Science.gov

Sample records for membrane antigen requires

  1. Conditional expression of apical membrane antigen 1 in Plasmodium falciparum shows it is required for erythrocyte invasion by merozoites

    PubMed Central

    Yap, Alan; Azevedo, Mauro F; Gilson, Paul R; Weiss, Greta E; O’Neill, Matthew T; Wilson, Danny W; Crabb, Brendan S; Cowman, Alan F

    2014-01-01

    Summary Malaria is caused by obligate intracellular parasites, of which Plasmodium falciparum is the most lethal species. In humans, P. falciparum merozoites (invasive forms of the parasite) employ a host of parasite proteins to rapidly invade erythrocytes. One of these is the P. falciparum apical membrane antigen 1 (PfAMA1) which forms a complex with rhoptry neck proteins at the tight junction. Here, we have placed the Pfama1 gene under conditional control using dimerizable Cre recombinase (DiCre) in P. falciparum. DiCre-mediated excision of the loxP-flanked Pfama1 gene results in approximately 80% decreased expression of the protein within one intraerythrocytic growth cycle. This reduces growth by 40%, due to decreased invasion efficiency characterized by a post-invasion defect in sealing of the parasitophorous vacuole. These results show that PfAMA1 is an essential protein for merozoite invasion in P. falciparum and either directly or indirectly plays a role in resealing of the red blood cell at the posterior end of the invasion event. PMID:24571085

  2. CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

    PubMed Central

    Niklinska, B B; Hou, D; June, C; Weissman, A M; Ashwell, J D

    1994-01-01

    T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates. Images PMID:7526153

  3. Actin reorganization is required for the formation of polarized BCR signalosomes in response to both soluble and membrane-associated antigens1

    PubMed Central

    Liu, Chaohong; Miller, Heather; Orlowski, Gregory; Hang, Haiyin; Upadhyaya, Arpita

    2012-01-01

    B-cells encounter both soluble (sAg) and membrane-associated antigens (mAg) in the secondary lymphoid tissue, yet how the physical form of Ag modulates B-cell activation remains unclear. This study compares actin reorganization and its role in BCR signalosome formation in mAg- and sAg-stimulated B-cells. Both mAg and sAg induce F-actin accumulation and actin polymerization at BCR microclusters and at the outer rim of BCR central clusters, but the kinetics and magnitude of F-actin accumulation in mAg-stimulated B-cells are greater than those in sAg-stimulated B-cells. Accordingly, the actin regulatory factors, cofilin and gelsolin, are recruited to BCR clusters in both mAg- and sAg-stimulated B-cells but with different kinetics and patterns of cellular redistribution. Inhibition of actin reorganization by stabilizing F-actin inhibits BCR clustering and tyrosine phosphorylation induced by both forms of Ag. Depolymerization of F-actin leads to unpolarized microclustering of BCRs and tyrosine phosphorylation in BCR microclusters without mAg and sAg, but in much slower kinetics than those induced by Ag. Therefore, actin reorganization, mediated via both polymerization and depolymerization, is required for the formation of BCR signalosomes in response to both mAg and sAg. PMID:22387556

  4. Characterization of the major outer membrane antigens of Treponema hyodysenteriae.

    PubMed Central

    Wannemuehler, M J; Hubbard, R D; Greer, J M

    1988-01-01

    Outer membrane extracts of Treponema hyodysenteriae were used to evaluate the antibody responses in immunized or convalescent pigs. Western blot (immunoblot) analysis identified antibodies in sera reactive with 14- to 19-kilodalton (kDa) antigens. Reactivity against these antigens could be removed only by absorption of sera with butanol-water-extracted endotoxin from the homologous strain of T. hyodysenteriae. Treatment of the outer membrane extracts with 0.1 M sodium meta-periodate, but not with proteinase K, abolished reactivity with both outer membrane and endotoxin antigens (14 and 19 kDa). These results indicate that swine vaccinated with the outer membrane extract of T. hyodysenteriae develop antibody responses to outer membrane antigens qualitatively similar to those of swine convalescing from active infection, especially antibodies against low-molecular-mass antigens. The nature of the 14- to 19-kDa antigens appears consistent with that of treponemal endotoxin and lipopolysaccharide. Images PMID:2460406

  5. The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore

    PubMed Central

    Boone, Kyle; Altiyev, Agamyrat; Puschhof, Jens; Sauter, Roland; Arigi, Emma; Ruiz, Blanca; Peng, Xiuli; Almeida, Igor; Sherman, Michael; Xiao, Chuan; Sun, Jianjun

    2015-01-01

    Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig) domain of the anthrax toxin receptor 2 (ANTXR2) inhibited the function of the protective antigen (PA) pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA) domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax. PMID:26107617

  6. PROSTATE SPECIFIC MEMBRANE ANTIGEN-BASED IMAGING

    PubMed Central

    Osborne, Joseph R.; Akhtar, Naveed H.; Vallabhajosula, Shankar; Anand, Alok; Deh, Kofi; Tagawa, Scott T.

    2012-01-01

    SUMMARY Prostate cancer (PC) is the most common non-cutaneous malignancy affecting men in North America. Despite significant efforts, conventional imaging of PC does not contribute to patient management as much as imaging performed for other common cancers. Given the lack of specificity in conventional imaging techniques, one possible solution is to screen for PC specific antigenic targets and generate agents able to specifically bind. Prostate specific membrane antigen (PSMA) is over-expressed in PC tissue, with low levels of expression in the small intestine, renal tubular cells and salivary gland. The first clinical agent for targeting PSMA was 111In-capromab, involving an antibody recognizing the internal domain of PSMA. The second- and third-generation humanized PSMA binding antibodies have the potential to overcome some of the limitations inherent to capromab pendetide i.e. inability to bind to live PC cells. One example is the humanized monoclonal antibody J591 (Hu mAb J591) that was developed primarily for therapeutic purposes but also has interesting imaging characteristics including the identification of bone metastases in PC. The major disadvantage of use of mAb for imaging is slow target recognition and background clearance in an appropriate timeframe for diagnostic imaging. Urea-based compounds such as small molecule inhibitors may also present promising agents for PC imaging with SPECT and PET. Two such small-molecule inhibitors targeting PSMA, MIP-1072 and MIP-1095, have exhibited high affinity for PSMA. The uptake of 123I-MIP-1072 and 123I-MIP-1095 in PC xenografts have imaged successfully with favorable properties amenable to human trials. While advances in conventional imaging will continue, Ab and small molecule imaging exemplified by PSMA targeting have the greatest potential to improve diagnostic sensitivity and specificity. PMID:22658884

  7. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein

    PubMed Central

    Ducken, Deirdre R.; Brown, Wendy C.; Alperin, Debra C.; Brayton, Kelly A.; Reif, Kathryn E.; Turse, Joshua E.; Palmer, Guy H.; Noh, Susan M.

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines

  8. Protein Stains to Detect Antigen on Membranes.

    PubMed

    Dsouza, Anil; Scofield, R Hal

    2015-01-01

    Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical. PMID:26139252

  9. Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

    PubMed

    Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

    2015-12-15

    Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation. PMID:26628378

  10. Initial characterization of Trypanosoma lewisi surface membrane antigen.

    PubMed

    Viyanant, V; Jones, A W

    1978-03-01

    The purified T. lewisi were subjected to hypotonic lysis plus freezing and thawing in acetone dry ice bath. The trypanosome ghosts were obtained after repeated washing and centrifugation. The homogenized ghost suspension was assayed for enzyme Na++K+ ATPase activity to ratify the presence of the trypanosome surface membrane. Membrane solubilized in sodium dodecyl sulfate were fractionated by gel filtration on Sephadex columns equilibrated with the detergent and electrophoresis in polyacrylamide gel. Crude trypanosome surface membrane antigens were tested for their immunogenicity, administered to rats in Fraund's complete adjuvant. The results of these experiments indicated that the protective immunogen is tightly bound to the membrane since the use of strong anionic detergent is necessary in its extraction. PMID:212837

  11. Bloodstream form Trypanosome plasma membrane proteins: antigenic variation and invariant antigens.

    PubMed

    Schwede, Angela; Carrington, Mark

    2010-12-01

    Trypanosoma brucei is exposed to the adaptive immune system and complement in the blood of its mammalian hosts. The aim of this review is to analyse the role and regulation of the proteins present on the external face of the plasma membrane in the long-term persistence of an infection and transmission. In particular, the following are addressed: (1) antigenic variation of the variant surface glycoprotein (VSG), (2) the formation of an effective VSG barrier shielding invariant surface proteins, and (3) the rapid uptake of VSG antibody complexes combined with degradation of the immunoglobulin and recycling of the VSG. PMID:20109254

  12. Delivery of foreign antigens by engineered outer membrane vesicle vaccines.

    PubMed

    Chen, David J; Osterrieder, Nikolaus; Metzger, Stephan M; Buckles, Elizabeth; Doody, Anne M; DeLisa, Matthew P; Putnam, David

    2010-02-16

    As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines. PMID:20133740

  13. Extraction of tumor-specific antigen from cells and plasma membranes of line-10 hepatoma.

    PubMed

    Leonard, E J; Richardson, A K; Hardy, A S; Rapp, H J

    1975-07-01

    Tumor-specific antigen was extracted with 3 M KCl from line-10 guinea pig hepatoma cells. The yield of antigenic activity, estimated by production of delayed cutaneous hypersensitivity reactions in line-10 immune guinea pigs, was 10-30% of the antigen present in intact cells. By ultracentrifugation criteria, the extracted antigen was soluble. Gel filtration, ion exchange chromatography, and salting-out studies showed that the antigen was heterogeneous in size and net charge. The possibility that 3 M KCl extracted a homogeneous population of molecules associating into polymers of various sizes at low ionic strength was ruled out by heterogeneity on Sephadex G-200 chromatography at high ionic strength. After osmotic lysis of sucrose-loaded line-10 cells, whole plasma membranes or large membrane fragments were obtained in a yield of about 20%. The isolation procedure did not cause detectable loss of membrane antigenic activity. The membranes had 33 skin test U/mg membrane protein, compared to the intact cell value of 1.7 skin test U/mg cell protein. Extracts of plasma membranes had 10-20% of the antigenic activity of the starting membrane material. In contrast to the wide variety of proteins liberated from intact cells, much of the protein extracted from the membranes was in the molecular weight range above 250,000. PMID:169367

  14. Auger Radiopharmaceutical Therapy Targeting Prostate-Specific Membrane Antigen

    PubMed Central

    Kiess, Ana P.; Hobbs, Robert; Sgouros, George; Mease, Ronnie C.; Pullambhatla, Mrudula; Shen, Colette J.; Foss, Catherine A.; Pomper, Martin G.

