to virulence in the setting of endocarditis. These results demonstrate that Asp2 is a bifunctional protein involved in both the post-translational modification and transport of SRR glycoproteins. In addition, these findings indicate that these processes are coordinated during the biogenesis of SRR glycoproteins, such that the adhesin is optimally modified for binding. This requirement for the coupling of modification and export may explain the co-evolution of the SRR

  17. Protective effect of Edaravone against hypoxia-induced cytotoxicity in osteoblasts MC3T3-E1 cells.

    Science.gov (United States)

    Cao, Bo; Chai, Chunxiang; Zhao, Sishun

    2015-12-01

    Edaravone is a newly developed clinical medicine for the treatment of acute cerebral infarction. Reduced blood supply to bones (hypoxia) has been involved in the pathological development of osteoporosis. In this study, we investigated the effect of Edaravone and its latent mechanism on hypoxia-induced cell toxicity in MC3T3-E1 cells. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were determined by the fluorescence dyes 2',7'-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), respectively. mRNA and proteins were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Edaravone significantly restored the hypoxia-induced reduction of MC3T3-E1 cell viability and inhibited lactate dehydrogenase release. In addition, we found that Edaravone inhibits the generation of ROS and NO. Hoechst staining results indicated that the nuclear condensation characteristic of apoptosis was increased in MC3T3-E1 cells after hypoxia exposure, which was significantly suppressed by Edaravone treatment. Mechanistically, we found that Edaravone markedly reduced the expression of cleaved caspase-3 and blunted the release of cytochrome c. These findings strongly suggested that Edaravone suppresses hypoxia-induced cytotoxicity in MC3T3-E1 cells. The pleiotropic effects of Edaravone on hypoxia exposure in osteoblasts suggest potential antiosteoporosis mechanisms of Edaravone. © 2015 International Union of Biochemistry and Molecular Biology.

  18. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan); Nishio, Hiroaki, E-mail: nishio@fupharm.fukuyama-u.ac.jp [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  19. THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

    Directory of Open Access Journals (Sweden)

    A. V. Shulkin

    2015-09-01

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

  20. 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.

  1. Mechanical circulatory support is associated with loss of platelet receptors glycoprotein Ibα and glycoprotein VI.

    Science.gov (United States)

    Lukito, P; Wong, A; Jing, J; Arthur, J F; Marasco, S F; Murphy, D A; Bergin, P J; Shaw, J A; Collecutt, M; Andrews, R K; Gardiner, E E; Davis, A K

    2016-11-01

    Essentials Relationship of acquired von Willebrand disease (VWD) and platelet dysfunction is explored. Patients with ventricular assist devices and on extracorporeal membrane oxygenation are investigated. Acquired VWD and platelet receptor shedding is demonstrated in the majority of patients. Loss of platelet adhesion receptors glycoprotein (GP) Ibα and GPVI may increase bleeding risk. Background Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. Objectives To investigate whether loss of platelet receptors occurs in vivo, and the relationship with acquired von Willebrand syndrome (AVWS). Methods Platelet counts, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 21 continuous flow VAD (CF-VAD), 20 ECMO, 12 heart failure and seven aortic stenosis patients. Levels of platelet receptors were measured by flow cytometry or ELISA. Results The loss of high molecular weight VWF multimers was observed in 18 of 19 CF-VAD and 14 of 20 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Conclusions These data link AVWS and increased platelet receptor shedding in patients with CF-VADs or ECMO for the first time. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CF-VAD and ECMO patients may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions. © 2016 International Society on Thrombosis and Haemostasis.

  2. Induction of mesenchymal cell phenotypes in lung epithelial cells by adenovirus E1A.

    Science.gov (United States)

    Behzad, A R; Morimoto, K; Gosselink, J; Green, J; Hogg, J C; Hayashi, S

    2006-12-01

    Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.

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

    Science.gov (United States)

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

    2006-02-17

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

  4. Enterasys Networks delivers 10-Gigabit ethernet for the enterprise with new matrix E1 switching family

    CERN Multimedia

    2001-01-01

    Enterasys Networks Inc., today announced its new Matrix E1 family of 10-Gigabit and Gigabit Ethernet switches. The Matrix E1 Optical Access Switch (OAS) enables organizations to deliver applications at 10-Gb speeds across a single fibre optic pair. Jacques Altaber, deputy leader of IT at CERN said "High-bandwith solutions are essential to leveraging more computing power, so 10-Gb Ethernet is the next logical step for us...The Matrix E1 allows us to provide the networking support that our scientists need and gives us a certain future for bandwidth and computing expansion".

  5. Synthesis of in vitro Co1E1 transcripts with 5'-terminal ribonucleotides that exhibit noncomplementarity with the DNA template

    International Nuclear Information System (INIS)

    Parker, R.C.

    1986-01-01

    A region that forms the S1 nuclease site in Co1E1 DNA is shown to code for an in vitro transcript, called S1 RNA-B, which contains a 5'-terminal GTP residue that exhibits noncomplementarity with the template's DNA sequence. The synthesis of S1 RNA-B initiates four bases upstream from the start point for S1 RNA-C. The initial four bases in S1 RNA-B and S1 RNA-C are identical. The relative synthesis of S1 RNA-B to S1 RNA-C is sensitive to the concentration of GTP, a substrate that is required for elongation past the +4 position in S1 RNA-C. Dinucleotides that are expected to only initiate synthesis of S1 RNA-C yield two transcripts that appear to initiate from the S1 RNA-C and S1 RNA-B start sites. In vitro studies involving other Co1E1 transcripts, RNA-B and RNA-C, provide similar observations concerning the noncomplementary initiation phenomenon. A model involving transcriptional slippage is suggested to explain the noncomplementary initiation phenomenon. The model proposes that the cycling reaction of Escherichia coli RNA polymerase produces tetranucleotides that are transposed to nearby upstream sequences for priming transcription

  6. Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

    Science.gov (United States)

    Nakatsu, Yoshihiro; Nakagawa, Fumio; Higashi, Sen; Ohsumi, Tomoko; Shiiba, Shunji; Watanabe, Seiji; Takeuchi, Hiroshi

    2018-02-01

    N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

  7. Flunarizine Prevents Hepatitis C Virus Membrane Fusion in a Genotype-dependent Manner by Targeting the Potential Fusion Peptide within E1

    Science.gov (United States)

    Perin, Paula M.; Haid, Sibylle; Brown, Richard J. P.; Doerrbecker, Juliane; Schulze, Kai; Zeilinger, Carsten; von Schaewen, Markus; Heller, Brigitte; Vercauteren, Koen; Luxenburger, Eva; Baktash, Yasmine M.; Vondran, Florian W. R.; Speerstra, Sietkse; Awadh, Abdullah; Mukhtarov, Furkat; Schang, Luis M; Kirschning, Andreas; Müller, Rolf; Guzman, Carlos A.; Kaderali, Lars; Randall, Glenn; Meuleman, Philip; Ploss, Alexander; Pietschmann, Thomas

    2015-01-01

    To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies. PMID:26248546

  8. Anomalous behaviors of E1/E2 deep level defects in 6H silicon carbide

    International Nuclear Information System (INIS)

    Chen, X.D.; Ling, C.C.; Gong, M.; Fung, S.; Beling, C.D.; Brauer, G.; Anwand, W.; Skorupa, W.

    2005-01-01

    Deep level defects E 1 /E 2 were observed in He-implanted, 0.3 and 1.7 MeV electron-irradiated n-type 6H-SiC. Similar to others' results, the behaviors of E 1 and E 2 (like the peak intensity ratio, the annealing behaviors or the introduction rates) often varied from sample to sample. This anomalous result is not expected of E 1 /E 2 being usually considered arising from the same defect located at the cubic and hexagonal sites respectively. The present study shows that this anomaly is due to another DLTS peak overlapping with the E 1 /E 2 . The activation energy and the capture cross section of this defect are E C -0.31 eV and σ∼8x10 -14 cm 2 , respectively

  9. Water promoted allylic nucleophilic substitution reactions of (E)-1,3 diphenylallyl acetate

    KAUST Repository

    Ghorpade, Seema Arun; Sawant, Dinesh N; Makki, Arwa; Sekar, N; Eppinger, Jö rg

    2017-01-01

    Transition metal free, water based, greener protocol for allylic alkylation, allylic amination, O-allylation of (E)-1,3-diphenylallyl acetate is described. The developed methodology is applicable for a wide range of nucleophiles furnishing excellent yields of corresponding products up to 87% under mild reaction conditions. A Distinct effect of water and base is explored for allylic nucleophilic substitution reactions of (E)-1,3-diphenylallyl acetate.

  10. Water promoted allylic nucleophilic substitution reactions of (E)-1,3 diphenylallyl acetate

    KAUST Repository

    Ghorpade, Seema Arun

    2017-11-30

    Transition metal free, water based, greener protocol for allylic alkylation, allylic amination, O-allylation of (E)-1,3-diphenylallyl acetate is described. The developed methodology is applicable for a wide range of nucleophiles furnishing excellent yields of corresponding products up to 87% under mild reaction conditions. A Distinct effect of water and base is explored for allylic nucleophilic substitution reactions of (E)-1,3-diphenylallyl acetate.

  11. [Pregnancy-specific beta-glycoprotein in the serum of women with a complicated early pregnancy].

    Science.gov (United States)

    Radikov, N

    1989-01-01

    The author determined pregnancy specific beta 1-glycoprotein in 109 women with threatened early pregnancy as 32 of the women suffered from abortus imminens with several unsuccessful pregnancies in the past as well as 67 women with abortus incipiens with bleeding ex utero. The author established that 87% of women with abortus imminens and preserved pregnancies had values of beta 1-glycoprotein close to those of normal pregnancy for the respective gestational week. 93% of women with abortus incipiens preserved pregnancies till term, but the specific glycoprotein was with in normal ranges. Spontaneous abortion occurred in 7% of women with low values under the 10th percentile. The present study show that examination of pregnancy specific beta 1-glycoprotein in women with threatened early pregnancy is of prognostic significance for the outcome of pregnancy.

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

    Science.gov (United States)

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

    2017-05-01

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

  13. Boronic Acid-Based Approach for Separation and Immobilization of Glycoproteins and Its Application in Sensing

    Directory of Open Access Journals (Sweden)

    Lin Liu

    2013-10-01

    Full Text Available Glycoproteins influence a broad spectrum of biological processes including cell-cell interaction, host-pathogen interaction, or protection of proteins against proteolytic degradation. The analysis of their glyco-structures and concentration levels are increasingly important in diagnosis and proteomics. Boronic acids can covalently react with cis-diols in the oligosaccharide chains of glycoproteins to form five- or six-membered cyclic esters. Based on this interaction, boronic acid-based ligands and materials have attracted much attention in both chemistry and biology as the recognition motif for enrichment and chemo/biosensing of glycoproteins in recent years. In this work, we reviewed the progress in the separation, immobilization and detection of glycoproteins with boronic acid-functionalized materials and addressed its application in sensing.

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

    Directory of Open Access Journals (Sweden)

    Yang Gao

    2016-11-01

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

  15. Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase

    International Nuclear Information System (INIS)

    Yamaguchi, Yoshiki; Hirao, Takeshi; Sakata, Eri; Kamiya, Yukiko; Kurimoto, Eiji; Yoshida, Yukiko; Suzuki, Tadashi; Tanaka, Keiji; Kato, Koichi

    2007-01-01

    Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF Fbs1 ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man 3 GlcNAc 2 by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol

  16. 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.

  17. Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts.

    Science.gov (United States)

    Bhargava, Arjun K; Rothlauf, Paul W; Krummenacher, Claude

    2016-12-01

    Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor's interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Murine P-glycoprotein deficiency alters intestinal injury repair and blunts lipopolysaccharide-induced radioprotection.

    Science.gov (United States)

    Staley, Elizabeth M; Yarbrough, Vanisha R; Schoeb, Trenton R; Daft, Joseph G; Tanner, Scott M; Steverson, Dennis; Lorenz, Robin G

    2012-09-01

    P-glycoprotein (P-gp) has been reported to increase stem cell proliferation and regulate apoptosis. Absence of P-gp results in decreased repair of intestinal epithelial cells after chemical injury. To further explore the mechanisms involved in the effects of P-gp on intestinal injury and repair, we used the well-characterized radiation injury model. In this model, injury repair is mediated by production of prostaglandins (PGE(2)) and lipopolysaccharide (LPS) has been shown to confer radioprotection. B6.mdr1a(-/-) mice and wild-type controls were subjected to 12 Gy total body X-ray irradiation and surviving crypts in the proximal jejunum and distal colon were evaluated 3.5 days after irradiation. B6.mdr1a(-/-) mice exhibited normal baseline stem cell proliferation and COX dependent crypt regeneration after irradiation. However, radiation induced apoptosis was increased and LPS-induced radioprotection was blunted in the C57BL6.mdr1a(-/-) distal colon, compared to B6 wild-type controls. The LPS treatment induced gene expression of the radioprotective cytokine IL-1α, in B6 wild-type controls but not in B6.mdr1a(-/-) animals. Lipopolysaccharid-induced radioprotection was absent in IL-1R1(-/-) animals, indicating a role for IL-1α in radioprotection, and demonstrating that P-gp deficiency interferes with IL-1α gene expression in response to systemic exposure to LPS.

  19. 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

  20. Transcallosal Projections Require Glycoprotein M6-Dependent Neurite Growth and Guidance.

    Science.gov (United States)

    Mita, Sakura; de Monasterio-Schrader, Patricia; Fünfschilling, Ursula; Kawasaki, Takahiko; Mizuno, Hidenobu; Iwasato, Takuji; Nave, Klaus-Armin; Werner, Hauke B; Hirata, Tatsumi

    2015-11-01

    The function of mature neurons critically relies on the developmental outgrowth and projection of their cellular processes. It has long been postulated that the neuronal glycoproteins M6a and M6b are involved in axon growth because these four-transmembrane domain-proteins of the proteolipid protein family are highly enriched on growth cones, but in vivo evidence has been lacking. Here, we report that the function of M6 proteins is required for normal axonal extension and guidance in vivo. In mice lacking both M6a and M6b, a severe hypoplasia of axon tracts was manifested. Most strikingly, the corpus callosum was reduced in thickness despite normal densities of cortical projection neurons. In single neuron tracing, many axons appeared shorter and disorganized in the double-mutant cortex, and some of them were even misdirected laterally toward the subcortex. Probst bundles were not observed. Upon culturing, double-mutant cortical and cerebellar neurons displayed impaired neurite outgrowth, indicating a cell-intrinsic function of M6 proteins. A rescue experiment showed that the intracellular loop of M6a is essential for the support of neurite extension. We propose that M6 proteins are required for proper extension and guidance of callosal axons that follow one of the most complex trajectories in the mammalian nervous system. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. The Sperm-surface glycoprotein, SGP, is necessary for fertilization in the frog, Xenopus laevis.

    Science.gov (United States)

    Nagai, Keita; Ishida, Takuya; Hashimoto, Takafumi; Harada, Yuichirou; Ueno, Shuichi; Ueda, Yasushi; Kubo, Hideo; Iwao, Yasuhiro

    2009-06-01

    To identify a molecule involved in sperm-egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm-surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti-SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti-SGP antibody recognized large molecules, with molecular masses of 65-150 kDa and minor smaller molecules with masses of 20-28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle-binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm-egg membrane binding and is responsible for the establishment of fertilization in Xenopus.

  2. Interaction of coenzyme Q10 with the intestinal drug transporter P-glycoprotein.

    Science.gov (United States)

    Itagaki, Shirou; Ochiai, Akiko; Kobayashi, Masaki; Sugawara, Mitsuru; Hirano, Takeshi; Iseki, Ken

    2008-08-27

    In clinical trials, patients usually take many kinds of drugs at the same time. Thus, drug-drug interactions can often directly affect the therapeutic safety and efficacy of many drugs. Oral delivery is the most desirable means of drug administration. Changes in the activity of drug transporters may substantially influence the absorption of administered drugs from the intestine. However, there have been a few studies on food-drug interactions involving transporters. It is important to be aware of the potential of food-drug interactions and to act in order to prevent undesirable and harmful clinical consequences. Coenzyme Q10 (CoQ10) is very widely consumed by humans as a food supplement because of its recognition by the public as an important nutrient in supporting human health. Since intestinal efflux transporter P-glycoprotein (P-gp) is one of the major factors in drug-drug interactions, we focused on this transporter. We report here for the first time that CoQ10, which is widely used as a food supplement, affects the transport activity of P-gp.

  3. 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.

  4. Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones

    International Nuclear Information System (INIS)

    Schulster, D.

    1977-01-01

    Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique. (author)

  5. Elite suppressor-derived HIV-1 envelope glycoproteins exhibit reduced entry efficiency and kinetics.

    Directory of Open Access Journals (Sweden)

    Kara G Lassen

    2009-04-01

    Full Text Available Elite suppressors (ES are a rare subset of HIV-1-infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor and CCR5 (co-receptor. In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals.

  6. Dysfunction of bovine endogenous retrovirus K2 envelope glycoprotein is related to unsuccessful intracellular trafficking.

    Science.gov (United States)

    Nakaya, Yuki; Miyazawa, Takayuki

    2014-06-01

    Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  8. The Frequency of Cytochrome P450 2E1 Polymorphisms in Black South Africans

    Directory of Open Access Journals (Sweden)

    Paul K. Chelule

    2006-01-01

    Full Text Available Polymorphisms in the promoter region of the Cytochrome P4502E1 (CYP2E1 gene reportedly modify the metabolic activity of CYP2E1 enzyme, and have been associated with increased susceptibility to squamous cell carcinoma (SCC of the oesophagus in high prevalence areas such as China. To assess the frequency of these polymorphisms in Black South Africans, a population with a high incidence of oesophageal SCC, this study examined genomic DNA from 331 subjects for restriction fragment length polymorphisms in the CYP2E1 (RsaI and PstI digestion. The frequency of the CYP2E1 c1/c1 and c1/c3 genotypes was 95% and 5% respectively. The frequency of the CYP2E1 allele distribution was found to be markedly different between Chinese and South African populations; hence it is important to place racial differences into consideration when proposing allelic variants as genetic markers for cancer.