    2015-01-01

    Auger electron emitters such as 125I have a high linear energy transfer and short range of emission (<10 μm), making them suitable for treating micrometastases while sparing normal tissues. We used a highly specific small molecule targeting the prostate-specific membrane antigen (PSMA) to deliver 125I to prostate cancer cells. Methods The PSMA-targeting Auger emitter 2-[3-[1-carboxy-5-(4-125I-iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid (125I-DCIBzL) was synthesized. DNA damage (via phosphorylated H2A histone family member X staining) and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human prostate cancer cells after treatment with 125I-DCIBzL. Subcellular drug distribution was assessed with confocal microscopy using a related fluorescent PSMA-targeting compound YC-36. In vivo antitumor efficacy was tested in nude mice bearing PSMA+ PC3 PIP or PSMA− PC3 flu flank xenografts. Animals were administered (intravenously) 111 MBq (3 mCi) of 125I-DCIBzL, 111 MBq (3 mCi) of 125I-NaI, an equivalent amount of nonradiolabeled DCIBzL, or saline. Results After treatment with 125I-DCIBzL, PSMA+ PC3 PIP cells exhibited increased DNA damage and decreased clonogenic survival when compared with PSMA− PC3 flu cells. Confocal microscopy of YC-36 showed drug distribution in the perinuclear area and plasma membrane. Animals bearing PSMA+ PC3 PIP tumors had significant tumor growth delay after treatment with 125I-DCIBzL, with only 1 mouse reaching 5 times the initial tumor volume by 60 d after treatment, compared with a median time to 5 times volume of less than 15 d for PSMA− PC3 flu tumors and all other treatment groups (P = 0.002 by log-rank test). Conclusion PSMA-targeted radiopharmaceutical therapy with the Auger emitter 125I-DCIBzL yielded highly specific antitumor efficacy in vivo, suggesting promise for treatment of prostate cancer micrometastases. PMID:26182968

  15. Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.

    PubMed Central

    Eismann, K; Mlejnek, K; Zipprich, D; Hoppert, M; Gerberding, H; Mayer, F

    1995-01-01

    Electron microscopic immunogold labeling experiments were performed with ultrathin sections of plasmolyzed cells of Alcaligenes eutrophus and "whole-mount" samples of spheroplasts and protoplasts. They demonstrated that antigenic determinants of the membrane-bound hydrogenase are exposed, at the outside of the cytoplasmic membrane, to the periplasm. PMID:7592402

  16. Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

    PubMed Central

    Dutta, Sheetij; Dlugosz, Lisa S.; Drew, Damien R.; Ge, Xiopeng; Ababacar, Diouf; Rovira, Yazmin I.; Moch, J. Kathleen; Shi, Meng; Long, Carole A.; Foley, Michael; Beeson, James G.; Anders, Robin F.; Miura, Kazutoyo; Haynes, J. David; Batchelor, Adrian H.

    2013-01-01

    Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal

  17. Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB

    PubMed Central

    Gerke, Christiane; Colucci, Anna Maria; Giannelli, Carlo; Sanzone, Silvia; Vitali, Claudia Giorgina; Sollai, Luigi; Rossi, Omar; Martin, Laura B.; Auerbach, Jochen; Di Cioccio, Vito; Saul, Allan

    2015-01-01

    Recently, we developed a high yield production process for outer membrane particles from genetically modified bacteria, called Generalized Modules of Membrane Antigens (GMMA), and the corresponding simple two step filtration purification, enabling economic manufacture of these particles for use as vaccines. Using a Shigella sonnei strain that was genetically modified to produce penta-acylated lipopolysaccharide (LPS) with reduced endotoxicity and to maintain the virulence plasmid encoding for the immunodominant O antigen component of the LPS, scale up of the process to GMP pilot scale was straightforward and gave high yields of GMMA with required purity and consistent results. GMMA were formulated with Alhydrogel and were highly immunogenic in mice and rabbits. In mice, a single immunization containing 29 ng protein and 1.75 ng of O antigen elicited substantial anti-LPS antibody levels. As GMMA contain LPS and lipoproteins, assessing potential reactogenicity was a key aspect of vaccine development. In an in vitro monocyte activation test, GMMA from the production strain showed a 600-fold lower stimulatory activity than GMMA with unmodified LPS. Two in vivo tests confirmed the low potential for reactogenicity. We established a modified rabbit pyrogenicity test based on the European Pharmacopoeia pyrogens method but using intramuscular administration of the full human dose (100 μg of protein). The vaccine elicited an average temperature rise of 0.5°C within four hours after administration, which was considered acceptable and showed that the test is able to detect a pyrogenic response. Furthermore, a repeat dose toxicology study in rabbits using intramuscular (100 μg/dose), intranasal (80 μg/dose), and intradermal (10 μg/dose) administration routes showed good tolerability of the vaccine by all routes and supported its suitability for use in humans. The S. sonnei GMMA vaccine is now in Phase 1 dose-escalation clinical trials. PMID:26248044

  18. Uncommon structural motifs dominate the antigen binding site in human autoantibodies reactive with basement membrane collagen.

    PubMed

    Foster, Mary H; Buckley, Elizabeth S; Chen, Benny J; Hwang, Kwan-Ki; Clark, Amy G

    2016-08-01

    Autoantibodies mediate organ destruction in multiple autoimmune diseases, yet their origins in patients remain poorly understood. To probe the genetic origins and structure of disease-associated autoantibodies, we engrafted immunodeficient mice with human CD34+ hematopoietic stem cells and immunized with the non-collagenous-1 (NC1) domain of the alpha3 chain of type IV collagen. This antigen is expressed in lungs and kidneys and is targeted by autoantibodies in anti-glomerular basement membrane (GBM) nephritis and Goodpasture syndrome (GPS), prototypic human organ-specific autoimmune diseases. Using Epstein Barr virus transformation and cell fusion, six human anti-alpha3(IV)NC1 collagen monoclonal autoantibodies (mAb) were recovered, including subsets reactive with human kidney and with epitopes recognized by patients' IgG. Sequence analysis reveals a long to exceptionally long heavy chain complementarity determining region3 (HCDR3), the major site of antigen binding, in all six mAb. Mean HCDR3 length is 25.5 amino acids (range 20-36), generated from inherently long DH and JH genes and extended regions of non-templated N-nucleotides. Long HCDR3 are suited to forming noncontiguous antigen contacts and to binding recessed, immunologically silent epitopes hidden from conventional antibodies, as seen with self-antigen crossreactive broadly neutralizing anti-HIV Ig (bnAb). The anti-alpha3(IV)NC1 collagen mAb also show preferential use of unmutated variable region genes that are enriched among human chronic lymphocytic leukemia antibodies that share features with natural polyreactive Ig. Our findings suggest unexpected relationships between pathogenic anti-collagen Ig, bnAb, and autoreactive Ig associated with malignancy, all of which arise from B cells expressing unconventional structural elements that may require transient escape from tolerance for successful expansion. PMID:27450516

  19. Modulation of Endotoxicity of Shigella Generalized Modules for Membrane Antigens (GMMA) by Genetic Lipid A Modifications

    PubMed Central

    Rossi, Omar; Pesce, Isabella; Giannelli, Carlo; Aprea, Susanna; Caboni, Mariaelena; Citiulo, Francesco; Valentini, Sara; Ferlenghi, Ilaria; MacLennan, Calman Alexander; D'Oro, Ugo; Saul, Allan; Gerke, Christiane

    2014-01-01

    Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development. PMID:25023285

  20. Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion

    PubMed Central

    Bargieri, Daniel Y.; Andenmatten, Nicole; Lagal, Vanessa; Thiberge, Sabine; Whitelaw, Jamie A.; Tardieux, Isabelle; Meissner, Markus; Ménard, Robert

    2013-01-01

    Apicomplexan parasites invade host cells by forming a ring-like junction with the cell surface and actively sliding through the junction inside an intracellular vacuole. Apical membrane antigen 1 is conserved in apicomplexans and a long-standing malaria vaccine candidate. It is considered to have multiple important roles during host cell penetration, primarily in structuring the junction by interacting with the rhoptry neck 2 protein and transducing the force generated by the parasite motor during internalization. Here, we generate Plasmodium sporozoites and merozoites and Toxoplasma tachyzoites lacking apical membrane antigen 1, and find that the latter two are impaired in host cell attachment but the three display normal host cell penetration through the junction. Therefore, apical membrane antigen 1, rather than an essential invasin, is a dispensable adhesin of apicomplexan zoites. These genetic data have implications on the use of apical membrane antigen 1 or the apical membrane antigen 1–rhoptry neck 2 interaction as targets of intervention strategies against malaria or other diseases caused by apicomplexans. PMID:24108241

  1. Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites.

    PubMed

    Kay, M M; Marchalonis, J J; Hughes, J; Watanabe, K; Schluter, S F

    1990-08-01

    Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and marks them for removal by the immune system in mammals. It is derived from band 3, a ubiquitous membrane transport protein found in diverse cell types and tissues. We have used synthetic peptides to identify aging antigenic sites on band 3, using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: (i) the active antigenic sites of the aging antigen reside on membrane protein band 3 residues that are extracellular regions implicated in anion transport (residues 538-554 and 788-827); (ii) a putative ankyrin-binding-region peptide is not involved in SCA activity; and (iii) carbohydrate moieties are not required for the antigenicity or recognition of SCA because synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. One of the putative transport sites that contributes to the aging antigen is located toward the carboxyl terminus. A model of band 3 is presented. Localization of the active antigenic site on the band 3 molecule facilitates definition of the molecular changes occurring during aging that initiate molecular as well as cellular degeneration. PMID:1696010

  2. Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

    PubMed Central

    Bueno, Lilian Lacerda; Lobo, Francisco Pereira; Morais, Cristiane Guimarães; Mourão, Luíza Carvalho; de Ávila, Ricardo Andrez Machado; Soares, Irene Silva; Fontes, Cor Jesus; Lacerda, Marcus Vinícius; Olórtegui, Carlos Chavez; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio; Braga, Érika Martins

    2011-01-01

    Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies. PMID:21713006

  3. The Presence of Tumour Specific Membrane Antigen in the Serum of Rats with Chemically Induced Sarcomata

    PubMed Central

    Thomson, D. M. P.; Steele, K.; Alexander, P.

    1973-01-01

    Antibodies to the tumour-specific transplantation type antigen (TSTA) of a transplanted methylcholanthrene-induced sarcoma (MC-1) in syngeneic rats were studied using the techniques of indirect membrane immunofluorescence and mixed haemadsorption with a 51Cr-labelled indicator cell. After tumour excision, anti-TSTA antibody was readily measurable in both serum and lymph. In contrast, the tumour-bearing animal had no measurable anti-TSTA antibody in the serum but low titres in the lymph. Consequently, we formed the hypothesis that in the presence of a growing tumour the serum contained antigen-antibody complexes with antigen in excess. To test this hypothesis, tumour-bearing serum was examined for the presence of free antigen and antigen-antibody complexes by 2 different methods. In the first method, tumour-bearing serum was cross-linked with glutaraldehyde and was found to absorb specifically the anti-TSTA antibody, indicating free circulating TSTA. Next, antigen-antibody complexes were split with salt or acid and separated into a low molecular weight (or “antigen”) fraction (<100,000) and a high molecular weight (or “antibody”) fraction (>100,000). The low M.W. fraction specifically inhibited the anti-TSTA antibody when tested by either membrane immunofluorescence or mixed haemadsorption, indicating the presence of antigen from antigen-antibody complexes in the tumour-bearing circulation. The possible effect on the host's immune response of circulating free tumour antigen and antigen-antibody complexes are discussed. PMID:4568460

  4. Purification of a native membrane-associated adenovirus tumor antigen.

    PubMed Central

    Persson, H; Katze, M G; Philipson, L

    1982-01-01

    A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro. Images PMID:7097863

  5. Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.

    PubMed Central

    Damjanovich, S; Trón, L; Szöllösi, J; Zidovetzki, R; Vaz, W L; Regateiro, F; Arndt-Jovin, D J; Jovin, T M

    1983-01-01

    The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different determinants on the H-2Kk antigen itself were determined by using fluorescence resonance energy-transfer measurements with a dual-laser flow sorter. From the lack of energy transfer between bound monoclonal anti-H-2Kk antibodies conjugated with fluorescein (donor) and rhodamine (acceptor), we conclude that the H-2Kk antigen exists without appreciable clustering on the cell surface. Substantial energy transfer between appropriately labeled Con A and antibodies bound to the H-2Kk antigen shows that the two populations are interspersed. Donor/acceptor pairs of monoclonal antibodies binding to different determinants on the same H-2Kk antigen exhibited a degree of energy transfer indicative of a mean separation of 8.6 nm between the sites. Time-resolved phosphorescence anisotropy measurements with anti-H-2Kk antibodies labeled with eosin or erythrosin yielded rotational mobility information for the antigen-antibody complexes on the cell membrane. The rotational correlation time of 10-20 mus and the finite residual anisotropy are compatible with an uniaxial mode of rotation of monomeric antigen around its transmembrane portion and, thus, provide additional evidence for an unclustered distribution. Capping by rabbit anti-mouse IgG immobilized the antigen-antibody complex. Fluorescence recovery after photobleaching was used to calculate an apparent lateral diffusion coefficient of 5 +/- 3 X 10(-10) cm2 . s-1 for the H-2Kk antigen labeled with fluoresceinated IgG or its corresponding Fab fragment. PMID:6351071

  6. Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

    PubMed Central

    Felix, Tristan; Soprova, Zora; ten Hagen-Jongman, Corinne M.; Vikström, David; Majlessi, Laleh; Beskers, Joep; Follmann, Frank; de Punder, Karin; van der Wel, Nicole N.; Baumgarten, Thomas; Pham, Thang V.; Piersma, Sander R.; Jiménez, Connie R.; van Ulsen, Peter; de Gier, Jan-Willem; Leclerc, Claude; Jong, Wouter S. P.

    2014-01-01

    Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development. PMID:25038093

  7. Cross-linking of anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial outer membrane proteins are the primary targets of a protective immune response. The specific characteristics of outer membrane-based immunogens, in terms of antigen content and context that are required for protective immunity remain unknown for a wide variety of bacterial pathogens. Usin...

  8. Use of antisera to epithelial membrane antigen for the cytodiagnosis of malignancy in serous effusions.

    PubMed Central

    To, A; Coleman, D V; Dearnaley, D P; Ormerod, M G; Steele, K; Neville, A M

    1981-01-01

    A new human antigen, designated epithelial membrane antigen (EMA), has recently been described on surface membranes of a wide variety of normal epithelium but not on connective tissue cells. The antigen is only weakly expressed on normal or reactive mesothelium. Increased expression of the antigen has been observed in most neoplasms of epithelial origin and in malignant mesothelioma. We have investigated the possibility of using this difference in the expression of the antigen to distinguish between mesothelial cells and malignant cells in cytological smears of serous effusions. This distinction cannot always be made on morphological grounds alone and problems of differential diagnosis are encountered in about 15% of all specimens of serous effusions sent for cytological examination. Using antisera to EMA we have applied an indirect immunoalkaline phosphatase technique to alcohol-fixed smears prepared from serous effusions and have found that intense staining of the antigen is confined to effusions from patients in whom there is either clinical or cytological evidence of malignancy. The technique proved to be especially useful in cytologically equivocal cases, where there were problems of differential diagnosis. Images PMID:7035498

  9. Antigen

    MedlinePlus

    An antigen is any substance that causes your immune system to produce antibodies against it. This means your immune ... and is trying to fight it off. An antigen may be a substance from the environment, such ...

  10. A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

    PubMed Central

    Buckley, K M; Schweitzer, E S; Miljanich, G P; Clift-O'Grady, L; Kushner, P D; Reichardt, L F; Kelly, R B

    1983-01-01

    The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft. Images PMID:6359167

  11. [Immunotropic and immunogenic properties of Burkholderia pseudomallei surface and membrane antigens].

    PubMed

    Piven', N N; Rybkin, V S; Plekhanova, N G; Zhukova, S I; Viktorov, D V; Avrorova, I V; Demediuk, E A; Drefs, N M; Popov, S F

    2001-01-01

    The immunotropic and immunogenic properties of some chromatographic fractions of B. pseudomallei surface antigenic complex, as well as the preparations of B. pseudomallei outer and cytoplasmic membranes, were studied. The difference between the biopolymers under study in cytotoxicity, humoral and cell-mediated immunity characteristics, phagocytic activity were established. Some antigenic fractions (B, C, C1, H) showed perceptible protective activity (25-60%) in experiments on mice infected with B. pseudomallei virulent strain. One of the preparations of cytoplasmic membrane (CM-1) was also found to have protective properties (30%). Complex immunization with the antigenic complexes under study, introduced in combination with the immunomodulating agent Bromantan, was shown to enhance the protective effect. PMID:11236497

  12. Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

    SciTech Connect

    Braylan, R.C.; Benson, N.A.; Nourse, V.; Kruth, H.S.

    1982-03-01

    Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.

  13. Evaluation of solubilized herpes simplex virus membrane antigen by enzyme-linked immunosorbent assay.

    PubMed Central

    Jeansson, S; Forsgren, M; Svennerholm, B

    1983-01-01

    An antigen prepared by solubilization of membranes from herpes simplex virus (HSV)-infected cells with deoxycholate was evaluated by enzyme-linked immunosorbent assay. The deoxycholate-solubilized antigen, previously shown to contain all major HSV glycoproteins, was noninfectious and adsorbed easily and reproducibly to a polystyrene surface at pH 9.6. The deoxycholate-solubilized antigen provided an enzyme-linked immunosorbent assay of high sensitivity and reproducibility with complete correlation with complement fixation for the diagnosis of acute HSV infection. The correlation with neutralization and immunofluorescence for the presence or absence of anti-HSV activity was very good. Comparison with an HSV envelope preparation yielded results slightly in favor of the deoxycholate-solubilized antigen. The assay seems to be useful for demonstration of intrathecal production of antibody activity in HSV encephalitis. PMID:6315767

  14. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  15. Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.