  9. Fragment E1 labeled with I-123 in the detection of venous thrombosis

    International Nuclear Information System (INIS)

    Knight, L.C.; Maurer, A.H.; Robbins, P.S.; Malmud, L.S.; Budzynski, A.Z.

    1985-01-01

    Fragment E1, which has been shown to have specific binding affinity for thrombi in an animal model, was investigated in humans for its safety and ability to bind to venous thrombi. Human Fragment E1 was labeled with I-123 and administered intravenously to patients with proved or suspected deep vein thrombosis. The vascular distribution of radioactivity was documented by obtaining gamma camera images of the patients' legs for 30 minutes following administration of I-123-Fragment E1. All patients (n = 5) with documented venous thrombi had rapid localization of labeled Fragment E1 in the area of thrombus. Patients without evidence of thrombi (n = 5) showed no focal localization, although two of these patients showed diffuse uptake along the length of the veins, due to superficial phlebitis. Analysis of blood samples in four patients indicated that disappearance of Fragment E1 from the circulation was more rapid in individuals with thrombosis (t 1/2 = 20 min) than in individuals without thrombosis (t 1/2 = 90 min), and a radiolabeled species of high molecular weight was found in patients with thrombosis but was absent from patients without thrombosis. These early results suggest that radiolabeled Fragment E1 is a safe and potentially valuable agent for the rapid detection of venous thrombosis

  10. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    Science.gov (United States)

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  11. Involving women.

    Science.gov (United States)

    Agbo, J

    1994-01-01

    I am a primary health care (PHC) coordinator working with the May Day Rural project, a local NGO involved in integrated approaches and programs with rural communities in the Ga District of the Greater-Accra region in Ghana. When we talk about the community development approach we must first and foremost recognize that we are talking about women, because in the developing world frequent childbirths mean that her burden of mortality is higher than a man's; her workload is extremely heavy--whether in gardening, farming, other household duties, caring for the sick, or the rearing of children; she has a key role in PHC and community development, because men are always looking for greener pastures elsewhere, leaving the women behind. Women's concerns are critical in most health care projects and women and children are their main beneficiaries. Why not include women in the management team, project design, implementation and evaluation processes? That is what the May Day Rural project is practicing, encouraging women's participation and creating a relationship of trust. full text

  12. Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

    OpenAIRE

    Puddington, L; Woodgett, C; Rose, J K

    1987-01-01

    The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

  13. Glycoproteins of axonal transport: affinity chromatography on fucose-specific lectins

    Energy Technology Data Exchange (ETDEWEB)

    Gustavsson, S.; Ohlson, C.; Karlsson, J.O.

    1982-03-01

    Rapidly transported fucose-labeled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The solubilized components were subjected to affinity chromatography on three different fucose-specific lectins. A recently characterized fucose-specific lectin from Aleuria aurantia bound reversibly approximately 60% of the applied protein-bound radioactivity. The lectins from Lotus tetragonolobus and Ulex europaeus bound are very small proportions of the labeled rapidly transported glycoproteins.

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

    Directory of Open Access Journals (Sweden)

    A.J. Parodi

    1998-05-01

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

  15. Changes in intestinal absorption of nutrients and brush border glycoproteins after total parenteral nutrition in rats.

    Science.gov (United States)

    Miura, S; Tanaka, S; Yoshioka, M; Serizawa, H; Tashiro, H; Shiozaki, H; Imaeda, H; Tsuchiya, M

    1992-01-01

    The effect of total parenteral nutrition on nutrients absorption and glycoprotein changes of brush border membrane was examined in rat small intestine. In total parenteral nutrition rats, a marked decrease in activity of brush border enzymes was observed mainly in the proximal and middle segments of the intestine. Galactose perfusion of jejunal segment showed that hexose absorption was significantly inhibited, while intestinal absorption of glycine or dipeptide, glycylglycine was not significantly affected by total parenteral nutrition treatment. When brush border membrane glycoprotein profile was examined by [3H]-glucosamine or [3H]-fucose incorporation into jejunal loops, significant changes were observed in the glycoprotein pattern of brush border membrane especially in the high molecular weight range over 120 kDa after total parenteral nutrition treatment, suggesting strong dependency of glycoprotein synthesis on luminal substances. Molecular weight of sucrase isomaltase in brush border membrane detected by specific antibody showed no significant difference, however, in total parenteral nutrition and control rats. Also, molecular weight of specific sodium glucose cotransporter of intestinal brush border membrane detected by selective photoaffinity labelling was not altered in total parenteral nutrition rats. It may be that prolonged absence of oral food intake may produce significant biochemical changes in brush border membrane glycoprotein and absorptive capacity of small intestine, but these changes were not observed in all brush border membrane glycoproteins. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1582592

  16. Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity

    Directory of Open Access Journals (Sweden)

    Yongke Lu

    2015-10-01

    Full Text Available Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT, CYP2E1 knockout (KO or CYP2E1 humanized transgenic knockin (KI, mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA, an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These

  17. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M; Melendy, Thomas; Archambault, Jacques

    2016-01-06

    The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural

  18. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    Science.gov (United States)

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  19. 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.

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

    International Nuclear Information System (INIS)

    Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J.

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Kerstin Gnirß

    2014-04-01

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

  2. Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins.

    Science.gov (United States)

    Weir, Dawn L; Smith, Ina L; Bossart, Katharine N; Wang, Lin-Fa; Broder, Christopher C

    2013-09-01

    Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s). Published by Elsevier Inc.

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

    Directory of Open Access Journals (Sweden)

    Victoria Rydengård

    2008-08-01

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

  4. Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform

    International Nuclear Information System (INIS)

    Gallagher, John R.; Torian, Udana; McCraw, Dustin M.; Harris, Audray K.

    2017-01-01

    While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanism of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines.

  5. The RSV F and G glycoproteins interact to form a complex on the surface of infected cells

    International Nuclear Information System (INIS)

    Low, Kit-Wei; Tan, Timothy; Ng, Ken; Tan, Boon-Huan; Sugrue, Richard J.

    2008-01-01

    In this study, the interaction between the respiratory syncytial virus (RSV) fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) proteins was examined. Immunoprecipitation analysis suggested that the F and G proteins exist as a protein complex on the surface of RSV-infected cells, and this conclusion was supported by ultracentrifugation analysis that demonstrated co-migration of surface-expressed F and G proteins. Although our analysis provided evidence for an interaction between the G and SH proteins, no evidence was obtained for a single protein complex involving all three of the virus proteins. These data suggest the existence of multiple virus glycoprotein complexes within the RSV envelope. Although the stimulus that drives RSV-mediated membrane fusion is unknown, the association between the G and F proteins suggest an indirect role for the G protein in this process

  6. Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform

    Energy Technology Data Exchange (ETDEWEB)

    Gallagher, John R.; Torian, Udana; McCraw, Dustin M.; Harris, Audray K., E-mail: harrisau@mail.nih.gov

    2017-02-15

    While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanism of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines.

  7. Crystal structure of the pestivirus envelope glycoprotein E(rns) and mechanistic analysis of its ribonuclease activity.

    Science.gov (United States)

    Krey, Thomas; Bontems, Francois; Vonrhein, Clemens; Vaney, Marie-Christine; Bricogne, Gerard; Rümenapf, Till; Rey, Félix A

    2012-05-09

    Pestiviruses, which belong to the Flaviviridae family of RNA viruses, are important agents of veterinary diseases causing substantial economical losses in animal farming worldwide. Pestivirus particles display three envelope glycoproteins at their surface: E(rns), E1, and E2. We report here the crystal structure of the catalytic domain of E(rns), the ribonucleolytic activity of which is believed to counteract the innate immunity of the host. The structure reveals a three-dimensional fold corresponding to T2 ribonucleases from plants and fungi. Cocrystallization experiments with mono- and oligonucleotides revealed the structural basis for substrate recognition at two binding sites previously identified for T2 RNases. A detailed analysis of poly-U cleavage products using (31)P-NMR and size exclusion chromatography, together with molecular docking studies, provides a comprehensive mechanistic picture of E(rns) activity on its substrates and reveals the presence of at least one additional nucleotide binding site. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. PprM is necessary for up-regulation of katE1, encoding the major catalase of Deinococcus radiodurans, under unstressed culture conditions.

    Science.gov (United States)

    Jeong, Sun-Wook; Seo, Ho Seong; Kim, Min-Kyu; Choi, Jong-Il; Lim, Heon-Man; Lim, Sangyong

    2016-06-01

    Deinococcus radiodurans is a poly-extremophilic organism, capable of tolerating a wide variety of different stresses, such as gamma/ultraviolet radiation, desiccation, and oxidative stress. PprM, a cold shock protein homolog, is involved in the radiation resistance of D. radiodurans, but its role in the oxidative stress response has not been investigated. In this study, we investigated the effect of pprM mutation on catalase gene expression. pprM disruption decreased the mRNA and protein levels of KatE1, which is the major catalase in D. radiodurans, under normal culture conditions. A pprM mutant strain (pprM MT) exhibited decreased catalase activity, and its resistance to hydrogen peroxide (H2O2) decreased accordingly compared with that of the wild-type strain. We confirmed that RecG helicase negatively regulates katE1 under normal culture conditions. Among katE1 transcriptional regulators, the positive regulator drRRA was not altered in pprM (-), while the negative regulators perR, dtxR, and recG were activated more than 2.5-fold in pprM MT. These findings suggest that PprM is necessary for KatE1 production under normal culture conditions by down-regulation of katE1 negative regulators.

  9. Sulfation of fulvestrant by human liver cytosols and recombinant SULT1A1 and SULT1E1

    Directory of Open Access Journals (Sweden)

    Edavana VK

    2011-11-01

    Full Text Available Vineetha Koroth Edavana1, Xinfeng Yu1, Ishwori B Dhakal1, Suzanne Williams1, Baitang Ning2, Ian T Cook3, David Caldwell1, Charles N Falany3, Susan Kadlubar11Division of Medical Genetics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA; 2Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA; 3Department of Pharmacology, University of Alabama, Birmingham, AL, USAAbstract: Fulvestrant (Faslodex™ is a pure antiestrogen that is approved to treat hormone receptor-positive metastatic breast cancer in postmenopausal women. Previous studies have demonstrated that fulvestrant metabolism in humans involves cytochromes P450 and UDP-glucuronosyltransferases (UGTs. To date, fulvestrant sulfation has not been characterized. This study examined fulvestrant sulfation with nine recombinant sulfotransferases and found that only SULT1A1 and SULT1E1 displayed catalytic activity toward this substrate, with Km of 4.2 ± 0.99 and 0.2 ± 0.16 µM, respectively. In vitro assays of 104 human liver cytosols revealed marked individual variability that was highly correlated with β-naphthol sulfation (SULT1A1 diagnostic substrate; r = 0.98, P < 0.0001, but not with 17ß-estradiol sulfation (SULT1E1 diagnostic substrate; r = 0.16, P = 0.10. Fulvestrant sulfation was correlated with both SULT1A1*1/2 genotype (P value = 0.023 and copy number (P < 0.0001. These studies suggest that factors influencing SULT1A1/1E1 tissue expression and/or enzymatic activity could influence the efficacy of fulvestrant therapy.Keywords: fulvestrant, sulfotransferase, genotype, copy number

  10. Inhibition of urethane-induced genotoxicity and cell proliferation in CYP2E1-null mice

    International Nuclear Information System (INIS)

    Hoffler, Undi; Dixon, Darlene; Peddada, Shyamal; Ghanayem, Burhan I.

    2005-01-01

    Urethane is a multi-site animal carcinogen and was classified as 'reasonably anticipated to be a human carcinogen.' Urethane is a fermentation by-product and found at appreciable levels in alcoholic beverages and foods such as bread and cheese. Recent work in this laboratory demonstrated for the first time that CYP2E1 is the principal enzyme responsible for urethane metabolism. The current studies were undertaken to assess the relationships between CYP2E1-mediated metabolism and urethane-induced genotoxicity and cell proliferation as determined by induction of micronucleated erythrocytes (MN) and expression of Ki-67, respectively, using CYP2E1-null and wild-type mice. Urethane was administered at 0 (vehicle), 1, 10, or 100 mg/kg/day (p.o.), 5 days/week for 6 weeks. A significant dose-dependent increase in MN was observed in wild-type mice; however, a slight increase was measured in the MN-polychromatic erythrocytes in CYP2E1-null mice treated with 100 mg/kg. A significant increase in the expression of Ki-67 was detected in the livers and the lungs (terminal bronchioles, alveoli, and bronchi) of wild-type mice administered 100 mg urethane/kg in comparison to controls. In contrast, CYP2E1-null mice administered this dose exhibited negligible alterations in Ki-67 expression in the livers and lungs compared to controls. Interestingly, while Ki-67 expression in the forestomach decreased in wild-type mice, it increased in CYP2E1-null mice. Subsequent comparative metabolism studies demonstrated that total urethane-derived radioactivity in the plasma, liver, and lung was significantly higher in CYP2E1-null versus wild-type mice and un-metabolized urethane constituted greater than 83% of the radioactivity in CYP2E1-null mice. Un-metabolized urethane was not detectable in the plasma, liver, and lung of wild-type mice. In conclusion, these data demonstrated that CYP2E1-mediated metabolism of urethane, presumably via epoxide formation, is necessary for the induction of

  11. Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA

    Science.gov (United States)

    Hershfield, Vickers; Boyer, Herbert W.; Yanofsky, Charles; Lovett, Michael A.; Helinski, Donald R.

    1974-01-01

    DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage ϕ80pt190 (trp+) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp- strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the ϕ80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes. Images PMID:4610576

  12. Cooperative effects for CYP2E1 differ between styrene and its metabolites

    Science.gov (United States)

    Hartman, Jessica H.; Boysen, Gunnar; Miller, Grover P.

    2014-01-01

    Cooperative interactions are frequently observed in the metabolism of drugs and pollutants by cytochrome P450s; nevertheless, the molecular determinants for cooperativity remain elusive. Previously, we demonstrated that steady-state styrene metabolism by CYP2E1 exhibits positive cooperativity.We hypothesized that styrene metabolites have lower affinity than styrene toward CYP2E1 and limited ability to induce cooperative effects during metabolism. To test the hypothesis, we determined the potency and mechanism of inhibition for styrene and its metabolites toward oxidation of 4-nitrophenol using CYP2E1 Supersomes® and human liver microsomes.Styrene inhibited the reaction through a mixed cooperative mechanism with high affinity for the catalytic site (67 μM) and lower affinity for the cooperative site (1100 μM), while increasing substrate turnover at high concentrations. Styrene oxide and 4-vinylphenol possessed similar affinity for CYP2E1. Styrene oxide behaved cooperatively like styrene, but 4-vinylphenol decreased turnover at high concentrations. Styrene glycol was a very poor competitive inhibitor. Among all compounds, there was a positive correlation with binding and hydrophobicity.Taken together, these findings for CYP2E1 further validate contributions of cooperative mechanisms to metabolic processes, demonstrate the role of molecular structure on those mechanisms and underscore the potential for heterotropic cooperative effects between different compounds. PMID:23327532

  13. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

    Directory of Open Access Journals (Sweden)

    Ann R Hunt

    2010-07-01

    Full Text Available Venezuelan equine encephalitis virus (VEEV is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion and E2 (binds receptor and elicits virus neutralizing antibodies. Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs. Six E2 epitopes (E2(c,d,e,f,g,h bound VEEV-neutralizing antibody and mapped to amino acids (aa 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration

  14. Analytical Pipeline for Discovery and Verification of Glycoproteins from Plasma-Derived Extracellular Vesicles as Breast Cancer Biomarkers.

    Science.gov (United States)

    Chen, I-Hsuan; Aguilar, Hillary Andaluz; Paez Paez, J Sebastian; Wu, Xiaofeng; Pan, Li; Wendt, Michael K; Iliuk, Anton B; Zhang, Ying; Tao, W Andy

    2018-05-15

    Glycoproteins comprise more than half of current FDA-approved protein cancer markers, but the development of new glycoproteins as disease biomarkers has been stagnant. Here we present a pipeline to develop glycoproteins from extracellular vesicles (EVs) through integrating quantitative glycoproteomics with a novel reverse phase glycoprotein array and then apply it to identify novel biomarkers for breast cancer. EV glycoproteomics show promise in circumventing the problems plaguing current serum/plasma glycoproteomics and allowed us to identify hundreds of glycoproteins that have not been identified in blood. We identified 1,453 unique glycopeptides representing 556 glycoproteins in EVs, among which 20 were verified significantly higher in individual breast cancer patients. We further applied a novel glyco-specific reverse phase protein array to quantify a subset of the candidates. Together, this study demonstrates the great potential of this integrated pipeline for biomarker discovery.

  15. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujise

    2004-01-01

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

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

    DEFF Research Database (Denmark)

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

    2001-01-01

    Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the v...