    PubMed

    Skraba, Cissiara Manetti; Pedroso, Raíssa Bocchi; Fiorini, Adriana; Rosado, Fábio Rogério; Aristides, Sandra Mara Alessi; Lonardoni, Maria Valdrinez Campana; Teixeira, Jorge Juarez Vieira; Silveira, Thaís Gomes Verzignassi

    2014-04-01

    This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients. PMID:24485589

  16. Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein.

    PubMed Central

    Buchanan, T M; Hildebrandt, J F

    1981-01-01

    Principal outer membrane protein (protein I) of Neisseria gonorrhoeae was prepared nearly free of lipopolysaccharide (LPS) and substantially purified from other membrane proteins by chromatography of partially purified gonococcal outer membranes over Sepharose 6B in the presence of deoxycholate at pH 9.0. This protein I of nine separate antigenic types was coated to polystyrene tubes and used in the enzyme-linked immunosorbent assay (ELISA) to measure antibody to protein I or in inhibition tests to quantitate protein I antigen. No significant inhibition of the ELISA test was produced by purified LPS from the strain used to prepare each of the protein I types or by whole gonococci bearing the same LPS but different protein I antigens as the strain used to produce a given protein I antigen. Of 125 strains of gonococci used as whole organisms to inhibit the protein I ELISA, 124 (99%) typed with one or more of the nine protein I types, and 35% of these typed with a single protein I serotype. Sixty-one of 65 (94%) strains from Seattle and Atlanta patients with disseminated gonococcal infection contained protein I serotype 1, and 16 of 24 (64%) strains from Seattle patients with salpingitis bore one or both of protein I serotypes 1 and 2. Images PMID:6166568

  17. Characterisation of a Babesia orientalis apical membrane antigen, and comparison of its orthologues among selected apicomplexans.

    PubMed

    He, Lan; Fan, Lizhe; Hu, Jinfang; Miao, Xiaoyan; Huang, Yuan; Zhou, Yanqin; Hu, Min; Zhao, Junlong

    2015-04-01

    In the present study, we identified and characterised the complete coding sequence of Babesia orientalis apical membrane antigen 1 (designated Bo-ama1); it is 1803bp in length and encodes a polypeptide of 601 amino acids (aa). The Bo-ama-1 gene product (Bo-AMA1) is predicted to be 67kDa in size and contains a signal peptide. Mature Bo-AMA1 is predicted to have one transmembrane region and a short cytoplasmic tail (C-terminal domain). The extracellular part of Bo-AMA1 has three functional domains (DI, DII and DIII) with 14 conserved cysteine residues. A Bo-AMA1 fragment containing all three of these domains (designated Bo-AMA1-DI/II/III) was cloned into the plasmid vector pET-28a and expressed as a recombinant (His-fusion) protein of 53kDa. Antibodies in the serum from a B. orientalis-infected water buffalo specifically recognised this protein in immunoblotting analysis. Rabbit antibodies raised against the recombinant protein were able to detect native Bo-AMA1 (67kDa) from erythrocytes of B. orientalis-infected water buffalo. Bo-AMA1 is a new member of the AMA1 family and might be a good antigen for the specific detection of antibodies produced in B. orientalis infected cattle. This protein is likely to play critical roles during host cell adherence and invasion by B. orientalis, as the AMA1s reported in other organisms such as Plasmodium falciparum and Toxoplasma gondii. Further research is required to explore the biological functions of this protein and to determine whether its immunisation can induce protective effects in water buffalo against B. orientalis infection. PMID:25732411

  18. Prostate-Specific Membrane Antigen PET/CT in Splenic Sarcoidosis.

    PubMed

    Kobe, Carsten; Maintz, David; Fischer, Thomas; Drzezga, Alexander; Chang, De-Hua

    2015-11-01

    A 65-year-old man who had prostate cancer presented with slightly progressive prostate-specific antigen values. In this situation of biochemical relapse, prostate-specific membrane antigen (PSMA) PET/CT has proven to be superior to choline PET. The Ga-PSMA PET/CT of our patient revealed PSMA-positive tissue in the spleen. Although the localization was not typical for metastases, metastasis could not be excluded because of the intense focal tracer uptake. A supplementary MRI was performed but also failed to rule out a malignant origin. Finally, biopsy confirmed benign disease in the spleen in the form of granulomatous disease. PMID:26018688

  19. The outer membrane, not a coat of host proteins, limits antigenicity of virulent Treponema pallidum.

    PubMed Central

    Cox, D L; Chang, P; McDowall, A W; Radolf, J D

    1992-01-01

    Virulent Treponema pallidum reacts poorly with the specific antibodies present in human and rabbit syphilitic sera, a phenomenon often attributed to an outer coat of host serum proteins. Here we present additional evidence that the limited antigenicity of virulent organisms actually is due to a paucity of proteins in the outer membrane. Initially, we used electron microscopy to demonstrate that the outer membrane is highly susceptible to damage from physical manipulation (i.e., centrifugation and resuspension) and nonionic detergents. Organisms with disrupted outer membranes were markedly more antigenic than intact treponemes as determined by immunoelectron microscopy (IEM) with rabbit syphilitic and antiendoflagellar antisera. Data obtained with a new radioimmunoassay, designated the T. pallidum surface-specific radioimmunoassay, corroborated these IEM findings by demonstrating that the major T. pallidum immunogens are not surface exposed; the assay also was unable to detect serum proteins, including fibronectin, on the surfaces of intact organisms. Furthermore, IEM of T. pallidum on ultrathin cryosections with monospecific anti-47-kDa-immunogen antiserum confirmed the intracellular location of the 47-kDa immunogen. On the basis of these and previous findings, we proposed a new model for T. pallidum ultrastructure in which the outer membrane contains a small number of transmembrane proteins and the major membrane immunogens are anchored by lipids to the periplasmic leaflet of the cytoplasmic membrane. This unique ultrastructure explains the remarkable ability of virulent organisms to evade the humoral immune response of the T. pallidum-infected host. Images PMID:1541522

  20. Lipid-Free Antigen B Subunits from Echinococcus granulosus: Oligomerization, Ligand Binding, and Membrane Interaction Properties

    PubMed Central

    Silva-Álvarez, Valeria; Franchini, Gisela R.; Pórfido, Jorge L.; Kennedy, Malcolm W.; Ferreira, Ana M.; Córsico, Betina

    2015-01-01

    Background The hydatid disease parasite Echinococcus granulosus has a restricted lipid metabolism, and needs to harvest essential lipids from the host. Antigen B (EgAgB), an abundant lipoprotein of the larval stage (hydatid cyst), is thought to be important in lipid storage and transport. It contains a wide variety of lipid classes, from highly hydrophobic compounds to phospholipids. Its protein component belongs to the cestode-specific Hydrophobic Ligand Binding Protein family, which includes five 8-kDa isoforms encoded by a multigene family (EgAgB1-EgAgB5). How lipid and protein components are assembled into EgAgB particles remains unknown. EgAgB apolipoproteins self-associate into large oligomers, but the functional contribution of lipids to oligomerization is uncertain. Furthermore, binding of fatty acids to some EgAgB subunits has been reported, but their ability to bind other lipids and transfer them to acceptor membranes has not been studied. Methodology/Principal Findings Lipid-free EgAgB subunits obtained by reverse-phase HPLC were used to analyse their oligomerization, ligand binding and membrane interaction properties. Size exclusion chromatography and cross-linking experiments showed that EgAgB8/2 and EgAgB8/3 can self-associate, suggesting that lipids are not required for oligomerization. Furthermore, using fluorescent probes, both subunits were found to bind fatty acids, but not cholesterol analogues. Analysis of fatty acid transfer to phospholipid vesicles demonstrated that EgAgB8/2 and EgAgB8/3 are potentially capable of transferring fatty acids to membranes, and that the efficiency of transfer is dependent on the surface charge of the vesicles. Conclusions/Significance We show that EgAgB apolipoproteins can oligomerize in the absence of lipids, and can bind and transfer fatty acids to phospholipid membranes. Since imported fatty acids are essential for Echinococcus granulosus, these findings provide a mechanism whereby EgAgB could engage in lipid

  1. Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

    PubMed Central

    Tiwari, Sapana; Kumar, Ashu; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-01-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  2. Splenunculus Masquerading as Prostate-specific Membrane Antigen-positive Lymph Node Metastasis in a Patient With Prostate-specific Antigen Relapse After Radical Prostatectomy.

    PubMed

    Froehner, Michael; Zöphel, Klaus; Hölscher, Tobias; Laniado, Michael; Wirth, Manfred P

    2016-08-01

    A 45-year-old patient presented with prostate-specific antigen relapse after radical prostatectomy. Diagnostic workup revealed a (68)Ga-labeled prostate-specific membrane antigen-targeted ligand tracer uptaking nodule that was initially interpreted as lymph node metastasis but eventually identified as a splenunculus by scintigraphy with (99m)Tc pertechnetate-labeled heat-altered erythrocytes. Awareness of this constellation may spare unnecessary diagnostic procedures and inappropriate treatment. PMID:27125881

  3. Binding of Streptococcus mutans antigens to heart and kidney basement membranes.

    PubMed Central

    Stinson, M W; Barua, P K; Bergey, E J; Nisengard, R J; Neiders, M E; Albini, B

    1984-01-01

    Using indirect immunofluorescence, alkali-extracted components of Streptococcus mutans were found to bind in vitro to capillary walls and sarcolemmal sheaths of monkey cardiac muscle and to glomerular and tubular basement membranes of monkey kidney. Adsorption of S. mutans components to tissue fragments was also detected by indirect radioimmunoassay and immunoblotting on nitrocellulose paper. Antibodies did not bind to untreated, control tissues in these experiments, proving that antigens shared by S. mutans and tissue components were not involved. Rabbit and monkey heart and kidney components bound S. mutans antigens of 24,000, 35,000, and 65,000 Mr. Monkey heart also bound molecules of 90,000 and 120,000 Mr. Rabbits immunized by intravenous injection of disrupted S. mutans cells developed severe nephritis that was characterized by the deposition of immunoglobulins, complement component C3, and S. mutans antigens in the glomeruli. Immunoglobulin G eluted from nephritic kidneys reacted in immunoblots with the 24,000, 35,000, and 65,000 Mr components of S. mutans extract, indicating that the antigens that bound to tissue in vitro also bound in vivo and reacted with antibodies in situ. Antibodies to other S. mutans antigens were not detected in the kidney eluate, although they were present in the serum of the same rabbit. Images PMID:6384042

  4. An outer membrane protein (porin) as an eliciting antigen for delayed-type hypersensitivity in murine salmonellosis.

    PubMed Central

    Udhayakumar, V; Muthukkaruppan, V R

    1987-01-01

    The porin, an outer membrane protein of Salmonella typhimurium, was found to be a suitable antigen for eliciting delayed-type hypersensitivity in mouse salmonellosis. Histological examination of the reaction site revealed that the porin was superior to other antigenic preparations in eliciting a typical delayed-type hypersensitivity reaction consisting of mononuclear cell infiltration without polymorphonuclear cell contamination. This study indicates the importance of using a suitable protein antigen from S. typhi for human application. Images PMID:3028963

  5. A Novel Treponema pallidum Antigen, TP0136, Is an Outer Membrane Protein That Binds Human Fibronectin▿

    PubMed Central

    Brinkman, Mary Beth; McGill, Melanie A.; Pettersson, Jonas; Rogers, Arthur; Matějková, Petra; Šmajs, David; Weinstock, George M.; Norris, Steven J.; Palzkill, Timothy

    2008-01-01

    The antigenicity, structural location, and function of the predicted lipoprotein TP0136 of Treponema pallidum subsp. pallidum were investigated based on previous screening studies indicating that anti-TP0136 antibodies are present in the sera of syphilis patients and experimentally infected rabbits. Recombinant TP0136 (rTP0136) protein was purified and shown to be strongly antigenic during human and experimental rabbit infection. The TP0136 protein was exposed on the surface of the bacterial outer membrane and bound to the host extracellular matrix glycoproteins fibronectin and laminin. In addition, the TP0136 open reading frame was shown to be highly polymorphic among T. pallidum subspecies and strains at the nucleotide and amino acid levels. Finally, the ability of rTP0136 protein to act as a protective antigen to subsequent challenge with infectious T. pallidum in the rabbit model of infection was assessed. Immunization with rTP0136 delayed ulceration but did not prevent infection or the formation of lesions. These results demonstrate that TP0136 is expressed on the outer membrane of the treponeme during infection and may be involved in attachment to host extracellular matrix components. PMID:18332212

  6. Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

    PubMed Central

    Kashtan, C; Fish, A J; Kleppel, M; Yoshioka, K; Michael, A F

    1986-01-01

    We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous globular domain (NC1) of type IV collagen from normal human GBM, while FNS identified only the 26,000-mol wt monomer. FNS reacted with EBM of 12 controls and nine unaffected male kindred members but not EBM of eight affected males. Five affected females exhibited interrupted reactivity of FNS with EBM. GPS showed variable reactivity with EBM and was not discriminating with respect to Alport-type FN. FNS did not stain renal basement members of five affected males. However, the EBM, tubular basement membrane, and Bowman's capsules of affected males contained antigens reactive with GPS. These immunochemical studies suggest that the FNS antigen is distinct from Goodpasture antigen(s). The expression of FNS antigen located on the NC1 domain of type IV collagen is altered in basement membranes of patients with Alport-type FN, and the distribution of this antigenic anomaly within kindreds suggests X-linked dominant transmission of a defective gene. Images PMID:2428839

  7. Effect of particulate adjuvant on the anthrax protective antigen dose required for effective nasal vaccination.

    PubMed

    Bento, Dulce; Staats, Herman F; Borges, Olga

    2015-07-17

    Successful vaccine development is dependent on the development of effective adjuvants since the poor immunogenicity of modern subunit vaccines typically requires the use of potent adjuvants and high antigen doses. In recent years, adjuvant formulations combining both immunopotentiators and delivery systems have emerged as a promising strategy to develop effective and improved vaccines. In this study we investigate if the association of the mast cell activating adjuvant compound 48/80 (C48/80) with chitosan nanoparticles would promote an antigen dose sparing effect when administered intranasally. Even though the induction of strong mucosal immunity required higher antigen doses, incorporation of C48/80 into nanoparticles provided significant dose sparing when compared to antigen and C48/80 in solution with no significant effect on serum neutralizing antibodies titers. These results suggest the potential of this novel adjuvant combination to improve the immunogenicity of a vaccine and decrease the antigen dose required for vaccination. PMID:26087299

  8. Urinary excretion of glomerular basement membrane antigens in Alport's syndrome. A new diagnostic approach.

    PubMed

    Lubec, G; Balzar, E; Weissenbacher, G; Syré, G

    1978-05-01

    Alport's syndrome is defined by the combination of hereditary nephropathy and neurosensory deafness, and is diagnosed from the family history combined with renal electron microscopy. Immunoelectrophoresis of the urine of 8 of 12 children suspected of Alport's syndrome showed a precipitation line moving into the beta-zone, applying an antiglomerular basement membrane antibody derived from an immunised rabbit. All patients who showed the typical pattern of Alport's syndrome on renal electron microscopy were among the 8 cases whose urine gave this immunoelectrophoresis pattern. Additionally, 5 of the mothers of the 8 children excreted the same antigen in their urine. The urine of 30 healthy children and of 10 patients with the idiopathic nephrotic syndrome did not show the presence of this antigen. This characteristic sign of Alport's syndrome may therefore be useful for its detection. PMID:666354

  9. A requirement for epsin in mitotic membrane and spindle organization

    PubMed Central

    2009-01-01

    Eukaryotic cells possess a sophisticated membrane system to facilitate diverse functions. Whereas much is known about the nature of membrane systems in interphase, the organization and function of the mitotic membrane system are less well understood. In this study, we show that epsin, an endocytic adapter protein, regulates mitotic membrane morphology and spindle integrity in HeLa cells. Using epsin that harbors point mutations in the epsin NH2-terminal homology domain and spindle assembly assays in Xenopus laevis egg extracts, we show that epsin-induced membrane curvature is required for proper spindle morphogenesis, independent of its function in endocytosis during interphase. Although several other membrane-interacting proteins, including clathrin, AP2, autosomal recessive hypercholesterolemia, and GRASP65, are implicated in the regulation of mitosis, whether they participate through regulation of membrane organization is unclear. Our study of epsin provides evidence that mitotic membrane organization influences spindle integrity. PMID:19704019

  10. Effect fixation on T and B lymphocyte surface membrane antigen demonstration in paraffin processed tissue.

    PubMed

    Holgate, C S; Jackson, P; Pollard, K; Lunny, D; Bird, C C

    1986-08-01

    The identification of lymphoid surface membrane antigens in tissue sections using immunohistochemical techniques is becoming increasingly important for the diagnosis and classification of lymphoproliferative disorders. Many of the lymphocyte specific monoclonal antibodies used, however, can only be applied to frozen tissue sections. In this paper we report the successful application of a number of these antibodies to paraffin processed tissue utilizing alternative fixatives and the highly sensitive immunogold-silver staining method. The best fixatives for this purpose were formol dichromate, periodate-lysine-paraformaldehyde (PLP) and a novel fixative formed from the addition of a dichromate solution to PLP. PMID:3020216

  11. Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane.

    PubMed

    Kurien, Biji T; Bachmann, Michael P

    2015-01-01

    Antibodies directed against ribonucleoprotein (RNP) particles are observed in systemic lupus erythematosus. Ro RNP particle is one such target. It is composed of a 60 kDa protein (Ro 60 or SS-A) that is non-covalently associated with at least one of the four short uridine-rich RNAs (the hY RNAs). Previously, we showed that multiple antigenic peptides (MAPs) made from the sequence of the Ro 60 autoantigen could be used, using double-immunodiffusion studies, enzyme-linked immunosorbant assay, affinity chromatography, and surface plasmon resonance, to show intramolecular and intermolecular protein-protein interaction within the Ro 60 RNP particle. We also observed that calcium is important in mediating this interaction. We hypothesized, therefore, that 60 kDa Ro is a calcium-binding protein. To investigate this, we electrophoresed 60 kDa Ro MAPs, transferred them to PVDF membrane, and assayed calcium binding using the Quin-2 system. Several Ro 60 MAPs were found to bind calcium using this assay, as well as bovine serum albumin, another calcium-binding protein. However, a MAP constructed from the Sm autoantigen did not bind to calcium. These data, along with our observation regarding the involvement of calcium in protein-protein interaction occurring between Ro 60 antigen and Ro 60 MAPs, makes us propose that Ro 60 antigen is a calcium-binding protein. PMID:26139264

  12. Antibody-Functionalized Peptidic Membranes for Neutralization of Allogeneic Skin Antigen-Presenting Cells

    PubMed Central

    Wen, Yi; Liu, Wen; Bagia, Christina; Zhang, Shaojuan; Bai, Mingfeng; Janjic, Jelena M.; Giannoukakis, Nick; Gawalt, Ellen S.; Meng, Wilson S.