  17. Calcification of MC3T3-E1 cells on titanium and zirconium.

    Science.gov (United States)

    Umezawa, Takayuki; Chen, Peng; Tsutsumi, Yusuke; Doi, Hisashi; Ashida, Maki; Suzuki, Shoichi; Moriyama, Keiji; Hanawa, Takao

    2015-01-01

    To confirm similarity of hard tissue compatibility between titanium and zirconium, calcification of MC3T3-E1 cells on titanium and zirconium was evaluated in this study. Mirror-polished titanium (Ti) and zirconium (Zr) disks and zirconium-sputter deposited titanium (Zr/Ti) were employed in this study. The surface of specimens were characterized using scanning electron microscopy and X-ray diffraction. Then, the cellular proliferation, differentiation and calcification of MC3T3-E1 cells on specimens were investigated. The surface of Zr/Ti was much smoother and cleaner than those of Ti and Zr. The proliferation of the cell was the same among three specimens, while the differentiation and calcification on Zr/Ti were faster than those on Ti and Zr. Therefore, Ti and Zr showed the identical hard tissue compatibility according to the evaluation with MC3T3-E1 cells. Sputter deposition may improve cytocompatibility.

  18. Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

    Science.gov (United States)

    Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

    2006-06-01

    Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

  19. Anti myelin oligodendrocyte glycoprotein associated immunoglobulin G (AntiMOG-IgG-associated neuromyelitis optica spectrum disorder with persistent disease activity and residual cognitive impairment

    Directory of Open Access Journals (Sweden)

    Lekha Pandit

    2017-01-01

    Full Text Available Antibodies targeting myelin oligodendrocyte glycoprotein (MOG have been recently reported in association with idiopathic inflammatory central nervous system disorders. Initially believed to be a benign disorder, anti MOG-IgG was noted to cause steroid responsive recurrent optic neuritis and isolated longitudinally extensive myelitis. However, there is growing evidence that the disease may be predominantly relapsing, often producing severe visual loss and involving regions other than the spinal cord and optic nerve. We report an adolescent male with an aggressive disease course previously undescribed in anti MOG-IgG-associated disease that left him with residual cognitive dysfunction.

  20. Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego.

    Science.gov (United States)

    Fairhead, Michael; Giannini, Silva; Gillam, Elizabeth M J; Gilardi, Gianfranco

    2005-12-01

    The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

  1. Effects of chronic ethanol administration on hepatic glycoprotein secretion in the rat

    International Nuclear Information System (INIS)

    Sorrell, M.F.; Nauss, J.M.; Donohue, T.M. Jr.; Tuma, D.J.

    1983-01-01

    The effects of chronic ethanol feeding on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Liver slices from rats fed ethanol for 4-5 wk showed a decreased ability to incorporate [ 14 C]glucosamine into medium trichloracetic acid-precipitable proteins when compared to the pair-fed controls; however, the labeling of hepatocellular glycoproteins was unaffected by chronic ethanol treatment. Immunoprecipitation of radiolabeled secretory (serum) glycoproteins with antiserum against rat serum proteins showed a similar marked inhibition in the appearance of glucosamine-labeled proteins in the medium of slices from ethanol-fed rats. Minimal effects, however, were noted in the labeling of intracellular secretory glycoproteins. Protein synthesis, as determined by measuring [ 14 C]leucine incorporation into medium and liver proteins, was decreased in liver slices from ethanol-fed rats as compared to the pair-fed controls. This was the case for both total proteins as well as immunoprecipitable secretory proteins, although the labeling of secretory proteins retained in the liver slices was reduced to a lesser extent than total radiolabeled hepatic proteins. When the terminal sugar, [ 14 C]fucose, was employed as a precursor in order to more closely focus on the final steps of hepatic glycoprotein secretion, liver slices obtained from chronic ethanol-fed rats exhibited impaired secretion of fucose-labeled proteins into the medium. When ethanol (5 or 10 mM) was added to the incubation medium containing liver slices from the ethanol-fed rats, the alterations in protein and glycoprotein synthesis and secretion caused by the chronic ethanol treatment were further potentiated. The results of this study indicate that liver slices prepared from chronic ethanol-fed rats exhibit both impaired synthesis and secretion of proteins and glycoproteins, and these defects are further potentiated by acute ethanol administration

  2. 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

  3. Divalent Metal Ions Induced Osteogenic Differentiation of MC3T3E1

    Science.gov (United States)

    Wang, Guoshou; Su, Wenta; Chen, Pohung; Huang, Teyang

    2017-12-01

    Biomaterial scaffolds blended with biochemical signal molecules with adequate osteoinductive and osteoconductive properties have attracted significant interest in bone tissue engineering regeneration. The divalent metal ions can gradually release from the scaffold into the culture medium and then induced osteoblastic differentiation of MC3T3E1. These MC3T3E1 cells expressed high activity of alkaline phosphatase, bone-related gene expression of collagen type I, Runx2, osteopontin, osteocalcin, and significantly enhanced deposited minerals on scaffold after 21 days of culture. This experiment provided a useful inducer for osteogenic differentiation in bone repair.

  4. Thionin-D4E1 chimeric protein protects plants against bacterial infections

    Science.gov (United States)

    Stover, Eddie W; Gupta, Goutam; Hao, Guixia

    2017-08-08

    The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

  5. Low-energy E1 transitions and octupole softness in odd-A deformed nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Hagemann, G B [Niels Bohr Inst., Copenhagen (Denmark); Hamamoto, I [Lund Univ. (Sweden). Dept. of Mathematical Physics; Kownacki, J; Satula, W [Warsaw Univ. (Poland)

    1992-08-01

    It is found that B(E1) values for yrast spectroscopy of deformed odd-A rare-earth nuclei calculated by using a model in which one quasiparticle is coupled to a rotor are more than an order of magnitude too small. Therefore, measured B(E1) values for {sup 169}Lu were analyzed by introducing parameters which effectively took octupole softness into account. Some preliminary results of the theoretical analysis which are presented in this paper still give do not agree completely with experiment. 4 refs., 1 tab., 5 figs.

  6. RNase H and replication of ColE1 DNA in Escherichia coli.

    OpenAIRE

    Naito, S; Uchida, H

    1986-01-01

    Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase ...

  7. A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection.

    Directory of Open Access Journals (Sweden)

    Takuya Kanno

    Full Text Available Therapeutic agents to the central nervous system (CNS need to be efficiently delivered to the target site of action at appropriate therapeutic levels. However, a limited number of effective drugs for the treatment of neurological diseases has been developed thus far. Further, the pharmacological mechanisms by which such therapeutic agents can protect neurons from cell death have not been fully understood. We have previously reported the novel small-molecule compound, 2-[mesityl(methylamino]-N-[4-(pyridin-2-yl-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316, as a unique neuroprotectant against oxidative injury and a highly promising remedy for the treatment of amyotrophic lateral sclerosis (ALS. One of the remarkable characteristics of WN1316 is that its efficacious doses in ALS mouse models are much less than those against oxidative injury in cultured human neuronal cells. It is also noted that the WN1316 cytoprotective activity observed in cultured cells is totally dependent upon the addition of fetal bovine serum in culture medium. These findings led us to postulate some serum factors being tightly linked to the WN1316 efficacy. In this study, we sieved through fetal bovine serum proteins and identified two N-linked glycoproteins, alpha-2-HS-glycoprotein (AHSG and hemopexin (HPX, requisites to exert the WN1316 cytoprotective activity against oxidative injury in neuronal cells in vitro. Notably, the removal of glycan chains from these molecules did not affect the WN1316 cytoprotective activity. Thus, two glycoproteins, AHSG and HPX, represent a pivotal glycoprotein of the cytoprotective activity for WN1316, showing a concrete evidence for the novel glycan-independent function of serum glycoproteins in neuroprotective drug efficacy.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Oliveira M.C.

    1999-01-01

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

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-08-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    OpenAIRE

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

    2014-01-01

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

  16. A regulatory variant in CYP2E1 affects the risk of lung squamous cell carcinoma.

    Science.gov (United States)

    Cao, Lei; Lin, Jia; He, Bing; Wang, Hongge; Rao, Juan; Liu, Yingwen; Zhang, Xuemei

    2014-01-01

    Cytochrome P450 2E1 (CYP2E1), an ethanol-inducible enzyme, has been shown to metabolically activate various carcinogens, which is critical for the development of cancers. It has been demonstrated that CYP2E1 polymorphisms alter the transcriptional activity. However, studies on the association between CYP2E1 -1239G>C polymorphism and non-small cell lung cancer have reported conflicting results. Thus, the gain of the present study was to investigate whether CYP2E1 -1239G>C polymorphism is associated with the development of non-small cell lung cancer in Chinese population. A case-control study was conducted in which CYP2E1 -1239G>C polymorphism was analyzed in 526 Chinese patients with non-small cell lung cancer and 526 age-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism. Odds ratios were estimated by multivariate logistic regression. A meta-analysis was conducted to evaluate the association of CYP2E1 -1239G>C polymorphism with the risk of lung cancer in Chinese population by calculating pooled odds ratio (OR). For CYP2E1 -1239G>C polymorphism, -1239C allele carriers (OR = 0.67; 95% confidence interval (CI) = 0.51-0.87; P = 0.002) were associated with a decreased risk of non-small cell lung cancer when compared with -1239GG homozygotes. In the group analyses by pathological types, for lung squamous cell carcinoma and other types, the ORs of the C allele carriers were 0.60 (95% CI = 0.41-0.88; P = 0.009) and 0.54 (95% CI = 0.30-0.99; P = 0.045). In the group analysis of smoking status, the OR for the -1239C allele-containing genotype was higher than that for -1239GG genotype (OR = 0.57; 95% CI = 0.40-0.81; P = 0.002) among smokers, but not among nonsmokers. Moreover, when the risk associated with CYP2E1 polymorphism was further evaluated within strata of C polymorphism and the risk of non-small cell lung cancer. Meta-analysis data also showed that the carriers of CYP2E1 -1239C allele

  17. The mitochondrial translocator protein, TSPO, inhibits HIV-1 envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway.

    Science.gov (United States)

    Zhou, Tao; Dang, Ying; Zheng, Yong-Hui

    2014-03-01

    The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4(+) T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ∼64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPR-associated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. The HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins

  18. Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

    Science.gov (United States)

    Komorek, Jessica; Kuppuswamy, Mohan; Subramanian, T; Vijayalingam, S; Lomonosova, Elena; Zhao, Ling-Jun; Mymryk, Joe S; Schmitt, Kimberly; Chinnadurai, G

    2010-03-01

    The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

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

    Science.gov (United States)

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

    2015-06-01

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

  20. 17 CFR 240.14e-1 - Unlawful tender offer practices.

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Unlawful tender offer... Securities Exchange Act of 1934 Regulation 14e § 240.14e-1 Unlawful tender offer practices. As a means... section 14(e) of the Act, no person who makes a tender offer shall: (a) Hold such tender offer open for...

  1. Inhibition of food stimulated acid secretion by misoprostol, an orally active synthetic E1 analogue prostaglandin.

    OpenAIRE

    Ramage, J K; Denton, A; Williams, J G

    1985-01-01

    The effect of 200 micrograms misoprostol (a synthetic prostaglandin E1 analogue) on food stimulated intragastric acidity has been monitored over a 9 h period in 16 normal volunteers. Misoprostol caused a significant inhibition of intragastric acidity for 2 h post-dosing, but no significant effect was seen thereafter on either basal or food stimulated acidity.

  2. 26 CFR 301.6511(e)-1 - Special rules applicable to manufactured sugar.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 18 2010-04-01 2010-04-01 false Special rules applicable to manufactured sugar... Assessment and Collection § 301.6511(e)-1 Special rules applicable to manufactured sugar. (a) Use as... the person entitled thereto. Such right accrues as of the date the manufactured sugar, or article...

  3. Performance simulation of the X-ray spectrometer in Chang'E-1 satellite payload

    International Nuclear Information System (INIS)

    Cao Xuelei; Wang Huanyu; Zhang Chengmo; Chen Yong; Yang Jiawei; Wang JInzhou; Liang Xiaohua; Gao Min; Zhang Jiayu; Ma Guofeng

    2007-01-01

    We discuss the performance simulation of the X-ray Spectrometer in Chang'E-1 satellite based on Geant4 system. It is divided in three parts, the efficiency simulation, the energy spectrum response and direction response. It provides the guidance on making the spectrometer. (authors)

  4. US NDC Modernization Iteration E1 Prototyping Report: Processing Control Framework

    Energy Technology Data Exchange (ETDEWEB)

    Prescott, Ryan [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Hamlet, Benjamin R. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-12-01

    During the first iteration of the US NDC Modernization Elaboration phase (E1), the SNL US NDC modernization project team developed an initial survey of applicable COTS solutions, and established exploratory prototyping related to the processing control framework in support of system architecture definition. This report summarizes these activities and discusses planned follow-on work.

  5. 26 CFR 301.6323(e)-1 - Priority of interest and expenses.

    Science.gov (United States)

    2010-04-01

    ... interest is not subrogated to the rights of the holder of the State sales tax lien. However, if the holder...)-1, to the rights of the holder of the sales tax lien, he will also be entitled to any additional....6323(e)-1 Priority of interest and expenses. (a) In general. If the lien imposed by section 6321 is not...

  6. The Adenovirus E1A C Terminus Suppresses a Delayed Antiviral Response and Modulates RAS Signaling.

    Science.gov (United States)

    Zemke, Nathan R; Berk, Arnold J

    2017-12-13

    The N-terminal half of adenovirus e1a assembles multimeric complexes with host proteins that repress innate immune responses and force host cells into S-phase. In contrast, the functions of e1a's C-terminal interactions with FOXK, DCAF7, and CtBP are unknown. We found that these interactions modulate RAS signaling, and that a single e1a molecule must bind all three of these host proteins to suppress activation of a subset of IFN-stimulated genes (ISGs). These ISGs were otherwise induced in primary respiratory epithelial cells at 12 hr p.i. This delayed activation of ISGs required IRF3 and coincided with an ∼10-fold increase in IRF3 from protein stabilization. The induced IRF3 bound to chromatin and localized to the promoters of activated ISGs. While IRF3, STAT1/2, and IRF9 all greatly increased in concentration, there were no corresponding mRNA increases, suggesting that e1a regulates the stabilities of these key activators of innate immune responses, as shown directly for IRF3. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. US NDC Modernization Iteration E1 Prototyping Report: User Interface Framework

    Energy Technology Data Exchange (ETDEWEB)

    Lober, Randall R. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-12-01

    During the first iteration of the US NDC Modernization Elaboration phase (E1), the SNL US NDC modernization project team completed an initial survey of applicable COTS solutions, and established exploratory prototyping related to the User Interface Framework (UIF) in support of system architecture definition. This report summarizes these activities and discusses planned follow-on work.

  8. PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Birk, Jesper Bratz; Sacchetti, Massimo

    2006-01-01

    . Phosphorylation had returned to resting levels at 3 h of exercise only in the control trial. Thus, an inverse association between PDH-E1a phosphorylation and PDHa activity exists. Short-term elevation in plasma FFA at rest increases PDH-E1a phosphorylation, but exercise overrules this effect of FFA on PDH-E1a...... extensor exercise at moderate intensity. During the 4-h resting period, activity of PDH in the active form (PDHa) did not change in either trial, yet phosphorylation of PDH-E1a site 1 (PDH-P1) and site 2 (PDH-P2) was elevated in the intralipid compared with the control trial. PDHa activity increased during...... exercise similarly in the two trials. After 3 h of exercise, PDHa activity remained elevated in the intralipid trial but returned to resting levels in the control trial. Accordingly, in both trials PDH-P1 and PDH-P2 decreased during exercise, and the decrease was more marked during intralipid infusion...

  9. US NDC Modernization Iteration E1 Prototyping Report: Common Object Interface

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Jennifer E. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Hess, Michael M. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2014-12-01

    During the first iteration of the US NDC Modernization Elaboration phase (E1), the SNL US NDC modernization project team completed an initial survey of applicable COTS solutions, and established exploratory prototyping related to the Common Object Interface (COI) in support of system architecture definition. This report summarizes these activities and discusses planned follow-on work.

  10. Emerging Concepts in the Resolution of Periodontal Inflammation: A Role for Resolvin E1

    Directory of Open Access Journals (Sweden)

    Maria G. Balta

    2017-12-01

    Full Text Available Inflammatory response is a protective biological process intended to eliminate the harmful effect of the insulting influx. Resolution of inflammation constitutes an active sequence of overlapping events mediated by specialized proresolving mediators, such as lipoxins, resolvins, protectins, and maresins, which originate from the enzymatic conversion of polyunsaturated fatty acids (PUFAs. An unresolved acute inflammatory response results in chronic inflammation, which is a leading cause of several common pathological conditions. Periodontitis is a biofilm-induced chronic inflammatory disease, which results in loss of periodontal connective tissue and alveolar bone support around the teeth, leading to tooth exfoliation. An inadequate proresolving host response may constitute a mechanism explaining the pathogenesis of periodontal disease. An emerging body of clinical and experimental evidence has focused on the underlying molecular mechanisms of resolvins and particularly Resolvin E1 (RvE1 in periodontitis. Recently, RvE1 has been directly correlated with the resolution of inflammation in periodontal disease. Herein, we provide a comprehensive overview of the literature regarding the role and possible mechanisms of action of RvE1 on different cell populations recruited in periodontal inflammation as well as its potential therapeutic implications. Along with recent data on the benefits of PUFAs supplementation in periodontal clinical parameters, we touch upon suggested future directions for research.

  11. Two-step method for curing Escherichia coli of ColE1-derived plasmids

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2008-01-01

    To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn10, fadAB::Tn10 or other...

  12. Transcriptional activation by the E1A regions of adenovirus types 40 and 41

    NARCIS (Netherlands)

    Loon, A.E. van; Gilardi, P.; Perricaudet, M.; Rozijn, Th. H.; Sussenbach, J.S.

    In order to establish whether the poor growth of the two fastidious adenoviruses types 40 and 41 (Ad40 and Ad41) in HeLa cells is due to a reduced trans-activation by the early region to (E1A), we have determined the trans-activating effect of this region on the expression of the chloramphenicol

  13. 11 CFR 101.1 - Candidate designations (2 U.S.C. 432(e)(1)).

    Science.gov (United States)

    2010-01-01

    ... 11 Federal Elections 1 2010-01-01 2010-01-01 false Candidate designations (2 U.S.C. 432(e)(1)). 101.1 Section 101.1 Federal Elections FEDERAL ELECTION COMMISSION GENERAL CANDIDATE STATUS AND... and address, party affiliation, and office sought, the District and State in which Federal office is...

  14. The expression and serological reactivity of recombinant canine herpesvirus 1 glycoprotein D

    Directory of Open Access Journals (Sweden)

    MarkéŽta Vaňkov‡á

    2016-01-01

    Full Text Available The aim of this work was to express recombinant glycoprotein D of canine herpesvirus 1 in bacterial cells and to evaluate its diagnostic sensitivity and specificity when compared to traditional serological methods. The gene fragment coding glycoprotein D of canine herpesvirus 1 was amplified by polymerase chain reaction, cloned into plasmid vector and expressed in Escherichia coli cells. Recombinant protein was then purified and used as an antigen in immunoblot for a detection of canine herpesvirus 1 specific antibodies. Antibody testing was performed on the panel of 100 canine sera by immunoblot with recombinant glycoprotein D as antigen and compared with indirect immunofluorescence assay. Serum samples were collected from 83 dogs with no history of canine herpesvirus 1 or reproductive disorders, and from 17 dogs from breeding kennels with a history of canine herpesvirus 1 related reproductive disorders. Sensitivity of glycoprotein D based immunoblot was 89.2% and specificity was 93%. Kappa value was calculated to be 0.8 between immunoblot and indirect immunofluorescence assay. Antibodies against canine herpesvirus 1 infection were detected in 33% of samples by immunoblot assay. Our study confirms that recombinant glycoprotein D expressed in bacterial cells could be used as a suitable and sensitive antigen for immunological tests and that herpesvirus infection seems to be common among the canine population in the Czech Republic.

  15. Study on the extraction and purification of glycoprotein from the yellow seahorse, Hippocampus kuda Bleeker

    Science.gov (United States)

    Su, Yuting; Xu, Yongjian

    2015-01-01

    The optimum parameters of extraction for glycoprotein from seahorse were examined and determined by Box-Behnken combined with ultrasonic extraction technology. Column chromatography of glycoprotein was used for further purification. The optimal extraction conditions of seahorse glycoprotein were extracting time 4.3 h, salt concentration 0.08 mol/L, extracting temperature 73°C, raw material, and water ratio 1:6. At the optimal conditions, the yield of saccharide reached to 1.123%, and the yield of protein reached to 5.898%. For purifying the crude glycoprotein, the stage renounces of DEAE-52 column chromatography were done, respectively, with 0.05, 0.1, 0.5 mol/L NaHCO3 solution, and further purification was done with Sephadex G-100 column chromatography. Finally, two pieces of seahorse glycoprotein were obtained by the column chromatography, that is, HG-11 and HG-21. The saccharide content was 56.7975% and 39.479%, the protein content was 30.5475% and 51.747%, respectively. PMID:26288722

  16. The effect of P-glycoprotein on methadone hydrochloride flux in equine intestinal mucosa.

    Science.gov (United States)

    Linardi, R L; Stokes, A M; Andrews, F M

    2013-02-01

    Methadone is an effective analgesic opioid that may have a place for the treatment of pain in horses. However, its absorption seems to be impaired by the presence of a transmembrane protein, P-glycoprotein, present in different tissues including the small intestine in other species. This study aims to determine the effect of the P-glycoprotein on methadone flux in the equine intestinal mucosa, as an indicator of in vivo drug absorption. Jejunum tissues from five horses were placed into the Ussing chambers and exposed to methadone solution in the presence or absence of Rhodamine 123 or verapamil. Electrical measurements demonstrated tissue viability for 120 min, and the flux of methadone across the jejunal membrane (mucosal to submucosal direction) was calculated based on the relative drug concentration measured by ELISA. The flux of methadone was significantly higher only in the presence of verapamil. P-glycoprotein was immunolocalized in the apical membrane of the jejunal epithelial cells (enterocytes), mainly located in the tip of the villi compared to cells of the crypts. P-glycoprotein is present in the equine jejunum and may possibly mediate the intestinal transport of methadone. This study suggests that P-glycoprotein may play a role in the poor intestinal absorption of methadone in vivo. © 2012 Blackwell Publishing Ltd.

  17. Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

    Directory of Open Access Journals (Sweden)

    Zong-Xiu Wang

    Full Text Available Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

  18. Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

    Directory of Open Access Journals (Sweden)

    Mahsa Mohseni

    2016-03-01

    Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1 to (15±2.6 nM and for vinblastine from (30±5.9 to (5±5.6 nM (P≤0.05.               P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001. Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05. Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents.  Conclusion:Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression.

  19. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    International Nuclear Information System (INIS)

    Kao, C T; Chen, C C; Cheong, U-I; Liu, S L; Huang, T H

    2014-01-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics. (paper)

  20. CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium.

    Science.gov (United States)

    Putkaradze, Natalia; Litzenburger, Martin; Abdulmughni, Ammar; Milhim, Mohammed; Brill, Elisa; Hannemann, Frank; Bernhardt, Rita

    2017-12-01

    CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6'β-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

  1. Identities of the deep level defects E1/E2 in 6H silicon carbide

    International Nuclear Information System (INIS)

    Ling, C.C.; Chen, X.D.; Beling, C.D.; Fung, S.; Lam, T.W.; Lam, C.H.; Gong, M.; Weng, H.M.; Hang, D.S.

    2004-01-01

    E 1 /E 2 (E C -0.36/0.44 eV) are deep level donors generally found in ion-implanted, electron and neutron irradiated n-type 6H-SiC materials. Their configurations are controversial and have been related to a negatively charged carbon vacancy, a divacancy or a V Si -complex. With positron lifetime technique, we have identified V Si and V C V Si in the Lely grown n-type 6H-SiC sample, with V Si annealed out at 650 C. Concentration of V C V Si persists at 1400 C annealing and significantly decreased after the 1600 C annealing. Considering the deep level transient spectroscopic (DLTS) results on the neutron irradiated n-type SiC epi sample that E 1 /E 2 completely disappeared after the 1400 C annealing, E 1 /E 2 is not the V C V Si defect. With positron annihilation techniques, A. A. Rempel et al (2002) have shown the energy dependence of vacancy generated by electron irradiation. With low irradiation energy of 0.3MeV, only V C was generated and at higher energy (0.5MeV), Si vacancy was detected. With focus to find the minimum energy for generating E 1 /E 2 , we have performed DLTS studies on n-type epi 6H-SiC materials irradiated by electrons with varying energies. Our results suggest that E 1 /E 2 have microstructure related to a carbon vacancy or a carbon interstitial. (orig.)

  2. Morbillivirus glycoprotein expression induces ER stress, alters Ca2+ homeostasis and results in the release of vasostatin.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Brunner

    Full Text Available Although the pathology of Morbillivirus in the central nervous system (CNS is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV that we inoculated into two different cell systems: a monkey cell line (Vero and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H markedly accumulated in the endoplasmic reticulum (ER. This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT, another ER resident chaperon critically involved in the response to misfolded proteins and in Ca(2+ homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca(2+ homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses.

  3. Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

    Directory of Open Access Journals (Sweden)

    Rafael Simó

    Full Text Available Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes and in vivo (C57BL6/mice experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not

  4. Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

    Science.gov (United States)

    Simó, Rafael; Hernández, Cristina; Sáez-López, Cristina; Soldevila, Berta; Puig-Domingo, Manel; Selva, David M

    2014-01-01

    Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight

  5. 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

  6. P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery.

    Science.gov (United States)

    Davis, Thomas P; Sanchez-Covarubias, Lucy; Tome, Margaret E

    2014-01-01

    The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription. © 2014 Elsevier Inc. All rights reserved.

  7. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    Science.gov (United States)

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Antibodies to myelin oligodendrocyte glycoprotein in idiopathic optic neuritis.

    Science.gov (United States)

    Nakajima, Hideki; Motomura, Masakatsu; Tanaka, Keiko; Fujikawa, Azusa; Nakata, Ruka; Maeda, Yasuhiro; Shima, Tomoaki; Mukaino, Akihiro; Yoshimura, Shunsuke; Miyazaki, Teiichiro; Shiraishi, Hirokazu; Kawakami, Atsushi; Tsujino, Akira

    2015-04-02

    To investigate the differences of clinical features, cerebrospinal fluid (CSF), MRI findings and response to steroid therapies between patients with optic neuritis (ON) who have myelin oligodendrocyte glycoprotein (MOG) antibodies and those who have seronegative ON. We recruited participants in the department of neurology and ophthalmology in our hospital in Japan. We retrospectively evaluated the clinical features and response to steroid therapies of patients with ON. Sera from patients were tested for antibodies to MOG and aquaporin-4 (AQP4) with a cell-based assay. Between April 2009 and March 2014, we enrolled serial 57 patients with ON (27 males, 30 females; age range 16-84 years) who ophthalmologists had diagnosed as having or suspected to have ON with acute visual impairment and declined critical flicker frequency, abnormal findings of brain MRI, optical coherence tomography and fluorescein fundus angiography at their onset or recurrence. We excluded those patients who fulfilled the diagnostic criteria of neuromyelitis optica (NMO)/NMO spectrum disorders (NMOSD), MS McDonald's criteria, and so on. Finally we defined 29 patients with idiopathic ON (14 males, 15 females, age range 16-84 years). 27.6% (8/29) were positive for MOG antibodies and 3.4% (1/29) were positive for AQP4. Among the eight patients with MOG antibodies, five had optic pain (p=0.001) and three had prodromal infection (p=0.179). Three of the eight MOG-positive patients showed significantly high CSF levels of myelin basic protein (p=0.021) and none were positive for oligoclonal band in CSF. On MRIs, seven MOG-positive patients showed high signal intensity on optic nerve, three had a cerebral lesion and one had a spinal cord lesion. Seven of the eight MOG-positive patients had a good response to steroid therapy. Although not proving primary pathogenicity of anti-MOG antibodies, the present results indicate that the measurement of MOG antibodies is useful in diagnosing and treating ON

  9. Complement inhibition enables tumor delivery of LCMV glycoprotein pseudotyped viruses in the presence of antiviral antibodies

    Directory of Open Access Journals (Sweden)

    Laura Evgin

    2016-01-01

    Full Text Available The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody.

  10. Identifying the Viral Genes Encoding Envelope Glycoproteins for Differentiation of Cyprinid herpesvirus 3 Isolates

    Directory of Open Access Journals (Sweden)

    Se Chang Park

    2013-01-01

    Full Text Available Cyprinid herpes virus 3 (CyHV-3 diseases have been reported around the world and are associated with high mortalities of koi (Cyprinus carpio. Although little work has been conducted on the molecular analysis of this virus, glycoprotein genes identified in the present study seem to be valuable targets for genetic comparison of this virus. Three envelope glycoprotein genes (ORF25, 65 and 116 of the CyHV-3 isolates from the USA, Israel, Japan and Korea were compared, and interestingly, sequence insertions or deletions were observed in these target regions. In addition, polymorphisms were presented in microsatellite zones from two glycoprotein genes (ORF65 and 116. In phylogenetic tree analysis, the Korean isolate was remarkably distinguished from USA, Israel, Japan isolates. These findings may be suitable for many applications including isolates differentiation and phylogeny studies.

  11. Identifying the Viral Genes Encoding Envelope Glycoproteins for Differentiation of Cyprinid herpesvirus 3 Isolates

    Science.gov (United States)

    Han, Jee Eun; Kim, Ji Hyung; Renault, Tristan; Choresca, Casiano; Shin, Sang Phil; Jun, Jin Woo; Park, Se Chang

    2013-01-01

    Cyprinid herpes virus 3 (CyHV-3) diseases have been reported around the world and are associated with high mortalities of koi (Cyprinus carpio). Although little work has been conducted on the molecular analysis of this virus, glycoprotein genes identified in the present study seem to be valuable targets for genetic comparison of this virus. Three envelope glycoprotein genes (ORF25, 65 and 116) of the CyHV-3 isolates from the USA, Israel, Japan and Korea were compared, and interestingly, sequence insertions or deletions were observed in these target regions. In addition, polymorphisms were presented in microsatellite zones from two glycoprotein genes (ORF65 and 116). In phylogenetic tree analysis, the Korean isolate was remarkably distinguished from USA, Israel, Japan isolates. These findings may be suitable for many applications including isolates differentiation and phylogeny studies. PMID:23435236

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

    Science.gov (United States)

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

    2010-03-01

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

  13. Intestinal CYP2E1: A mediator of alcohol-induced gut leakiness

    Directory of Open Access Journals (Sweden)

    Christopher B. Forsyth

    2014-01-01

    Full Text Available Chronic alcohol use can result in many pathological effects including alcoholic liver disease (ALD. While alcohol is necessary for the development of ALD, only 20–30% of alcoholics develop alcoholic steatohepatitis (ASH with progressive liver disease leading to cirrhosis and liver failure (ALD. This suggests that while chronic alcohol consumption is necessary it is not sufficient to induce clinically relevant liver damage in the absence of a secondary risk factor. Studies in rodent models and alcoholic patients show that increased intestinal permeability to microbial products like endotoxin play a critical role in promoting liver inflammation in ALD pathogenesis. Therefore identifying mechanisms of alcohol-induced intestinal permeability is important in identifying mechanisms of ALD and for designing new avenues for therapy. Cyp2e1 is a cytochrome P450 enzyme that metabolizes alcohol has been shown to be upregulated by chronic alcohol use and to be a major source of oxidative stress and liver injury in alcoholics and in animal and in vitro models of chronic alcohol use. Because Cyp2e1 is also expressed in the intestine and is upregulated by chronic alcohol use, we hypothesized it could play a role in alcohol-induced intestinal hyperpermeability. Our in vitro studies with intestinal Caco-2 cells and in mice fed alcohol showed that circadian clock proteins CLOCK and PER2 are required for alcohol-induced permeability. We also showed that alcohol increases Cyp2e1 protein and activity but not mRNA in Caco-2 cells and that an inhibitor of oxidative stress or siRNA knockdown of Cyp2e1 prevents the increase in CLOCK or PER2 proteins and prevents alcohol-induced hyperpermeability. With our collaborators we have also shown that Cyp2e1 knockout mice are resistant to alcohol-induced gut leakiness and liver inflammation. Taken together our data support a novel Cyp2e1-circadian clock protein mechanism for alcohol-induced gut leakiness that could provide new

  14. Relevance of P-glycoprotein on CXCR4+ B cells to organ manifestation in highly active rheumatoid arthritis.

    Science.gov (United States)

    Tsujimura, Shizuyo; Adachi, Tomoko; Saito, Kazuyoshi; Kawabe, Akio; Tanaka, Yoshiya

    2018-03-01

    In rheumatoid arthritis (RA), P-glycoprotein (P-gp) expression on activated B cells is associated with active efflux of intracellular drugs, resulting in drug resistance. CXCR4 is associated with migration of B cells. This study was designed to elucidate the relevance of P-gp expression on CXCR4 + B cells to clinical manifestations in refractory RA. CD19 + B cells were analyzed using flow cytometry and immunohistochemistry. P-gp was highly expressed especially on CXCR4 + CD19 + B cells in RA. The proportion of P-gp-expressing CXCR4 + B cells correlated with disease activity, estimated by Simplified Disease Activity Index (SDAI), and showed marked expansion in RA patients with high SDAI and extra-articular involvement. In highly active RA, massive infiltration of P-gp + CXCR4 + CD19 + B cells was noted in CXCL12-expressing inflammatory lesions of RA synovitis and RA-associated interstitial pneumonitis. In RA patient with active extra-articular involvement, intracellular dexamethasone level (IDL) in lymphocytes diminished with expansion of P-gp + CXCR4 + CD19 + B cells. Adalimumab reduced P-gp + CXCR4 + CD19 + B cells, increased IDL in lymphocytes, and improved the clinical manifestation and allowed tapering of concomitant medications. Expansion of P-gp + CXCR4 + B cells seems to be associated with drug resistance, disease activity and progressive destructive arthritis with extra-articular involvement in RA.

  15. Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G

    International Nuclear Information System (INIS)

    Baquero, Eduard; Buonocore, Linda; Rose, John K.; Bressanelli, Stéphane; Gaudin, Yves; Albertini, Aurélie A.

    2012-01-01

    Chandipura virus glycoprotein ectodomain (Gth) was purified and crystallized at pH 7.5. X-ray diffraction data set was collected to a resolution of 3.1 Å. Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV G th ) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV G th obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal

  16. Glycan structures contain information for the spatial arrangement of glycoproteins in the plasma membrane.

    Directory of Open Access Journals (Sweden)

    M Kristen Hall

    Full Text Available Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells.