    2014-01-01

    We report herein application of an in situ material strategy to attenuate allograft T cell responses in a skin transplant mouse model. Functionalized peptidic membranes were used to impede trafficking of donor antigen-presenting cells (dAPCs) from skin allografts in recipient mice. Membranes formed by self-assembling peptides (SAPs) presenting antibodies were found to remain underneath grafted skins for up to 6 days. At the host-graft interface, dAPCs were targeted by using a monoclonal antibody that binds to a class II MHC molecule (IAd) expressed exclusively by donor cells. Using a novel cell labeling near-infrared nanoemulsion, we found more dAPCs remained in allografts treated with membranes loaded with aI-Ad than without. In vitro, dAPCs released from skin explants were found adsorbed preferentially on aI-Ad membranes. Recipient T cells from these mice produced lower concentrations of interferon-gamma cultured ex vivo with donor cells. Taken together, the data indicate that the strategy has the potential to alter the natural course of rejection immune mechanisms in stringent allogeneic models. PMID:25117952

  13. T-cell-specific membrane antigens in the Mexican axolotl (urodele amphibian).

    PubMed

    Kerfourn, F; Guillet, F; Charlemagne, J; Tournefier, A

    1992-01-01

    Comparative analysis of SDS-PAGE patterns of axolotl spleen cells membrane detergent lysates showed important discrepancies between control and thymectomized animals. Among these, a 38-kD protein band, which appeared as a major protein in controls, was not or poorly expressed after thymectomy. A rabbit antiserum (L12) raised against the 38-kD eluted band labeled in indirect immunofluorescence 80-86% of thymocytes and 40-46% of mIg- lymphoid cells in the spleen. The anti-38-kD antibodies stained in Western blotting two antigenically related polypeptides of 38- and 36-kD on splenocyte membrane lysates. Two-dimensional NEPHGE-PAGE analysis indicated that the anti-38-kD antibodies reacted in the spleen with several gathered spots in the 7.8-8.2 pI range, corresponding to 38-36-kD microheterogeneous polypeptides. Most of these spots are not further expressed in thymectomized animals. These results support evidence that the 38-kD surface antigens can be considered as specific surface markers of the axolotl thymus-derived lymphocytes. PMID:1627952

  14. A Phase 1 Study of a Vaccine Targeting Preferentially Expressed Antigen in Melanoma and Prostate-specific Membrane Antigen in Patients With Advanced Solid Tumors

    PubMed Central

    Weber, Jeffrey S.; Vogelzang, Nicholas J.; Ernstoff, Marc S.; Goodman, Oscar B.; Cranmer, Lee D.; Marshall, John L.; Miles, Sabrina; Rosario, Dar; Diamond, David C.; Qiu, Zhiyong; Obrocea, Mihail; Bot, Adrian

    2013-01-01

    Summary Preferentially expressed antigen in melanoma (PRAME) and prostate-specific membrane antigen (PSMA) are tumor-associated antigens implicated in cellular differentiation, genetic stability, and angiogenesis. MKC1106-PP is an immunotherapeutic regimen cotargeting PRAME and PSMA, comprised of a recombinant plasmid (pPRA-PSM encoding fragments derived from both antigens) and 2 peptides (E-PRA and E-PSM derived from PRAME and PSMA, respectively). This multicenter study evaluated MKC1106-PP with a fixed plasmid dose and 2 different peptide doses, administered by intralymph node injection in a prime-boost sequence in human leukocyte antigen-A*0201 and tumor-antigen-positive patients with progressing metastatic solid tumors who had failed standard therapy. Immune monitoring was done by tetramer and enzymatic-linked immune spot analysis. The treatment was well tolerated, with no significant differences in safety, immune response, and clinical outcome relative to peptide doses. Fifteen of 24 evaluable patients showed an immune response, as defined by the expansion of PRAME-specific or PSMA-specific T cells in the blood. There were no partial or complete responses by the Response Evaluation Criteria in Solid Tumors. Seven patients showed stable disease (SD) for 6 months or longer, or prostate specific antigen decline: 4 of 10 with prostate carcinoma, 2 of 2 with renal clear cell carcinoma, and 1 of 10 with metastatic melanoma. In addition, there was an association between the induction and persistence of antigen-specific T cells in blood above baseline levels and disease control, defined as SD for 6 months or longer. These results support further development of MKC1106-PP in specific clinical indications. PMID:21760528

  15. Protective efficacy of bacterial membranes containing surface-exposed BM95 antigenic peptides for the control of cattle tick infestations.

    PubMed

    Canales, Mario; Labruna, Marcelo B; Soares, João F; Prudencio, Carlos R; de la Fuente, José

    2009-12-01

    The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that the recombinant chimeric protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region for presentation on the Escherichia coli membrane was protective against R. microplus infestations in rabbits. This system provides a novel and simple approach for the production of tick protective antigens by surface display of antigenic protein chimera on live E. coli and suggests the possibility of using recombinant bacterial membrane fractions for vaccination against cattle tick infestations. PMID:19835826

  16. Mixed enzyme-linked immunosorbent assay (MELISA) for HLA class I antigen: a plasma membrane marker.

    PubMed

    Bjerrum, O W; Borregaard, N

    1990-03-01

    This study introduces a simple, reproducible assay for HLA class I antigen using antibodies against beta 2-microglobulin and the heavy chain on HLA. The sandwich technique was named mixed enzyme-linked immunosorbent assay (MELISA), and was designed for identification of plasma membranes in neutrophil subcellular fractions. The subcellular localization of HLA was identical to that of other plasma membrane markers, [3H]concanavalin A and detergent-independent alkaline phosphatase, and was unchanged by stimulation of cells by weak and strong secretagogues. In addition to the presence as part of the HLA complex in the plasma membrane uncomplexed beta 2-microglobulin is present in the specific granules of neutrophils. However, the release of beta 2-microglobulin from intact neutrophils stimulated with formyl-methionylleucylphenylalanine was much higher than could be explained by exocytosis of specific granules. Subcellular fractionation studies demonstrated that beta 2-microglobulin is localized in fractions characterized by latent alkaline phosphatase and released from this novel secretory compartment in response to stimulation with formyl-methionylleucylphenylalanine. PMID:2181625

  17. Antigens of the basement membranes of the seminiferous tubules induce autoimmunity in Wistar rats.

    PubMed

    Lustig, L; Satz, M L; Sztein, M B; Denduchis, B

    1982-05-01

    A preparation enriched in basement membranes from seminiferous tubules was isolated from rat testes (STBM) and injected with complete Freund's adjuvant into Wistar rats. In 60% of animals a mild multifocal orchitis was observed. In damaged areas, perivascular and peritubular mononuclear cell infiltrates and different degrees of cell sloughing of some seminiferous tubules were observed. Electron microscopy revealed focal thickenings and delamination of the basement membrane of the seminiferous tubules as well as vacuolization of Sertoli cell cytoplasm. Using immunofluorescence discontinuous linear deposits of IgG were detected along the seminiferous tubular wall. Moreover, the same pattern of immunofluorescence was observed when the IgG eluted from the testes of the immunized rats was layered on sections of normal rat testis. Circulating antibodies to STBM were detected using passive haemagglutination in approximately 45% of the immunized rats, with titers ranging from 1:20 to 1:80. Leukocyte migration was inhibited when the spleen cells of the immunized rats were incubated with antigens from the basement membrane of seminiferous tubules, whilst a negative reaction was obtained when the soluble fraction of testis homogenate was used. PMID:7050376

  18. Control of tick infestations in cattle vaccinated with bacterial membranes containing surface-exposed tick protective antigens.