  17. Allelic Variations at Four Major Maturity E Genes and Transcriptional Abundance of the E1 Gene Are Associated with Flowering Time and Maturity of Soybean Cultivars

    Science.gov (United States)

    Wang, Yueqiang; Chen, Xin; Ren, Haixiang; Yang, Jiayin; Cheng, Wen; Zong, Chunmei; Gu, Heping; Qiu, Hongmei; Wu, Hongyan; Zhang, Xingzheng; Cui, Tingting; Xia, Zhengjun

    2014-01-01

    The time to flowering and maturity are ecologically and agronomically important traits for soybean landrace and cultivar adaptation. As a typical short-day crop, long day conditions in the high-latitude regions require soybean cultivars with photoperiod insensitivity that can mature before frost. Although the molecular basis of four major E loci (E1 to E4) have been deciphered, it is not quite clear whether, or to what degree, genetic variation and the expression level of the four E genes are associated with the time to flowering and maturity of soybean cultivars. In this study, we genotyped 180 cultivars at E1 to E4 genes, meanwhile, the time to flowering and maturity of those cultivars were investigated at six geographic locations in China from 2011 to 2012 and further confirmed in 2013. The percentages of recessive alleles at E1, E2, E3 and E4 loci were 38.34%, 84.45%, 36.33%, and 7.20%, respectively. Statistical analysis showed that allelic variations at each of four loci had a significant effect on flowering time as well as maturity. We classified the 180 cultivars into eight genotypic groups based on allelic variations of the four major E loci. The genetic group of e1-nf representing dysfunctional alleles at the E1 locus flowered earliest in all the geographic locations. In contrast, cultivars in the E1E2E3E4 group originated from the southern areas flowered very late or did not flower before frost at high latitude locations. The transcriptional abundance of functional E1 gene was significantly associated with flowering time. However, the ranges of time to flowering and maturity were quite large within some genotypic groups, implying the presence of some other unknown genetic factors that are involved in control of flowering time or maturity. Known genes (e.g. E3 and E4) and other unknown factors may function, at least partially, through regulation of the expression of the E1 gene. PMID:24830458

  18. A hepatitis C virus (HCV) vaccine comprising envelope glycoproteins gpE1/gpE2 derived from a single isolate elicits broad cross-genotype neutralizing antibodies in humans

    DEFF Research Database (Denmark)

    Law, John Lok Man; Chen, Chao; Wong, Jason

    2013-01-01

    of genotype 1a). Cross neutralization was tested in Huh-7.5 human hepatoma cell cultures using infectious recombinant HCV (HCVcc) expressing structural proteins of heterologous HCV strains from all known major genotypes, 1-7. Vaccination induced significant neutralizing antibodies against heterologous HCV...... genotype 1a virus which represents the most common genotype in North America. Of the 16 vaccinees tested, 3 were selected on the basis of strong 1a virus neutralization for testing of broad cross-neutralizing responses. At least 1 vaccinee was shown to elicit broad cross-neutralization against all HCV...

  19. Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model

    DEFF Research Database (Denmark)

    Andersson, O.; Badisco, L.; Hansen, A. H.

    2014-01-01

    In earlier studies insects were proposed as suitable models for vertebrate blood–brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain b...... has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery.......In earlier studies insects were proposed as suitable models for vertebrate blood–brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain...

  20. 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.

  1. Design and synthesis of an antigenic mimic of the Ebola glycoprotein

    OpenAIRE

    Rutledge, Ryan D.; Huffman, Brian J.; Cliffel, David E.; Wright, David W.

    2008-01-01

    An antigenic mimic of the Ebola glycoprotein was synthesized and tested for its ability to be recognized by an anti-Ebola glycoprotein antibody. Epitope-mapping procedures yielded a suitable epitope that, when presented on the surface of a nanoparticle, forms a structure that is recognized by an antibody specific for the native protein. This mimic-antibody interaction has been quantitated through ELISA and QCM-based methods and yielded an affinity (Kd = 12 × 10−6 M) within two orders of magni...

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

    DEFF Research Database (Denmark)

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

    1990-01-01

    Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human...... enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs....

  3. 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.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...

  5. P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments

    DEFF Research Database (Denmark)

    Ejsing, Thomas B.; Pedersen, Anne D.; Linnet, Kristian

    2005-01-01

    P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice......P-glycoprotein, risperidone, nortriptyline, cyclosporine A, drug-drug interaction, blood-brain barrier, knock-out mice...

  6. Forbidden Raman scattering processes. I. General considerations and E1--M1 scattering

    International Nuclear Information System (INIS)

    Harney, R.C.

    1979-01-01

    The generalized theory of forbidden Raman scattering processes is developed in terms of the multipole expansion of the electromagnetic interaction Hamiltonian. Using the general expressions, the theory of electric dipole--magnetic dipole (E1--M1) Raman scattering is derived in detail. The 1 S 0 → 3 P 1 E1--M1 Raman scattering cross section in atomic magnesium is calculated for two applicable laser wavelengths using published f-value data. Since resonantly enhanced cross sections larger than 10 -29 cm 2 /sr are predicted it should be possible to experimentally observe this scattering phenomenon. In addition, by measuring the frequency dependence of the cross section near resonance, it may be possible to directly determine the relative magnitudes of the Axp and AxA contributions to the scattering cross section. Finally, possible applications of the effect in atomic and molecular physics are discussed

  7. Cytochrome P450 2E1 polymorphism and nasopharyngeal carcinoma development in Thailand: a correlative study

    International Nuclear Information System (INIS)

    Kongruttanachok, Narisorn; Sukdikul, Sairoong; Setavarin, Surachai; Kerekhjanarong, Verachai; Supiyaphun, Pakpoom; Voravud, Narin; Poovorawan, Yong; Mutirangura, Apiwat

    2001-01-01

    Nasopharyngeal carcinoma (NPC) is a rare tumor in most parts of the world but occurs at relatively high frequency among people of Chinese descent. The cytochrome P450 2E1 enzyme (CYP2E1) is responsible for the metabolic activation of nitrosamines, and has been shown to be a susceptibility gene for NPC development in Taiwan [RR = 2.6; 95%CI = 1.2-5.7]. Since there has been only one report of this link, it was decided to investigate the susceptibility of CYP2E1 to NPC development in other populations. Therefore, the correlation between the RsaI polymorphism of this gene and NPC was studied in-patients including Thai and Chinese in Thailand. The present study comprised 217 cases diagnosed with NPC and 297 healthy controls. Similar to the result found in Taiwanese, a homozygous uncut genotype demonstrated a higher relative risk both when all cases were analyzed [RR = 2.19; 95%CI = 0.62-8.68] or individual racial groups, Thai [RR = 1.51; 95%CI = 0.08-90.06] or Chinese [RR = 1.99; 95%CI = 0.39-10.87]. The ethnicity-adjusted odds ratio is 2.39 with 95%CI, 0.72-7.89. Though our finding was not statistically significant due to the moderate sample size of the study, similarity to the study in Taiwan with only a slight loss in precision was demonstrated. The higher RR found for the same genotype in distinct populations confirmed that CYP2E1 is one of several NPC susceptibility genes and that the RsaI minus variant is one mutation that affects phenotype

  8. Cooperativity in CYP2E1 Metabolism of Acetaminophen and Styrene Mixtures

    OpenAIRE

    Hartman, Jessica H.; Letzig, Lynda G.; Robertsc, Dean W.; James, Laura P.; Fifer, E. Kim; Miller, Grover P.

    2015-01-01

    Risk assessment for exposure to mixtures of drugs and pollutants relies heavily on in vitro characterization of their bioactivation and/or metabolism individually and extrapolation to mixtures assuming no interaction. Herein, we demonstrated that in vitro CYP2E1 metabolic activation of acetaminophen and styrene mixtures could not be explained through the Michaelis-Menten mechanism or any models relying on that premise. As a baseline for mixture studies with styrene, steady-state analysis of a...

  9. Aminotriazole alleviates acetaminophen poisoning via downregulating P450 2E1 and suppressing inflammation.

    Directory of Open Access Journals (Sweden)

    Yuping Jing

    Full Text Available Aminotriazole (ATZ is commonly used as a catalase (CAT inhibitor. We previously found ATZ attenuated oxidative liver injury, but the underlying mechanisms remain unknown. Acetaminophen (APAP overdose frequently induces life-threatening oxidative hepatitis. In the present study, the potential hepatoprotective effects of ATZ on oxidative liver injury and the underlying mechanisms were further investigated in a mouse model with APAP poisoning. The experimental data indicated that pretreatment with ATZ dose- and time-dependently suppressed the elevation of plasma aminotransferases in APAP exposed mice, these effects were accompanied with alleviated histological abnormality and improved survival rate of APAP-challenged mice. In mice exposed to APAP, ATZ pretreatment decreased the CAT activities, hydrogen peroxide (H2O2 levels, malondialdehyde (MDA contents, myeloperoxidase (MPO levels in liver and reduced TNF-α levels in plasma. Pretreatment with ATZ also downregulated APAP-induced cytochrome P450 2E1 (CYP2E1 expression and JNK phosphorylation. In addition, posttreatment with ATZ after APAP challenge decreased the levels of plasma aminotransferases and increased the survival rate of experimental animals. Posttreatment with ATZ had no effects on CYP2E1 expression or JNK phosphorylation, but it significantly decreased the levels of plasma TNF-α. Our data indicated that the LD50 of ATZ in mice was 5367.4 mg/kg body weight, which is much higher than the therapeutic dose of ATZ in the present study. These data suggested that ATZ might be effective and safe in protect mice against APAP-induced hepatotoxicity, the beneficial effects might resulted from downregulation of CYP2E1 and inhibiton of inflammation.

  10. Purification of supercoiled DNA of plasmid Col E1 by RPC-5 chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Best, A.N.; Allison, D.P.; Novelli, G.D.

    1981-07-01

    Col E1 DNA can be purified to a high degree by RPC-5 chromatography of a partially purified cell lysate with a very shallow linear NaC1 gradient at pH 7.8. Electron micrographs demonstrated that the purest fractions were composed of 93% supercoiled (form I) DNA and 7% open circular (form II) DNA. The actual chromatography can be accomplished in 13 to 14 h and is designed for the production of several milligrams of plasmid DNA.

  11. 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

  12. Ovarian Hormone Estrone Glucuronide (E1G) quantification-impedimetric electrochemical spectroscopy approach

    KAUST Repository

    Zia, Asif I.; Mukhopadhyay, Subhas Chandra; Yu, Paklam; Al-Bahadly, Ibrahim H.; Yudhana, Anton; Gooneratne, Chinthaka Pasan; Kosel, Jü rgen

    2013-01-01

    A study was conducted on detection and concentration measurement of estrone glucuronide (E1G), an important metabolite of the ovarian hormone estradiol, by using Electrochemical Impedance Spectroscopy (EIS) technique. A miniature planar Inter-digital (ID) capacitive sensor fabricated on single crystal silicon substrate with sputtered gold electrodes coupled with EIS was used to measure conductivity, permeability and dielectric properties of the said hormone metabolite. A thin film of Silicon Nitride (50 um) was coated on the sensor as passivation layer to avoid Faradic currents through the sensor. Impedance spectrums were obtained with various concentrations of E1G in buffer solution by exposing the samples to electrical perturbations at certain frequency range. Relationship of sample conductance with E1G concentration was studied on basis Randle's equivalent circuit model and results were analyzed to deduce Constant Phase Equivalent (CPE) Circuit model in order to evaluate the double layer capacitance produced at the solution-electrode interface due to kinetic processes taking place in the electrochemical cell. The sensitivity of the sensor was evaluated against concentration. The result analysis confirmed that fabricated ID sensor together with EIS can provide a rapid and successful low cost sensing system which can help a lay user to determine peak time for feminine reproductive fertility at home without submitting samples for an expensive and time consuming laboratory test. © 2013 IEEE.

  13. Ovarian Hormone Estrone Glucuronide (E1G) quantification-impedimetric electrochemical spectroscopy approach

    KAUST Repository

    Zia, Asif I.

    2013-12-01

    A study was conducted on detection and concentration measurement of estrone glucuronide (E1G), an important metabolite of the ovarian hormone estradiol, by using Electrochemical Impedance Spectroscopy (EIS) technique. A miniature planar Inter-digital (ID) capacitive sensor fabricated on single crystal silicon substrate with sputtered gold electrodes coupled with EIS was used to measure conductivity, permeability and dielectric properties of the said hormone metabolite. A thin film of Silicon Nitride (50 um) was coated on the sensor as passivation layer to avoid Faradic currents through the sensor. Impedance spectrums were obtained with various concentrations of E1G in buffer solution by exposing the samples to electrical perturbations at certain frequency range. Relationship of sample conductance with E1G concentration was studied on basis Randle\\'s equivalent circuit model and results were analyzed to deduce Constant Phase Equivalent (CPE) Circuit model in order to evaluate the double layer capacitance produced at the solution-electrode interface due to kinetic processes taking place in the electrochemical cell. The sensitivity of the sensor was evaluated against concentration. The result analysis confirmed that fabricated ID sensor together with EIS can provide a rapid and successful low cost sensing system which can help a lay user to determine peak time for feminine reproductive fertility at home without submitting samples for an expensive and time consuming laboratory test. © 2013 IEEE.

  14. Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

    International Nuclear Information System (INIS)

    Kibe, A.; Shimada, K.; Tagaki, Y.

    1979-01-01

    Hybrid ColE1 plasmids called ColE1-cos lambda-guaA or ColE1-cos lambda-gal can be efficiently transduced into various E.coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E.coli K-12 mutants. The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos lambda-guaA. Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. ColE1-cos lambda-guaA and ColE1-cos lambda-gal DNAs could temporarily but not stably co-exist in E.coli K-12 recA cells. The presence of ColE1-cos lambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos lambda-guaA about 7-fold. The same ColE1-cos lambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA + gene function(s) and suggest that ColE1 plasmit itself provides no recA + -like functions. (orig.) [de

  15. Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kibe, A; Shimada, K; Tagaki, Y [Kyushu Univ., Fukuoka (Japan). Dept. of Biochemistry

    1979-01-01

    Hybrid ColE1 plasmids called ColE1-cos lambda-guaA or ColE1-cos lambda-gal can be efficiently transduced into various E.coli K-12 cells through packaging into lambda phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E.coli K-12 mutants. The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos lambda-guaA. Pre-existing hybrid ColE1 plasmids had no effect on the frequency of lambda phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. ColE1-cos lambda-guaA and ColE1-cos lambda-gal DNAs could temporarily but not stably co-exist in E.coli K-12 recA cells. The presence of ColE1-cos lambda-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos lambda-guaA about 7-fold. The same ColE1-cos lambda-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA/sup +/ gene function(s) and suggest that ColE1 plasmit itself provides no recA/sup +/-like functions.

  16. Chemoenzymatic site-specific labeling of influenza glycoproteins as a tool to observe virus budding in real time.

    Directory of Open Access Journals (Sweden)

    Maximilian Wei-Lin Popp

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

  17. Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

    Directory of Open Access Journals (Sweden)

    Yamauchi Mika

    2007-11-01

    Full Text Available Abstract Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1 mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR, in the cells. AdipoR1 small interfering RNA (siRNA transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

  18. Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH.

    Science.gov (United States)

    Vallbracht, Melina; Rehwaldt, Sascha; Klupp, Barbara G; Mettenleiter, Thomas C; Fuchs, Walter

    2018-05-01

    Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide

  19. Envelope gene sequences encoding variable regions 3 and 4 are involved in macrophage tropism of feline immunodeficiency virus

    NARCIS (Netherlands)

    Horzinek, M.C.; Vahlenkamp, T.W.; Ronde, A. de; Schuurman, N.M.P.; Vliet, A.L.W. van; Drunen, J. van; Egberink, H.F.

    1999-01-01

    The envelope is of cardinal importance for the entry of feline immunodeficiency virus (FIV) into its host cells, which consist of cells of the immune system including macrophages. To characterize the envelope glycoprotein determinants involved in macrophage tropism, chimeric infectious molecular

  20. Molecular mechanisms involved in modulation of p-glycoprotein expression from squamous cell carcinoma by low dose fractionated radiation (LDFR)

    International Nuclear Information System (INIS)

    Shajahan; Shahin; Shareef, Mohammed M.; Sathishkumar, Sabapathi; Mohiuddin, Mohammed; Ahmed, Mansoor M.; Brown, Brandee C.; Jones, Raleigh; Spring, Paul M.

    2004-01-01

    In the present study, two squamous cell carcinoma oral cavity cells (SCCOC), T-167 (p53 wild type) and T-409 (p53 mutant), were exposed to either clinically relevant dose (2 Gy), high dose (7Gy) or fractionated low dose (LDFR) (0.5 Gy x 4) and the expression of Mdr1 gene was assessed by real time RT-PCR, semiquantitative 32 P RT-PCR and luciferase reporter assay

  1. The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

    Science.gov (United States)

    Sedbrook, John C.; Carroll, Kathleen L.; Hung, Kai F.; Masson, Patrick H.; Somerville, Chris R.

    2002-01-01

    To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

  2. Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr1a P-glycoprotein

    NARCIS (Netherlands)

    Mayer, U; Wagenaar, E; Beijnen, J.H; Smit, J.W; Meijer, D.K F; van Asperen, J.; Borst, P; Schinkel, A.H

    1 We have used mice with a disrupted mdrla P-glycoprotein gene (mdrIa (-/-) mice) to study the role of P-glycoprotein in the pharmacokinetics of digoxin, a model P-glycoprotein substrate. 2 [K-3]-digoxin at a dose of 0.2 mg kg(-1) was administered as a single i.v. or oral bolus injection. We

  3. Evolution of camel CYP2E1 and its associated power of binding toxic industrial chemicals and drugs.

    Science.gov (United States)

    Kandeel, Mahmoud; Altaher, Abdullah; Kitade, Yukio; Abdelaziz, Magdi; Alnazawi, Mohamed; Elshazli, Kamal

    2016-10-01

    Camels are raised in harsh desert environment for hundreds of years ago. By modernization of live and the growing industrial revolution in camels rearing areas, camels are exposed to considerable amount of chemicals, industrial waste, environmental pollutions and drugs. Furthermore, camels have unique gene evolution of some genes to withstand living in harsh environments. In this work, the camel cytochrome P450 2E1 (CYP2E1) is compromised to detect its evolution rate and its power to bind with various chemicals, protoxins, procarcinogens, industrial toxins and drugs. In comparison with human CYP2E1, camel CYP2E1 more efficiently binds to small toxins as aniline, benzene, catechol, amides, butadiene, toluene and acrylamide. Larger compounds were more preferentially bound to the human CYP2E1 in comparison with camel CYP2E1. The binding of inhalant anesthetics was almost similar in both camel and human CYP2E1 coinciding with similar anesthetic effect as well as toxicity profiles. Furthermore, evolutionary analysis indicated the high evolution rate of camel CYP2E1 in comparison with human, farm and companion animals. The evolution rate of camel CYP2E1 was among the highest evolution rate in a subset of 57 different organisms. These results indicate rapid evolution and potent toxin binding power of camel CYP2E1. Copyright © 2016. Published by Elsevier Ltd.

  4. Cuscuta chinensis seeds water extraction protecting murine osteoblastic MC3T3-E1 cells against tertiary butyl hydroperoxide induced injury.

    Science.gov (United States)

    Gao, Jian-mei; Li, Ran; Zhang, Lei; Jia, Li-long; Ying, Xi-xiang; Dou, De-qiang; Li, Jian-chun; Li, Hai-bo

    2013-07-09

    Cuscuta chinensis (C. chinensis) is a well-known traditional Chinese herb that has been used to treat heart disease, diabetes, liver injury, cancer, and aging. Murine osteoblastic MC3T3-E1 cells were treated with various concentrations of C. chinensis water extraction at different time intervals. The antioxidant effect of C. chinensis on MC3T3-E1 cells was evaluated using MTT and TUNEL assays. The effect of C. chinensis on cell cycle was analyzed by flow cytometry with propidium iodide. Lipid peroxidation was measured by the HPLC method. The cellular redox status was determined from the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) and the enzymes involved in glutathione metabolism, including glutathione reductase (GR), Glutathione S-transferase (GST), and Glucose-6-phosphate dehydrogenase (G6PD). The changes in relative mitochondrial transmembrane potential (ΔΨm) in the MC3T3-E1 cells were analyzed with rhodamine 123 staining. Western blot analysis was used to evaluate the levels of cytochrome c (cyto c), Bax, Bcl-2, caspase 3, Sirt3, and IDH2 expressions. The C. chinensis water extraction protects tertiary butyl hydroperoxide (TBHP)-treated MC3T3-E1 cells from death in a dose-dependent manner. C. chinensis treatment significantly inhibited the reactive oxygen species (ROS) generation, malondialdehyde (MDA) production, and increased the activity of superoxide dismutase (SOD), GR, GST, and G6PD. The release of cyto c from mitochondria was reduced by C. chinensis, which increased the expression of antiapoptotic IDH2, Sirt3, and Bcl-2 and decreased the expression of Bax, cyto c, and caspase 3. C. chinensis modulated the oxidative stress-induced apoptosis in MC3T3-E1 cells, probably due to its antioxidant activity and functioning via mitochondria-dependent pathways. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. N-Hydroxylation of 4-Aminobiphenyl by CYP2E1 Produces Oxidative Stress in a Mouse Model of Chemically Induced Liver Cancer

    Science.gov (United States)

    Wang, Shuang; Sugamori, Kim S.; Tung, Aveline; McPherson, J. Peter; Grant, Denis M.

    2015-01-01

    4-Aminobiphenyl (ABP) is a trace component of cigarette smoke and hair dyes, a suspected human carcinogen and a potent rodent liver carcinogen. Postnatal exposure of mice to ABP results in a higher incidence of liver tumors in males than in females, paralleling the sex difference in human liver cancer incidence. A traditional model of ABP tumorigenesis involves initial CYP1A2-mediated N-hydroxylation, which eventually leads to production of mutagenic ABP-DNA adducts that initiate tumor growth. However, several studies have found no correlation between sex or CYP1A2 function and the DNA-damaging, mutagenic, or tumorigenic effects of ABP. Oxidative stress may be an important etiological factor for liver cancer, and it has also been linked to ABP exposure. The goals of this study were to identify novel enzyme(s) that contribute to ABP N-oxidation, and to investigate a potential role for oxidative stress in ABP liver tumorigenicity. Isozyme-selective inhibition experiments using liver microsomes from wild-type and genetically modified mice identified CYP2E1 as a major ABP N-hydroxylating enzyme. The N-hydroxylation of ABP by transiently expressed CYP2E1 produced oxidative stress in cultured mouse hepatoma cells. In vivo postnatal exposure of mice to a tumorigenic dose of ABP also produced oxidative stress in male wild-type mice, but not in male Cyp2e1(−/−) mice or in female mice. However, a stronger NRF2-associated antioxidant response was observed in females. Our results identify CYP2E1 as a novel ABP-N-oxidizing enzyme, and suggest that sex differences in CYP2E1-dependent oxidative stress and antioxidant responses to ABP may contribute to the observed sex difference in tumor incidence. PMID:25601990

  6. Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima

    DEFF Research Database (Denmark)

    Belli, Sabina I; Wallach, Michael G; Luxford, Catherine

    2003-01-01

    infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage...... (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography......-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode...

  7. Transport of radiolabelled glycoprotein to cell surface and lysosome-like bodies of absorptive cells in cultured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease

    International Nuclear Information System (INIS)

    Ginsel, L.A.; Onderwater, J.J.M.; Daems, W.T.

    1979-01-01

    The transport of 3 H-fucose and 3 H-glucosamine-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue was investigated with light- and electron-microscopical autoradiography. The findings showed that these glycoproteins were completed in the Golgi apparatus and transported in small vesicular structures to the apical cytoplasm of these cells. Since this material arrived in the cell coat on the microvilli and in the lysosome-like bodies simultaneously, a crinophagic function of these organelles in the regulation of the transport or secretion of cell-coat material was supported. In the absorptive cells of patients with fucosidosis or Hunter's type of lysosomal storage disease, a similar transport of cell-coat material to the lysosome-like bodies and a congenital defect of a lysosomal hydrolase normally involved in the degradation of cell-coat material, can explain the accumulation of this material in the dense bodies. (orig.) [de

  8. Human intestinal P-glycoprotein activity estimated by the model substrate digoxin

    DEFF Research Database (Denmark)

    Larsen, U L; Hyldahl Olesen, L; Nyvold, Charlotte Guldborg

    2007-01-01

    P-glycoprotein (Pgp) plays a part in the intestinal uptake of xenobiotics and has been associated with susceptibility to ulcerative colitis. The aim of this study was to examine Pgp activity in relation to age, gender, medical treatment (rifampicin or ketoconazole) and the multidrug resistance (MDR...

  9. Generating Isoform-Specific Antibodies : Lessons from Nucleocytoplasmic Glycoprotein Skp1

    NARCIS (Netherlands)

    West, Christopher M.; Van Der Wel, Hanke; Chinoy, Zoiesha; Boons, Geert Jan; Gauthier, Ted J.; Taylor, Carol M.; Xu, Yuechi

    2015-01-01

    Antibodies that discriminate protein isoforms differing by modifications at specific amino acids have revolutionized studies of their functions. Skp1 is a novel nucleocytoplasmic glycoprotein that is hydroxylated at proline-143 and then O-glycosylated by a pentasaccharide attached via a GlcNAcα1,

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

    Science.gov (United States)

    Kim, Dae-Won; Kim, You-Jin; Park, Sung Han; Yun, Mi-Ran; Yang, Jeong-Sun; Kang, Hae Ji; Han, Young Woo; Lee, Han Saem; Kim, Heui Man; Kim, Hak; Kim, A-Reum; Heo, Deok Rim; Kim, Su Jin; Jeon, Jun Ho; Park, Deokbum; Kim, Joo Ae; Cheong, Hyang-Min; Nam, Jeong-Gu; Kim, Kisoon; Kim, Sung Soon

    2016-01-01

    An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

  11. Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

    NARCIS (Netherlands)

    C.H.J. Siebelink (Kees); E.J. Tijhaar (Edwin); R.C. Huisman (Robin); W. Huisman (Willem); A. de Ronde; I.H. Darby; M.J. Francis; G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Albert)

    1995-01-01

    textabstractCats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein

  12. 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

  13. St. John's Wort constituents modulate P-glycoprotein transport activity at the blood-brain barrier.

    NARCIS (Netherlands)

    Ott, M.; Huls, M.; Cornelius, M.G.; Fricker, G.

    2010-01-01

    PURPOSE: The purpose of this study was to investigate the short-term signaling effects of St. John's Wort (SJW) extract and selected SJW constituents on the blood-brain barrier transporter P-glycoprotein and to describe the role of PKC in the signaling. METHODS: Cultured porcine brain capillary

  14. Evolutionary conservation of the lipopolysaccharide binding site of β₂-glycoprotein I

    NARCIS (Netherlands)

    Ağar, Çetin; de Groot, Philip G.; Marquart, J. Arnoud; Meijers, Joost C. M.

    2011-01-01

    β₂-Glycoprotein I (β₂GPI) is a highly abundant plasma protein and the major antigen for autoantibodies in the antiphospholipid syndrome. Recently, we have described a novel function of β₂GPI as scavenger of lipopolysaccharide (LPS). With this in mind we investigated the conservation of β₂GPI in

  15. Do asparagine-linked carbohydrate chains in glycoproteins have a preference for beta-bends?

    NARCIS (Netherlands)

    Beintema, Jaap J.

    X-ray structures of the conformation of carbohydrate moieties and connected regions of glycoproteins are summarized. Evidence is presented that there is some preference for carbohydrate attachment at β-bends. Evolution may have favored glycosylation to occur at bends to ensure free mobility of the

  16. 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

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

    DEFF Research Database (Denmark)

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

    1997-01-01

    Three glycoforms of alpha 1-acid glycoprotein (AGP) were biotinylated to examine their binding in mouse testis by light microscopy. The transition from one stage to another in the spermatogenic cycle is marked with an appearance of a receptor for the Concanavalin A (Con A) non-reactive glycoform...

  18. Cytomegalovirus glycoprotein B genotyping in ocular fluids and blood of AIDS patients with cytomegalovirus retinitis

    NARCIS (Netherlands)

    Peek, R.; Verbraak, F.; Bruinenberg, M.; van der Lelij, A.; van den Horn, G.; Kijlstra, A.

    1998-01-01

    To determine the frequency of cytomegalovirus glycoprotein B (gB) genotypes in clinical samples of ocular fluids of patients with acquired immune deficiency syndrome (AIDS) who have cytomegalovirus retinitis and to compare these with the cytomegalovirus gB genotype in paired peripheral blood

  19. The combination of simple MALDI matrices for the improvement of intact glycoproteins and glycans analysis

    Czech Academy of Sciences Publication Activity Database

    Laštovičková, Markéta; Chmelík, Josef; Bobálová, Janette

    2009-01-01

    Roč. 281, 1-2 (2009), s. 82-88 ISSN 1387-3806 R&D Projects: GA AV ČR IAA600040701; GA MŠk 1M0570 Institutional research plan: CEZ:AV0Z40310501 Keywords : glycoproteins * binary matrices * MALDI-TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.117, year: 2009

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

    DEFF Research Database (Denmark)

    Thaysen-Andersen, Morten; Thøgersen, Ida; Lademann, Ulrik Axel

    2008-01-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  2. 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

  3. Structural analysis of the carbohydrate chains of glycoproteins by 500-MHz 1H-NMR spectroscopy

    International Nuclear Information System (INIS)

    Mutsaers, J.H.G.M.

    1986-01-01

    This thesis deals with the structural analysis by 500-MHz 1 H-NMR spectroscopy of carbohydrate chains obtained from glycoproteins. In the chapters 1 to 6 the structural analysis of N-glycosidically linked carbohydrate chains is described. The chapters 7 to 10 describe the structural analysis of O-glycosidically linked carbohydrate chains. 381 refs.; 44 figs.; 24 tabs.; 7 schemes

  4. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  5. Low rate doses effects of gamma radiation on glycoproteins of transmembrane junctions in fibroblasts

    International Nuclear Information System (INIS)

    Bringas, J.E.; Caceres, J.L.

    1996-01-01

    Glycoproteins of trans-membrane junctions are molecules that help to bind cells with the extracellular matrix. Integrins are the most important trans-membrane molecules among others. The damage of gamma radiation on those proteins could be an important early event that causes membrane abnormalities which may lead to cell malfunction and cancer induced by radiation due to cell dissociation. Randomized blocks with 3 repetitions of mouse embryo fibroblast cultures, were irradiated with Cobalt-60 gamma rays, during 20 days. Biological damage to glycoproteins and integrins was evaluated by cellular growth and fibroblast proliferative capacity. Integrins damage was studied by isolation by column immunoaffinity chromatography migrated on SDS-Page under reducing and non reducing conditions, and inhibition of integrins extracellular matrix adhesion by monoclonal antibodies effect. The dose/rate (0.05 Gy/day-0.2 Gy/day) of gamma given to cells did not show damage evidence on glycoproteins and integrins. If damage happened, it was repaired by cells very soon, was delayed by continuous cellular division or by glycoproteins characteristic of being multiple extracellular ligatures. Bio effects became more evident with an irradiation time greater than 20 days or a high dose/rate. (authors). 6 refs

  6. Development of oligoclonal nanobodies for targeting the tumor-associated glycoprotein 72 antigen

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali

    2013-01-01

    The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specific...

  7. 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

  8. Serum-SP/sub 1/-pregnancy-specific-. beta. -glycoprotein in choriocarcinoma and other neoplastic disease

    Energy Technology Data Exchange (ETDEWEB)

    Searle, F; Leake, B A; Bagshawe, K D; Dent, J [Charing Cross Group of Hospitals, London (UK)

    1978-03-18

    A radioimmunoassay for a placental glycoprotein, ..beta../sub 1/SP/sub 1/, capable of detecting 2 ..mu..g/l of the glycoprotein in serum was used to measure concentrations of ..beta../sub 1/SP/sub 1/ in patients with choriocarcinoma, teratoma, colonic cancer, breast cancer, and ovarian cancer. Twelve out of 94 (13%) healthy men and healthy non-pregnant women had detectable serum-..beta../sub 1/SP/sub 1/ concentrations. Concentrations up to 50,000 ..mu..g/l were found in the sera of patients with hydatidiform mole, invasive mole, choriocarcinoma, and malignant teratoma. ..beta../sub 1/-glycoprotein concentrations were generally much lower than corresponding concentrations of chorionic gonadotrophin which is the most reliable marker for trophoblastic tumors. In a few cases, however, ..beta../sub 1/-glycoprotein measurements may be useful in the detection of minimal residual tumor. The slightly raised values found in some patients with carcinoma of the colon, breast, or ovary seem unlikely to be useful for diagnostic purposes or for monitoring the course of these cancers.

  9. Characterization of glycoprotein C of HSZP strain of herpes simplex virus 1

    NARCIS (Netherlands)

    Oravcova, [No Value; Kudelova, M; Mlcuchova, J; Matis, J; Bystricka, M; Westra, DF; Welling-Wester, S; Rajcani, J

    Sequences of UL44 genes of strains HSZP, KOS and 17 of herpes simplex virus 1 (HSV-1) were determined and the amino acid sequences of corresponding glycoproteins (gC) were deduced. In comparison with the 17 strain, the HSZP strain showed specific changes in 3 nucleotides and in 2 amino acids (aa 139

  10. Studies on the subunits of human glycoprotein hormones in relation to reproduction

    International Nuclear Information System (INIS)

    Hagen, C.

    1977-01-01

    In this review summarising present knowledge of the biological and immunological activity of the subunits of human glycoprotein hormones, the specificity of the α-subunit and β-subunit radioimmunoassays are discussed. The crossreaction studies performed with the α-subunit radioimmunoassays are aummarised in one table while those with the β-subunit radioimmunoassays are presented in a second table. (JIW)

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

    Science.gov (United States)

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

  12. Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections

    NARCIS (Netherlands)

    Xiong, Xiaoli; Tortorici, M Alejandra; Snijder, Joost|info:eu-repo/dai/nl/338018328; Yoshioka, Craig; Walls, Alexandra C; Li, Wentao|info:eu-repo/dai/nl/411296272; McGuire, Andrew T; Rey, Félix A; Bosch, Berend-Jan|info:eu-repo/dai/nl/273306049; Veesler, David

    2017-01-01

    Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    -glycosylated proteins in body fluids and other complex samples. An approach for identification of N-glycosylated proteins and mapping of their glycosylation sites is described. In this approach, glycoproteins are initially selectively purified by lectin chromatography. Following tryptic digestion, glycopeptides...

  14. Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells.

    Science.gov (United States)

    Fatokun, Amos A; Tome, Mercedes; Smith, Robert A; Darlington, L Gail; Stone, Trevor W

    2015-01-01

    Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.

  15. Low energy electron irradiation induced deep level defects in 6H-SiC: The implication for the microstructure of the deep levels E1/E2

    International Nuclear Information System (INIS)

    Chen, X.D.; Fung, S.; Beling, C.D.; Lui, M.K.; Ling, C.C.; Yang, C.L.; Ge, W.K.; Wang, J.N.; Gong, M.