    PubMed

    Almazán, Consuelo; Moreno-Cantú, Orlando; Moreno-Cid, Juan A; Galindo, Ruth C; Canales, Mario; Villar, Margarita; de la Fuente, José

    2012-01-01

    Vaccines containing the Rhipicephalus (Boophilus) microplus BM86 and BM95 antigens protect cattle against tick infestations. Tick subolesin (SUB), elongation factor 1a (EF1a) and ubiquitin (UBQ) are new candidate protective antigens for the control of cattle tick infestations. Previous studies showed that R. microplus BM95 immunogenic peptides fused to the Anaplasma marginale major surface protein (MSP) 1a N-terminal region (BM95-MSP1a) for presentation on the Escherichia coli membrane were protective against R. microplus infestations in rabbits. In this study, we extended these results by expressing SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusion proteins on the E. coli membrane using this system and demonstrating that bacterial membranes containing the chimeric proteins BM95-MSP1a and SUB-MSP1a were protective (>60% vaccine efficacy) against experimental R. microplus and Rhipicephalus annulatus infestations in cattle. This system provides a novel, simple and cost-effective approach for the production of tick protective antigens by surface display of antigenic protein chimera on the E. coli membrane and demonstrates the possibility of using recombinant bacterial membrane fractions in vaccine preparations to protect cattle against tick infestations. PMID:22085549

  19. The pathogenic antigen of Heymann nephritis is a membrane glycoprotein of the renal proximal tubule brush border.

    PubMed Central

    Kerjaschki, D; Farquhar, M G

    1982-01-01

    Purified brush border fractions prepared from rat kidneys were solubilized in detergent, iodinated, and subjected to immunoprecipitation to identify the pathogenic antigen present in brush border membranes that is responsible for the production of Heymann nephritis (HN). Purified IgG prepared from the sera of rabbits or rats immunized with a crude cortical preparation, known as Fx1A, precipitated multiple peptides, whereas IgG eluted from glomeruli of rats with active or passive HN specifically immunoprecipitated a single large glycoprotein (Mr = 330,000). This protein (gp330) was subsequently purified by gel filtration and lentil lectin affinity chromatography from detergent-solubilized brush border membranes. When rats were immunized with purified gp330, they developed anti-brush border antibodies and active HN. IgG prepared from the serum of rats with active HN caused passive HN when injected into normal recipients. Rats immunized against brush border membrane proteins depleted of gp330 developed anti-brush border antibodies but did not develop HN. Further analysis of gp330 indicated that it is solubilized by detergent treatment of isolated brush border microvilli, and its antigenic component is released from intact microvilli by trypsin. By immunoperoxidase staining it was localized to the luminal side of the brush border membranes. These results indicate that (i) gp330 is the pathogenic antigen of HN; (ii) the antigen is a glycoprotein of the brush border membrane; and (iii) it is disposed with its pathogenic domain(s) facing the tubule lumen. Images PMID:6752952

  20. Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer protection against colonization.

    PubMed

    Kuipers, Kirsten; Daleke-Schermerhorn, Maria H; Jong, Wouter S P; ten Hagen-Jongman, Corinne M; van Opzeeland, Fred; Simonetti, Elles; Luirink, Joen; de Jonge, Marien I

    2015-04-21

    Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because they have intrinsic immunostimulatory properties. In principle, heterologous antigens incorporated into OMVs will elicit specific immune responses, especially if presented at the vesicle surface and thus optimally exposed to the immune system. In this study, we explored the feasibility of our recently developed autotransporter Hbp platform, designed to efficiently and simultaneously display multiple antigens at the surface of bacterial OMVs, for vaccine development. Using two Streptococcus pneumoniae proteins as model antigens, we showed that intranasally administered Salmonella OMVs displaying high levels of antigens at the surface induced strong protection in a murine model of pneumococcal colonization, without the need for a mucosal adjuvant. Importantly, reduction in bacterial recovery from the nasal cavity was correlated with local production of antigen-specific IL-17A. Furthermore, the protective efficacy and the production of antigen-specific IL-17A, and local and systemic IgGs, were all improved at increased concentrations of the displayed antigen. This discovery highlights the importance of an adequate antigen expression system for development of recombinant OMV vaccines. In conclusion, our findings demonstrate the suitability of the Hbp platform for development of a new generation of OMV vaccines, and illustrate the potential of using this approach to develop a broadly protective mucosal pneumococcal vaccine. PMID:25776921

  1. Concentration of membrane antigens by forward transport and trapping in neuronal growth cones.

    PubMed

    Sheetz, M P; Baumrind, N L; Wayne, D B; Pearlman, A L

    1990-04-20

    Formation of the nervous system requires that neuronal growth cones follow specific paths and then stop at recognition signals, sensed at the growth cone's leading edge. We used antibody-coated gold particles viewed by video-enhanced differential interference contrast microscopy to observe the distribution and movement of two cell surface molecules, N-CAM and the 2A1 antigen, on growth cones of cultured cortical neurons. Gold particles are occasionally transported forward at 1-2 microns/s to the leading edge where they are trapped but continue to move. Concentration at the edge persists after cytochalasin D treatment or ATP depletion, but active movements to and along edges cease. We also observed a novel outward movement of small cytoplasmic aggregates at 1.8 microns/s in filopodia. We suggest that active forward transport and trapping involve reversible attachment of antigens to and transport along cytoskeletal elements localized to edges of growth cones. PMID:2331749

  2. Characteristics of antisera against periodate-resistant membrane antigens from Neisseria gonorrhoeae.

    PubMed

    Røe, S F; Eggset, G; Iversen, O J; Maeland, J A

    1980-12-01

    Crude outer membrane (OM) was prepared by extraction of bacteria of the Neisseria gonorrhoeae strains 8551. V, and VII, with an EDTA-containing buffer. The preparations contained the lipopolysaccharide (LPS) and at least 10 proteins as shown by SDS-polyacrylamide gel electrophoresis. Immunization of rabbits with untreated OM resulted in production of antibodies against several antigens, including LPS. Antisera raised against periodate-treated OM did not contain antibodies against LPS. These latter antisera agglutinated heat-treated (100 degrees C, 60 min) gonoccal cells by means of antibodies to one or more common agglutinogens and against a strain-specific agglutinogen that was susceptible to digestion with proteolytic enzymes. Both side agglutination and a plate agglutination test could be used to detect antibodies against these agglutinogens. PMID:6261525

  3. Prostate Specific Membrane Antigen-Targeted Photodynamic Therapy Induces Rapid Cytoskeletal Disruption

    PubMed Central

    Liu, Tiancheng; Wu, Lisa Y.; Berkman, Clifford E.

    2010-01-01

    Prostate-specific membrane antigen (PSMA), an established enzyme-biomarker for prostate cancer, has attracted considerable attention as a target for imaging and therapeutic applications. We aimed to determine the effects of PSMA-targeted photodynamic therapy (PDT) on cytoskeletal networks in prostate cancer cells. PSMA-targeted PDT resulted in rapid disruption of microtubules (α-/β-tubulin), microfilaments (actin), and intermediate filaments (cytokeratin 8/18) in the cytoplasm of LNCaP cells. The collapse of cytoplasmic microtubules and the later nuclear translocation of α-/β-tubulin were the most dramatic alternation. It is likely that these early changes of cytoskeletal networks are partly involved in the initiation of cell death. PMID:20452720

  4. 68Ga Prostate-Specific Membrane Antigen Uptake in Renal Cell Cancer Lymph Node Metastases.

    PubMed

    Einspieler, Ingo; Tauber, Robert; Maurer, Tobias; Schwaiger, Markus; Eiber, Matthias

    2016-05-01

    Ga prostate-specific membrane antigen (PSMA)-HBED-CC PET/CT in a patient with a history of both prostate cancer (PC) and renal cell cancer (RCC) shows high PSMA expression in the residual right seminal vesicle suggestive of local recurrence of PC as well as suspected PSMA-positive mediastinal, retroperitoneal, and iliac lymph nodes. Regarding the latter, biopsy revealed lymph node metastases from RCC excluding PC metastases. This case exemplarily demonstrates that high PSMA expression in RCC metastases can potentially mimic PC metastases. Thus, for accurate interpretation of imaging results in PC patients with additional primary tumors, knowledge of PSMA expression of non-PC tissue is necessary. PMID:26859205

  5. Naturally-Acquired Immune Response against Plasmodium vivax Rhoptry-Associated Membrane Antigen

    PubMed Central

    Changrob, Siriruk; Wang, Bo; Han, Jin-Hee; Lee, Seong-Kyun; Nyunt, Myat Htut; Lim, Chae Seung; Tsuboi, Takafumi; Chootong, Patchanee; Han, Eun-Taek

    2016-01-01

    Rhoptry-associated membrane antigen (RAMA) is an abundant glycophosphatidylinositol (GPI)-anchored protein that is embedded within the lipid bilayer and is implicated in parasite invasion. Antibody responses against rhoptry proteins are produced by individuals living in a malaria-endemic area, suggesting the immunogenicity of Plasmodium vivax RAMA (PvRAMA) for induction of immune responses during P. vivax infection. To determine whether PvRAMA contributes to the acquisition of immunity to malaria and could be a rational candidate for a vaccine, the presence of memory T cells and the stability of the antibody response against PvRAMA were evaluated in P. vivax-exposed individuals. The immunogenicity of PvRAMA for the induction of T cell responses was evaluated by in vitro stimulation of peripheral blood mononuclear cells (PBMCs). High levels of interferon (IFN)-γ and interleukin (IL)-10 cytokines were detected in the culture supernatant of PBMCs, and the CD4+ T cells predominantly produced IL-10 cytokine. The levels of total anti-PvRAMA immunoglobulin G (IgG) antibody were significantly elevated, and these antibodies persisted over the 12 months of the study. Interestingly, IgG1, IgG2 and IgG3 were the major antibody subtypes in the response to PvRAMA. The frequency of IgG3 in specific to PvRAMA antigen maintained over 12 months. These data could explain the immunogenicity of PvRAMA antigen in induction of both cell-mediated and antibody-mediated immunity in natural P. vivax infection, in which IFN-γ helps antibody class switching toward the IgG1, IgG2 and IgG3 isotypes and IL-10 supports PvRAMA-specific antibody production. PMID:26886867

  6. Enhanced detection of bladder cancer using the epithelial surface marker epithelial membrane antigen: a preliminary report.