    2004-01-01

    N-type 6H-SiC samples irradiated with electrons having energies of E e =0.2, 0.3, 0.5, and 1.7 were studied by deep level transient technique. No deep level was detected at below 0.2 MeV irradiation energy while for E e ≥0.3 MeV, deep levels ED1, E 1 /E 2 , and E i appeared. By considering the minimum energy required to displace the C atom or the Si atom in the SiC lattice, it is concluded that generation of the deep levels E 1 /E 2 , as well as ED1 and E i , involves the displacement of the C atom in the SiC lattice

  16. Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo

    International Nuclear Information System (INIS)

    Bošnjak, Ivana; Borra, Marco; Iamunno, Franco; Benvenuto, Giovanna; Ujević, Ivana; Bušelić, Ivana; Roje-Busatto, Romana; Mladineo, Ivona

    2014-01-01

    Highlights: • Effects of BPA on embryonic development of Paracentrotus lividus were determined. • Transport assay, intracellular BPA measurements and gene expression surveys were made. • Multidrug efflux transporter P-gp/ABCB1 is involved in BPA elimination. • Endocrine disruption is inferred by orphan steroid hormone receptor (shr2) upregulation. • BPA delayed mitosis, inducing aberrant karyokinesis and dysfunctional microfilaments. - Abstract: Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation

  17. Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo

    Energy Technology Data Exchange (ETDEWEB)

    Bošnjak, Ivana [Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, Zagreb (Croatia); Borra, Marco [Molecular Biology Service, Stazione Zoologica Anton Dohrn, Villa Comunale 80121, Napoli (Italy); Iamunno, Franco; Benvenuto, Giovanna [Electron Microscopy Service, Stazione Zoologica Anton Dohrn, Villa Comunale 80121, Napoli (Italy); Ujević, Ivana [Laboratory of Plankton and Shellfish Toxicity, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Bušelić, Ivana [Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Roje-Busatto, Romana [Laboratory of Plankton and Shellfish Toxicity, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Mladineo, Ivona, E-mail: mladineo@izor.hr [Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Assemble Marine Laboratory, Stazione Zoological Anton Dohrn, Villa Comunale, Naples (Italy)

    2014-11-15

    Highlights: • Effects of BPA on embryonic development of Paracentrotus lividus were determined. • Transport assay, intracellular BPA measurements and gene expression surveys were made. • Multidrug efflux transporter P-gp/ABCB1 is involved in BPA elimination. • Endocrine disruption is inferred by orphan steroid hormone receptor (shr2) upregulation. • BPA delayed mitosis, inducing aberrant karyokinesis and dysfunctional microfilaments. - Abstract: Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation.

  18. Effects of ethanol on CYP2E1 levels and related oxidative stress using a standard balanced diet.

    Science.gov (United States)

    Azzalis, Ligia A; Fonseca, Fernando L A; Simon, Karin A; Schindler, Fernanda; Giavarotti, Leandro; Monteiro, Hugo P; Videla, Luis A; Junqueira, Virgínia B C

    2012-07-01

    Expression of cytochrome P4502E1 (CYP2E1) is very much influenced by nutritional factors, especially carbohydrate consumption, and various results concerning the expression of CYP2E1 were obtained with a low-carbohydrate diet. This study describes the effects of ethanol treatment on CYP2E1 levels and its relationship with oxidative stress using a balanced standard diet to avoid low or high carbohydrate consumption. Rats were fed for 1, 2, 3, or 4 weeks a commercial diet plus an ethanol-sucrose solution. The results have shown that ethanol administration was associated with CYP2E1 induction and stabilization without related oxidative stress. Our findings suggest that experimental models with a low-carbohydrate/high-fat diet produce some undesirable CYP2E1 changes that are not present when a balanced standard diet is given.

  19. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    Energy Technology Data Exchange (ETDEWEB)

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm J.

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1 > LPA4 > LPA2 >> LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G protein-coupled receptor LPA1.

  20. Modelaje Molecular de E-1-[4’-(3-(sustituido) acriloil) fenil]-3-tosilureas

    OpenAIRE

    León, Caritza; Colman, Trina; Domínguez, José

    2012-01-01

    Se presentan resultados del estudio de modelado molecular de una serie de compuestos del tipo E-1-[4’-(3-(sustituido) acriloil)fenil]-3- tosilureas, para las cuales se ha reportado actividad antimalárica como inhibidores del desarrollo in vitro de cepas de P. falciparum resistentes a la Cloroquina. El estudio revela que los compuestos más activos biológicamente muestran una clara superposición en el fragmento comprendido entre el carbono carbonilo del sistema α,β-insaturado y el átomo de azuf...

  1. The relation between the photonuclear E1 sum rule and the effective orbital g-factor

    Energy Technology Data Exchange (ETDEWEB)

    Bentz, Wolfgang E-mail: bentz@keyaki.cc.u-tokai.ac.jp; Arima, Akito

    2004-05-17

    The connection between the enhancement factor (1+{kappa}) of the photonuclear E1 sum rule and the orbital angular momentum g-factor (g{sub l}) of a bound nucleon is investigated in the framework of the Landau-Migdal theory for isospin asymmetric nuclear matter. Special emphasis is put on the role of gauge invariance to establish the {kappa}-g{sub l} relation. By identifying the physical processes which are taken into account in {kappa} and g{sub l}, the validity and limitations of this relation will be discussed. The connections to the collective excitations and to nuclear Compton scattering are also shown.

  2. Chlorido{(E-1-[(2-methoxyphenyldiazenyl]naphthalen-2-olato}palladium(II

    Directory of Open Access Journals (Sweden)

    Assia Mili

    2016-04-01

    Full Text Available In the title complex, [Pd(C17H13N2O2Cl], the PdII atom is tetracoordinated by an N and two O atoms of an (E-1-[(2-methoxyphenyldiazenyl]naphthalen-2-olate ligand and by a Cl atom, and has a square-planar coordination. In the crystal, molecules are linked by pairs of C—H...Cl hydrogen bonds, forming inversion dimers. The dimers are linked via offset π–π interactions [intercentroid distance = 3.546 (3 Å], forming chains running parallel to [100].

  3. Brucella abortus ΔrpoE1 confers protective immunity against wild type challenge in a mouse model of brucellosis.

    Science.gov (United States)

    Willett, Jonathan W; Herrou, Julien; Czyz, Daniel M; Cheng, Jason X; Crosson, Sean

    2016-09-30

    The Brucella abortus general stress response (GSR) system regulates activity of the alternative sigma factor, σ(E1), which controls transcription of approximately 100 genes and is required for persistence in a BALB/c mouse chronic infection model. We evaluated the host response to infection by a B. abortus strain lacking σ(E1) (ΔrpoE1), and identified pathological and immunological features that distinguish ΔrpoE1-infected mice from wild-type (WT), and that correspond with clearance of ΔrpoE1 from the host. ΔrpoE1 infection was indistinguishable from WT in terms of splenic bacterial burden, inflammation and histopathology up to 6weeks post-infection. However, Brucella-specific serum IgG levels in ΔrpoE1-infected mice were 5 times higher than WT by 4weeks post-infection, and remained significantly higher throughout the course of a 12-week infection. Total IgG and Brucella-specific IgG levels peaked strongly in ΔrpoE1-infected mice at 6weeks, which correlated with reduced splenomegaly and bacterial burden relative to WT-infected mice. Given the difference in immune response to infection with wild-type and ΔrpoE1, we tested whether ΔrpoE1 confers protective immunity to wild-type challenge. Mice immunized with ΔrpoE1 completely resisted WT infection and had significantly higher serum titers of Brucella-specific IgG, IgG2a and IFN-γ after WT challenge relative to age-matched naïve mice. We conclude that immunization of BALB/c mice with the B. abortus GSR pathway mutant, ΔrpoE1, elicits an adaptive immune response that confers significant protective immunity against WT infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Global analysis of glycoproteins identifies markers of endotoxin tolerant monocytes and GPR84 as a modulator of TNFα expression.

    Science.gov (United States)

    Müller, Mario M; Lehmann, Roland; Klassert, Tilman E; Reifenstein, Stella; Conrad, Theresia; Moore, Christoph; Kuhn, Anna; Behnert, Andrea; Guthke, Reinhard; Driesch, Dominik; Slevogt, Hortense

    2017-04-12

    Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

  5. Identification, isolation, and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Nicotiana alata.

    Science.gov (United States)

    Jahnen, W; Batterham, M P; Clarke, A E; Moritz, R L; Simpson, R J

    1989-05-01

    S-Gene-associated glycoproteins (S-glycoproteins) from styles of Nicotiana alata, identified by non-equilibrium two-dimensional electrophoresis, were purified by cation exchange fast protein liquid chromatography with yields of 0.5 to 8 micrograms of protein per style, depending on the S-genotype of the plant. The method relies on the highly basic nature of the S-glycoproteins. The elution profiles of the different S-glycoproteins from the fast protein liquid chromatography column were characteristic of each S-glycoprotein, and could be used to establish the S-genotype of plants in outbreeding populations. In all cases, the S-genotype predicted from the style protein profile corresponded to that predicted from DNA gel blot analysis using S-allele-specific DNA probes and to that established by conventional breeding tests. Amino-terminal sequences of five purified S-glycoproteins showed a high degree of homology with the previously published sequences of N. alata and Lycopersicon esculentum S-glycoproteins.

  6. Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum

    International Nuclear Information System (INIS)

    Leberer, E.; Charuk, J.H.M.; MacLennan, D.H.; Green, N.M.

    1989-01-01

    Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1,308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH 2 -terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca 2+ . The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45 C 2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca 2+ with the membrane. The authors propose that this lumenal Ca 2+ binding glycoprotein of the sarcoplasmic reticulum be designated sarcalumenin

  7. Collective vector method for calculation of E1 moments in atomic transition arrays

    International Nuclear Information System (INIS)

    Bloom, S.D.; Goldberg, A.

    1985-10-01

    The CV (collective vector) method for calculating E1 moments for a transition array is described and applied in two cases, herein denoted Z26A and Z26B, pertaining to two different configurations of iron VI. The basic idea of the method is to create a CV from each of the parent (''initial state'') state-vectors of the transition array by application of the E1 operator. The moments of each of these CV's, referred to the parent energy, are then the rigorous moments for that parent, requiring no state decomposition of the manifold of daughter state-vectors. Since, in cases of practical interest, the daughter manifold can be orders of magnitude larger in size than the parent manifold, this makes possible the calculation of many moments higher than the second in situations hitherto unattainable via standard methods. The combination of the moments of all the parents, with proper statistical weighting, then yields the transition array moments from which the transition strength distribution can be derived by various procedures. We describe two of these procedures: (1) The well-known GC (Gram-Charlier) expansion in terms of Hermite polynomials, (2) The Lanczos algorithm or Stieltjes imaging method, also called herein the delta expansion. Application is made in the cases of Z26A (50 lines) and Z26B (5523 lines) and the relative merits and shortcomings of the two procedures are discussed. 10 refs., 15 figs., 2 tabs

  8. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    International Nuclear Information System (INIS)

    Anzenbacherova, Eva; Hudecek, Jiri; Murgida, Daniel; Hildebrandt, Peter; Marchal, Stephane; Lange, Reinhard; Anzenbacher, Pavel

    2005-01-01

    Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics

  9. Comparison of chlorzoxazone one-sample methods to estimate CYP2E1 activity in humans

    DEFF Research Database (Denmark)

    Kramer, Iza; Dalhoff, Kim; Clemmesen, Jens O

    2003-01-01

    OBJECTIVE: Comparison of a one-sample with a multi-sample method (the metabolic fractional clearance) to estimate CYP2E1 activity in humans. METHODS: Healthy, male Caucasians ( n=19) were included. The multi-sample fractional clearance (Cl(fe)) of chlorzoxazone was compared with one...... estimates, Cl(est) at 3 h or 6 h, and MR at 3 h, can serve as reliable markers of CYP2E1 activity. The one-sample clearance method is an accurate, renal function-independent measure of the intrinsic activity; it is simple to use and easily applicable to humans.......-time-point clearance estimation (Cl(est)) at 3, 4, 5 and 6 h. Furthermore, the metabolite/drug ratios (MRs) estimated from one-time-point samples at 1, 2, 3, 4, 5 and 6 h were compared with Cl(fe). RESULTS: The concordance between Cl(est) and Cl(fe) was highest at 6 h. The minimal mean prediction error (MPE) of Cl...

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

    Directory of Open Access Journals (Sweden)

    Sandra Murphy

    2015-06-01

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

  11. Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5)

    Science.gov (United States)

    Zhang, Wenshuai; Zeng, Xiaoyan; Zhang, Li; Peng, Haiyan; Jiao, Yongjun; Zeng, Jun; Treutlein, Herbert R.

    2013-06-01

    In this work, we have developed a new approach to predict the epitopes of antigens that are recognized by a specific antibody. Our method is based on the "multiple copy simultaneous search" (MCSS) approach which identifies optimal locations of small chemical functional groups on the surfaces of the antibody, and identifying sequence patterns of peptides that can bind to the surface of the antibody. The identified sequence patterns are then used to search the amino-acid sequence of the antigen protein. The approach was validated by reproducing the binding epitope of HIV gp120 envelop glycoprotein for the human neutralizing antibody as revealed in the available crystal structure. Our method was then applied to predict the epitopes of two glycoproteins of a newly discovered bunyavirus recognized by an antibody named MAb 4-5. These predicted epitopes can be verified by experimental methods. We also discuss the involvement of different amino acids in the antigen-antibody recognition based on the distributions of MCSS minima of different functional groups.

  12. Molecular organization in bacterial cell membranes. Specific labelling and topological distribution of glycoproteins and proteins in Streptomyces albus membranes

    Energy Technology Data Exchange (ETDEWEB)

    Larraga, V; Munoz, E [Consejo Superior de Investigaciones Cientificas, Madrid (Spain). Instituto de Biologia Celular

    1975-05-01

    The paper reports about an investigation into the question of the specific labelling and topological distribution of glycoproteins and proteins in Streptomyces albus membranes. The method of sample preparation is described: Tritium labelling of glycoproteins in protoplasts and membranes, iodination of proteins, trypsin treatment and polyacrylamide gel electrophoresis. The findings suggest an asymmetrical distribution of the glycoproteins in membranes and a weak accessibility to iodine label. A structural model of the plasma membranes of Streptomyces albus is proposed similar to the general 'fluid mosaic' model of Singer and Nicholson.

  13. Neural glycoprotein M6a is released in extracellular vesicles and modulated by chronic stressors in blood

    OpenAIRE

    Monteleone, Melisa C.; Billi, Silvia C.; Brocco, Marcela A.; Frasch, Alberto C.

    2017-01-01

    Membrane neuronal glycoprotein M6a is highly expressed in the brain and contributes to neural plasticity promoting neurite growth and spine and synapse formation. We have previously showed that chronic stressors alter hippocampal M6a mRNA levels in rodents and tree shrews. We now show that M6a glycoprotein can be detected in mouse blood. M6a is a transmembrane glycoprotein and, as such, unlikely to be free in blood. Here we demonstrate that, in blood, M6a is transported in extracellular vesic...

  14. Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

    Directory of Open Access Journals (Sweden)

    Koh Eunhee

    2007-07-01

    Full Text Available Abstract Background EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Conclusion Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

  15. Immunolocalization and Changes of Hydroxyproline-Rich Glycoproteins During Symbiotic Germination of Dendrobium officinale.

    Science.gov (United States)

    Li, Yuan-Yuan; Chen, Xiao-Mei; Zhang, Ying; Cho, Yu-Hsiu; Wang, Ai-Rong; Yeung, Edward C; Zeng, Xu; Guo, Shun-Xing; Lee, Yung-I

    2018-01-01

    Hydroxyproline-rich glycoproteins (HRGPs) are abundant cell wall components involved in mycorrhizal symbiosis, but little is known about their function in orchid mycorrhizal association. To gain further insight into the role of HRGPs in orchid symbiosis, the location and function of HRGPs were investigated during symbiotic germination of Dendrobium officinale . The presence of JIM11 epitope in developing protocorms was determined using immunodot blots and immunohistochemical staining procedures. Real-time PCR was also employed to verify the expression patterns of genes coding for extensin-like genes selected from the transcriptomic database. The importance of HRGPs in symbiotic germination was further investigated using 3,4-dehydro-L-proline (3,4-DHP), an inhibitor of HRGP biosynthesis. In symbiotic cultures, immunodot blots of JIM11 signals were moderate in mature seeds, and the signals became stronger in swollen embryos. After germination, signal intensities decreased in developing protocorms. In contrast, in asymbiotic cultures, JIM11 signals were much lower as compared with those stages in symbiotic cultures. Immunofluorescence staining enabled the visualization of JIM11 epitope in mature embryo and protocorm cells. Positive signals were initially localized in the larger cells near the basal (suspensor) end of uninfected embryos, marking the future colonization site of fungal hyphae. After 1 week of inoculation, the basal end of embryos had been colonized, and a strong signal was detected mostly at the mid- and basal regions of the enlarging protocorm. As protocorm development progressed, the signal was concentrated in the colonized cells at the basal end. In colonized cells, signals were present in the walls and intracellularly associated with hyphae and the pelotons. The precise localization of JIM11 epitope is further examined by immunogold labeling. In the colonized cells, gold particles were found mainly in the cell wall and the interfacial matrix near the

  16. Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function

    Directory of Open Access Journals (Sweden)

    Schapira Marc

    2007-09-01

    Full Text Available Abstract Background P-selectin glycoprotein ligand-1 (PSGL-1 plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog and examined mammalian PSGL-1 interactions with human selectins. Results A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR. Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P

  17. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D. [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, Ontario (Canada)

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  18. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    International Nuclear Information System (INIS)

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2014-01-01

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ( 258 RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP 289 ). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved

  19. E1A FUNCTIONS AS A COACTIVATOR OF RETINOIC ACID-DEPENDENT RETINOIC ACID RECEPTOR-BETA-2 PROMOTER ACTIVATION

    NARCIS (Netherlands)

    KRUYT, FAE; FOLKERS, GE; WALHOUT, AJM; VANDERLEEDE, BM; VANDERSAAG, PT; Kruyt, Frank

    The retinoic acid (RA) receptor (RAR) beta2 promoter is strongly activated by RA in embryonal carcinoma (EC) cells. We examined this activation in the P19 EC-derived END-2 cell line and in E1A-expressing counterparts and found strong RA-dependent RARbeta2 promoter activation in the E1A-expressing

  20. Modeling interchild differences in pharmacokinetics on the basis of subject-specific data on physiology and hepatic CYP2E1 levels: A case study with toluene

    International Nuclear Information System (INIS)

    Nong, A.; McCarver, D.G.; Hines, R.N.; Krishnan, K.

    2006-01-01

    The objective of the present study was to evaluate the magnitude of interindividual variability in the internal dose of toluene in children of various age groups, on the basis of subject-specific hepatic CYP2E1 content and physiology. The methodology involved the use of a previously validated physiologically based pharmacokinetic (PBPK) model, in which the intrinsic clearance for hepatic metabolism (CL int ) was expressed in terms of the CYP2E1 content. The adult toluene PBPK model, with enzyme content-normalized CL int , facilitated the calculation of child-specific CL int based on knowledge of hepatic CYP2E1 protein levels. The child-specific physiological parameters, except liver volume, were computed with knowledge of age and body weight, whereas physicochemical parameters for toluene were kept age-invariant based on available data. The actual individual-specific liver volume (autopsy data) was also included in the model. The resulting model was used to simulate the blood concentration profiles in children exposed by inhalation, to 1 ppm toluene for 24 h. For this exposure scenario, the area under the venous blood concentration vs. time curve (AUC) ranged from 0.30 to 1.01 μg/ml x h in neonates with low CYP2E1 concentration (<3.69 pmol/mg protein). The simulations indicated that neonates with higher levels of CYP2E1 (4.33 to 55.93 pmol/mg protein) as well as older children would have lower AUC (0.16 to 0.43 μg/ml x h). The latter values were closer to those simulated for adults. Similar results were also obtained for 7 h exposure to 17 ppm toluene, a scenario previously evaluated in human volunteers. The interindividual variability factor for each subgroup of children and adults, calculated as the ratio of the 95th and 50th percentile values of AUC, was within a factor of 2. The 95th percentile value of the low metabolizing neonate group, however, was greater than the mean adult AUC by a factor of 3.9. This study demonstrates the feasibility of incorporating

  1. SMOG programme version for np (E=1-5 GeV) experiment

    International Nuclear Information System (INIS)

    Kotorobaj, F.

    1975-01-01

    The SMOG program, which can be used for NPD robot scanning in the described experiment, is described. A new modifiaction, set up so as to ensure its reliable use on the CDC-1604A, is considered. The program was designed for and used in processing the np (E=1-5 GeV) experiment. SMOG uses three magnetic tapes at the input (one at the extension) and one at the output containing the assembled collected events. A new SMOG variant with origination of errors according to parity during the recording time is attempted in order to continue to write the given zone five times. A short description of the work with the new SMOG program version is given

  2. Time series data correction for the Chang'E-1 gamma-ray spectrometer

    International Nuclear Information System (INIS)

    Zhang Liyan; Zou Yongliao; Liu Jianzhong; Liu Jianjun; Shen Ji; Mu Lingli; Ren Xin; Wen Weibin; Li Chunlai

    2011-01-01

    The main goal of the gamma-ray spectrometer (GRS) onboard Chang'E-1 (CE-1) is to acquire global maps of elemental abundances and their distributions on the moon, since such maps will significantly improve our understanding of lunar formation and evolution. To derive the elemental maps and enable research on lunar formation and evolution, raw data that are received directly from the spacecraft must be converted into time series corrected gamma-ray spectra. The data correction procedures for the CE-1 GRS time series data are thoroughly described. The processing procedures to create the time series gamma-ray spectra described here include channel processing, optimal data selection, energy calibration, gain correction, dead time correction, geometric correction, orbit altitude normalization, eliminating unusable data and galactic cosmic ray correction. Finally, descriptions are also given on data measurement uncertainties, which will help the interested scientists to understand and estimate various uncertainties associated with the above data processing. (research papers)

  3. Radiative rates for E1, E2, M1 and M2 transitions in Fe X

    International Nuclear Information System (INIS)

    Aggarwal, K.M.; Keenan, F.P.

    2004-01-01

    Energies of the 54 levels belonging to the (1s 2 2s 2 2p 6 ) 3s 2 3p 5 , 3s3p 6 , 3s 2 3p 4 3d and 3s3p 5 3d configurations of Fe X have been calculated using the GRASP code of Dyall and colleagues (1989). Additionally, radiative rates, oscillator strengths, and line strengths are calculated for all electric dipole (E1), magnetic dipole (M1), electric quadrupole (E2), and magnetic quadrupole (M2) transitions among these levels. Comparisons are made with results available in the literature, and the accuracy of the data is assessed. Our energy levels are estimated to be accurate to better than 3%, whereas results for other parameters are probably accurate to better than 20% . Additionally, the agreement between measured and calculated lifetimes is better than 10%. (authors)

  4. Observation of unexpectedly small E1 moments in sup 224 Ra

    Energy Technology Data Exchange (ETDEWEB)

    Poynter, R.J.; Butler, P.A.; Clarkson, N.; Hoare, T.H.; Jones, G.D.; White, C.A. (Liverpool Univ. (UK). Oliver Lodge Lab.); Cline, D. (Rochester Univ., NY (USA). Nuclear Structure Research Lab.); Connell, K.A.; Cunningham, R.A.; Simpson, J. (Science and Engineering Research Council, Daresbury (UK). Daresbury Lab.); Goettig, L. (Warsaw Univ. (Poland). Inst. Fizyki Doswiadczalnej); Hughes, J.R.; Jarvis, N.S.; Mullins, S.M.; Wadsworth, R.; Watson, D.L. (York Univ. (UK). Dept. of Physics); Juutinen, S. (Jyvaeskylae Univ. (Finland). Dept. of Physics); Pass, C.N. (Oxford Univ. (UK). Dept. of Nuclear Physics)

    1989-12-14

    The {alpha}-decay of {sup 228}Th and the reaction {sup 226}Ra({sup 58}Ni, {sup 60}Ni){sup 224}Ra have been employed to populate states in {sup 224}Ra up to I{sup {pi}}=12{sup +}. Values of the intrinsic electric dipole moment vertical strokeQ{sub 1}vertical stroke have been deduced from the observed B(E1)/B(E2) branching ratios. These values are < or approx.0.1 e fm for spins up to I{sup {pi}}=9{sup -}, and are thus smaller than those in {sup 218,220}Ra or {sup 226}Ra. The behaviour of Q{sub 1} suggests that shell effects are important in this mass region, although theoretical calculations which include these effects do not reproduce the observed pattern. (orig.).

  5. MicroRNA-16 Modulates HuR Regulation of Cyclin E1 in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xun Guo

    2015-03-01

    Full Text Available RNA binding protein (RBPs and microRNAs (miRNAs or miRs are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC. Despite this, miR-16 and HuR do not affect the other’s expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

  6. A Case–control and a family-based association study revealing an association between CYP2E1 polymorphisms and nasopharyngeal carcinoma risk in Cantonese

    Science.gov (United States)

    Jia, Wei-Hua; Pan, Qing-Hua; Qin, Hai-De; Xu, Ya-Fei; Shen, Guo-Ping; Chen, Lina; Chen, Li-Zhen; Feng, Qi-Sheng; Hong, Ming-Huang; Zeng, Yi-Xin; Shugart, Yin Yao

    2009-01-01

    Nasopharyngeal carcinoma (NPC) is rare in most parts of the world but is more prevalent in Southern China, especially in Guangdong. The cytochrome P450 2E1 (CYP2E1) has been recognized as one of the critically important enzymes involved in oxidizing carcinogens and is probably to be associated with NPC carcinogenesis. To systematically investigate the association between genetic variants in CYP2E1 and NPC risk in Cantonese, two independent studies, a family-based association study and a case–control study, were conducted using the haplotype-tagging single-nucleotide polymorphism approach. A total of 2499 individuals from 546 nuclear families were initially genotyped for the family-based association study. Single-nucleotide polymorphisms (SNPs) rs9418990, rs915908, rs8192780, rs1536826, rs3827688 and one haplotype h2 (CGTGTTAA) were revealed to be significantly associated with the NPC phenotype (P = 0.045–0.003 and P = 0.003, respectively). To follow up the initial study, a case–control study including 755 cases and 755 controls was conducted. Similar results were observed in the case–control study in individuals <46 years of age and had a history of cigarette smoking, with odds ratios (ORs) of specific genotypes ranging from 1.88 to 2.99 corresponding to SNP rs9418990, rs3813865, rs915906, rs2249695, rs8192780, rs1536826, rs3827688 and of haplotypes h2 with OR = 1.65 (P = 0.026), h5 (CCCGTTAA) with OR = 2.58 (P = 0.007). The values of false-positive report probability were <0.015 for six SNPs, suggesting that the reported associations are less probably to be false. This study provides robust evidence for associations between genetic variants of CYP2E1 and NPC risk. PMID:19805575

  7. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    Science.gov (United States)

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-09

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

  8. Characterization of the Fusion and Attachment Glycoproteins of Human Metapneumovirus and Human Serosurvey to Determine Reinfection Rates

    Science.gov (United States)

    2007-06-27

    Metapneumovirus genus. The Paramyxoviridae are in the taxonomical order Mononegavirales which includes Bornaviridae, Rhabdoviridae and Filoviridae which... Rhabdoviridae plant virus, replicate in the cytoplasm (66). The Paramyxoviridae are enveloped viruses and have been defined by the fusion glycoprotein

  9. Different regulation of P-glycoprotein function between Caco-2 and Caki-1 cells by ezrin, radixin and moesin proteins.

    Science.gov (United States)

    Yano, Kentaro; Otsuka, Kyoma; Kato, Yuko; Kawabata, Hideaki; Ohmori, Shinya; Arakawa, Hiroshi; Ogihara, Takuo

    2016-03-01

    P-glycoprotein (P-gp) mediates efflux of many xenobiotics, including therapeutic drugs, from normal and tumour tissues, and its functional localization on the plasma membrane of cells is regulated by scaffold proteins, such as ezrin, radixin and moesin (ERM proteins). We previously reported that radixin is involved in post-translational regulation of P-gp in hepatocellular carcinoma HepG2 cells and mouse small intestine, but not in mouse kidney. Here, we investigated whether the role of ERM proteins in regulation of P-gp transport activity in cancers is the same as that in the corresponding normal tissues, using human colon adenocarcinoma (Caco-2) cells and renal carcinoma (Caki-1) cells. In Caco-2 cells, radixin silencing alone reduced the P-gp-mediated intracellular accumulation of rhodamine123 (Rho123), while the mRNA level of P-gp was unchanged. Thus, it appears that only radixin among the ERMs regulates P-gp activity in Caco-2 cells. On the other hand, none of the ERM proteins influenced P-gp activity in Caki-1 cells. The regulation of P-gp by ERM proteins is different between Caco-2 and Caki-1 cells. Moreover, these regulatory properties are the same as those of the corresponding normal tissues, and suggest that tissue-specific differences in the regulation of P-gp by ERM proteins are retained in cancerous tissues. © 2016 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology.

  10. Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1

    International Nuclear Information System (INIS)

    Eisenberg, R.J.; Long, D.; Pereira, L.; Hampar, B.; Zweig, M.; Cohen, G.H.

    1982-01-01

    We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide

  11. Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

    Science.gov (United States)

    Dunn, Amy R; Stout, Kristen A; Ozawa, Minagi; Lohr, Kelly M; Hoffman, Carlie A; Bernstein, Alison I; Li, Yingjie; Wang, Minzheng; Sgobio, Carmelo; Sastry, Namratha; Cai, Huaibin; Caudle, W Michael; Miller, Gary W

    2017-03-14

    Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

  12. Functional analysis of P-glycoprotein and multidrug resistance associated protein related multidrug resistance in AML-blasts.

    Science.gov (United States)

    Brügger, D; Herbart, H; Gekeler, V; Seitz, G; Liu, C; Klingebiel, T; Orlikowsky, T; Einsele, H; Denzlinger, C; Bader, P; Niethammer, D; Beck, J F

    1999-05-01

    Despite the high effectiveness of various P-glycoprotein (P-gp) modulating substances in vitro their clinical value e.g. for combination treatment of acute myelogenous leukemias (AML) remains still unclear. This might be explainable by recent findings that other factors than P-gp (e.g. the multidrug resistance associated protein (MRP)) may also be involved in clinical occurring drug resistance. To study P-gp and MRP mediated MDR in AML blasts from patients with relapses at the functional level we measured rhodamine 123 (RHO) efflux in combination with a P-gp specific (SDZ PSC 833) or a MRP specific (MK571) modulator, respectively. Furthermore, direct antineoplastic drug action was monitored by determination of damaged cell fraction of a blast population using flow cytometry. We generally found strongly modulated RHO efflux by SDZ PSC 833 but slight RHO-efflux modulation by MK571 in blasts from relapsed states of AML expressing MDR1 or MRP mRNA at various levels. We could not demonstrate, though, significant PSC 833 or MK571 mediated modulation of the cytotoxic effects of etoposide. The results point to the possibility that combination of etoposide and a modulator might not improve responses to chemotherapy by targeting P-gp or MRP exclusively.

  13. 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.

  14. The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for Inflammatory Response Induced by Ebola Virus Glycoprotein.

    Directory of Open Access Journals (Sweden)

    Dianyuan Zhao

    2016-03-01

    Full Text Available Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP is involved in this process through activating dendritic cells (DCs and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12 and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response.

  15. The nectin-1α transmembrane domain, but not the cytoplasmic tail, influences cell fusion induced by HSV-1 glycoproteins

    International Nuclear Information System (INIS)

    Subramanian, Ravi P.; Dunn, Jennifer E.; Geraghty, Robert J.

    2005-01-01

    Nectin-1 is a receptor for herpes simplex virus (HSV), a member of the immunoglobulin superfamily, and a cellular adhesion molecule. To study domains of nectin-1α involved in cell fusion, we measured the ability of nectin-1α/nectin-2α chimeras, nectin-1α/CD4 chimeras, and transmembrane domain and cytoplasmic tail mutants of nectin-1α to promote cell fusion induced by HSV-1 glycoproteins. Our results demonstrate that only chimeras and mutants containing the entire V-like domain and a link to the plasma membrane conferred cell-fusion activity. The transmembrane domain and cytoplasmic tail of nectin-1 were not required for any viral receptor or cell adhesion function tested. Cellular cytoplasmic factors that bind to the nectin-1α cytoplasmic tail, therefore, did not influence virus entry or cell fusion. Interestingly, the efficiency of cell fusion was reduced when membrane-spanning domains of nectin-1α and gD were replaced by glycosylphosphatidylinositol tethers, indicating that transmembrane domains may play a modulatory role in the gD/nectin-1α interaction in fusion

  16. Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo.

    Science.gov (United States)

    Bošnjak, Ivana; Borra, Marco; Iamunno, Franco; Benvenuto, Giovanna; Ujević, Ivana; Bušelić, Ivana; Roje-Busatto, Romana; Mladineo, Ivona

    2014-11-01

    Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100nM and 4μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junin virus envelope glycoprotein

    International Nuclear Information System (INIS)

    York, Joanne; Nunberg, Jack H.

    2007-01-01

    The arenavirus envelope glycoprotein (GP-C) retains a cleaved and stable signal peptide (SSP) as an essential subunit of the mature complex. This 58-amino-acid residue peptide serves as a signal sequence and is additionally required to enable transit of the assembled GP-C complex to the Golgi, and for pH-dependent membrane fusion activity. We have investigated the C-terminal region of the Junin virus SSP to study the role of the cellular signal peptidase (SPase) in generating SSP. Site-directed mutagenesis at the cleavage site (positions - 1 and - 3) reveals a pattern of side-chain preferences consistent with those of SPase. Although position - 2 is degenerate for SPase cleavage, this residue in the arenavirus SSP is invariably a cysteine. In the Junin virus, this cysteine is not involved in disulfide bonding. We show that replacement with alanine or serine is tolerated for SPase cleavage but prevents the mutant SSP from associating with GP-C and enabling transport to the cell surface. Conversely, an arginine mutation at position - 1 that prevents SPase cleavage is fully compatible with GP-C-mediated membrane fusion activity when the mutant SSP is provided in trans. These results point to distinct roles of SSP sequences in SPase cleavage and GP-C biogenesis. Further studies of the unique structural organization of the GP-C complex will be important in identifying novel opportunities for antiviral intervention against arenaviral hemorrhagic disease

  18. Cancer cell–derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo

    Science.gov (United States)

    Thomas, Grace M.; Panicot-Dubois, Laurence; Lacroix, Romaric; Dignat-George, Françoise; Lombardo, Dominique

    2009-01-01

    Recent publications have demonstrated the presence of tissue factor (TF)–bearing microparticles (MPs) in the blood of patients suffering from cancer. However, whether these MPs are involved in thrombosis remains unknown. We show that pancreatic and lung cancer cells produce MPs that express active TF and P-selectin glycoprotein ligand 1 (PSGL-1). Cancer cell–derived MPs aggregate platelets via a TF-dependent pathway. In vivo, cancer cell–derived MPs, but not their parent cells, infused into a living mouse accumulate at the site of injury and reduce tail bleeding time and the time to occlusion of venules and arterioles. This thrombotic state is also observed in mice developing tumors. In such mice, the amount of circulating platelet-, endothelial cell–, and cancer cell–derived MPs is increased. Endogenous cancer cell–derived MPs shed from the growing tumor are able to accumulate at the site of injury. Infusion of a blocking P-selectin antibody abolishes the thrombotic state observed after injection of MPs or in mice developing a tumor. Collectively, our results indicate that cancer cell–derived MPs bearing PSGL-1 and TF play a key role in thrombus formation in vivo. Targeting these MPs could be of clinical interest in the prevention of thrombosis and to limit formation of metastasis in cancer patients. PMID:19667060

  19. 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.

  20. Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach.

    Science.gov (United States)

    Ben Azoun, Safa; Kallel, Héla

    2017-08-01

    Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV-G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV-G in P. pastoris. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.