    PubMed

    Ring, K S; Karp, F; Benson, M C

    1990-09-01

    The flow cytometry (FCM) technique allows for the rapid quantitative analysis of the DNA content of individual cells. In a variety of genitourinary tumors, DNA ploidy has a significant impact upon prognosis and ultimate patient survival. In patients having transitional cell cancer (TCC) of the bladder, FCM of voided urine and bladder barbotage specimens is highly correlated with cytologic analysis in the detection of malignant cells. One problem with this technique has been decreased sensitivity in samples containing large numbers of inflammatory cells. To improve FCM detection of TCC in bladder wash specimens, we developed a technique using a monoclonal antibody (Mab) specific to human, epithelial membrane antigen (EMA). The EMA cell-surface marker enabled us to differentiate bladder epithelial cells from lymphocytes and cellular debris. In combination with DNA analysis using propidium iodide, the EMA Mab increased the sensitivity and specificity of FCM compared to conventional analysis using propidium iodide alone. We conclude that epithelial cell-surface antigen staining using both EMA Mab and DNA staining can increase the FCM detection of TCC in bladder wash specimens. PMID:2074517

  7. Theranostic Agents for Photodynamic Therapy of Prostate Cancer by Targeting Prostate-Specific Membrane Antigen.

    PubMed

    Wang, Xinning; Tsui, Brian; Ramamurthy, Gopolakrishnan; Zhang, Ping; Meyers, Joseph; Kenney, Malcolm E; Kiechle, Jonathan; Ponsky, Lee; Basilion, James P

    2016-08-01

    Prostatectomy has been the mainstay treatment for men with localized prostate cancer. Surgery, however, often can result in major side effects, which are caused from damage and removal of nerves and muscles surrounding the prostate. A technology that can help surgeons more precisely identify and remove prostate cancer resulting in a more complete prostatectomy is needed. Prostate-specific membrane antigen (PSMA), a type II membrane antigen highly expressed in prostate cancer, has been an attractive target for imaging and therapy. The objective of this study is to develop low molecular weight PSMA-targeted photodynamic therapy (PDT) agents, which would provide image guidance for prostate tumor resection and allow for subsequent PDT to eliminate unresectable or remaining cancer cells. On the basis of our highly negatively charged, urea-based PSMA ligand PSMA-1, we synthesized two PSMA-targeting PDT conjugates named PSMA-1-Pc413 and PSMA-1-IR700. In in vitro cellular uptake experiments and in vivo animal imaging experiments, the two conjugates demonstrated selective and specific uptake in PSMA-positive PC3pip cells/tumors, but not in PSMA-negative PC3flu cells/tumors. Further in vivo photodynamic treatment proved that the two PSMA-1-PDT conjugates can effectively inhibit PC3pip tumor progression. The two PSMA-1-PDT conjugates reported here may have the potential to aid in the detection and resection of prostate cancers. It may also allow for the identification of unresectable cancer tissue and PDT ablation of such tissue after surgical resection with potentially less damage to surrounding tissues. Mol Cancer Ther; 15(8); 1834-44. ©2016 AACR. PMID:27297866

  8. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

    PubMed

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-01-01

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen. PMID:26563565

  9. Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism

    PubMed Central

    Guan, Hong-Hsiang; Yoshimura, Masato; Chuankhayan, Phimonphan; Lin, Chien-Chih; Chen, Nai-Chi; Yang, Ming-Chi; Ismail, Asma; Fun, Hoong-Kun; Chen, Chun-Jung

    2015-01-01

    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOTTM). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen. PMID:26563565

  10. The kinetics of antibody binding to Plasmodium falciparum VAR2CSA PfEMP1 antigen and modelling of PfEMP1 antigen packing on the membrane knobs

    PubMed Central

    2010-01-01

    Background Infected humans make protective antibody responses to the PfEMP1 adhesion antigens exported by Plasmodium falciparum parasites to the erythrocyte membrane, but little is known about the kinetics of this antibody-receptor binding reaction or how the topology of PfEMP1 on the parasitized erythrocyte membrane influences antibody association with, and dissociation from, its antigenic target. Methods A Quartz Crystal Microbalance biosensor was used to measure the association and dissociation kinetics of VAR2CSA PfEMP1 binding to human monoclonal antibodies. Immuno-fluorescence microscopy was used to visualize antibody-mediated adhesion between the surfaces of live infected erythrocytes and atomic force microscopy was used to obtain higher resolution images of the membrane knobs on the infected erythrocyte to estimate knob surface areas and model VAR2CSA packing density on the knob. Results Kinetic analysis indicates that antibody dissociation from the VAR2CSA PfEMP1 antigen is extremely slow when there is a high avidity interaction. High avidity binding to PfEMP1 antigens on the surface of P. falciparum-infected erythrocytes in turn requires bivalent cross-linking of epitopes positioned within the distance that can be bridged by antibody. Calculations of the surface area of the knobs and the possible densities of PfEMP1 packing on the knobs indicate that high-avidity cross-linking antibody reactions are constrained by the architecture of the knobs and the large size of PfEMP1 molecules. Conclusions High avidity is required to achieve the strongest binding to VAR2CSA PfEMP1, but the structures that display PfEMP1 also tend to inhibit cross-linking between PfEMP1 antigens, by holding many binding epitopes at distances beyond the 15-18 nm sweep radius of an antibody. The large size of PfEMP1 will also constrain intra-knob cross-linking interactions. This analysis indicates that effective vaccines targeting the parasite's vulnerable adhesion receptors should

  11. The prostate-specific membrane antigen: Lessons and current clinical implications from 20 years of research

    PubMed Central

    Ristau, Benjamin T.; O’Keefe, Denise S.; Bacich, Dean J.

    2014-01-01

    Objective Despite a multitude of detection and treatment advances in the past two decades, prostate cancer remains the second leading cause of cancer death among men in the United States. Technological evolution and expanding knowledge of tumor biomarkers have invigorated exploration in prostate cancer therapeutics. Prostate-specific membrane antigen (PSMA) was one of the first prostate cancer biomarkers successfully cloned. Since that time, it has been characterized as the prototypical cell-surface marker for prostate cancer and has been the subject of intense clinical inquiry. We review the relevant research in PSMA on the 20th anniversary of its cloning. Methods and materials A PubMed® search using the keywords “prostate-specific membrane antigen” or “glutamate carboxypeptidase II” provided 1019 results. An additional 3 abstracts were included from scientific meetings. Articles were vetted by title and abstract with emphasis placed on those with clinically relevant findings. Results Sixty articles were selected for inclusion. PSMA was discovered and cloned in 1993. Its structure and function were further delineated in the ensuing decade. Consensus sites of expression in normal physiology are prostate, kidney, nervous system, and small intestine. PSMA has been implicated in the neovasculature of several tumors including urothelial and renal cell carcinomas. In prostate cancer, expression of PSMA is directly related to Gleason grade. PSMA has been tested both in imaging and therapeutics in a number of prostate cancer clinical trials. Several recent approaches to target PSMA include use of small molecule inhibitors, PSMA-based immunotherapy, RNA aptamer conjugates, and PSMA-targeted prodrug therapy. Future study of PSMA in prostate cancer might focus on its intracellular functions and possible role in tumor neurogenesis. Conclusions Twenty years from its discovery, PSMA represents a viable biomarker and treatment target in prostate cancer. Research to

  12. Bacterial histo-blood group antigens contributing to genotype-dependent removal of human noroviruses with a microfiltration membrane.

    PubMed

    Amarasiri, Mohan; Hashiba, Satoshi; Miura, Takayuki; Nakagomi, Toyoko; Nakagomi, Osamu; Ishii, Satoshi; Okabe, Satoshi; Sano, Daisuke

    2016-05-15

    We demonstrated the genotype-dependent removal of human norovirus particles with a microfiltration (MF) membrane in the presence of bacteria bearing histo-blood group antigens (HBGAs). Three genotypes (GII.3, GII.4, and GII.6) of norovirus-like particles (NoVLPs) were mixed with three bacterial strains (Enterobacter sp. SENG-6, Escherichia coli O86:K61:B7, and Staphylococcus epidermidis), respectively, and the mixture was filtered with an MF membrane having a nominal pore size of 0.45 μm. All NoVLP genotypes were rejected by the MF membrane in the presence of Enterobacter sp. SENG-6, which excreted HBGAs as extracellular polymeric substances (EPS). This MF membrane removal of NoVLPs was not significant when EPS was removed from cells of Enterobacter sp. SENG-6. GII.6 NoVLP was not rejected with the MF membrane in the presence of E. coli O86:K61:B7, but the removal of EPS of E. coli O86:K61:B7 increased the removal efficiency due to the interaction of NoVLPs with the exposed B-antigen in lipopolysaccharide (LPS) of E. coli O86:K61:B7. No MF membrane removal of all three genotypes was observed when S. epidermidis, an HBGA-negative strain, was mixed with NoVLPs. These results demonstrate that the location of HBGAs on bacterial cells is an important factor in determining the genotype-dependent removal efficiency of norovirus particles with the MF membrane. The presence of HBGAs in mixed liquor suspended solids from a membrane bioreactor (MBR) pilot plant was confirmed by immune-transmission electron microscopy, which implies that bacterial HBGAs can contribute to the genotype-dependent removal of human noroviruses with MBR using MF membrane. PMID:27095709

  13. RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization

    PubMed Central

    Zou, Wei; Yadav, Smita; DeVault, Laura; Jan, Yuh Nung; Sherwood, David R.

    2015-01-01

    Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth. PMID:26394140

  14. PET Imaging in Prostate Cancer: Focus on Prostate-Specific Membrane Antigen

    PubMed Central

    Mease, Ronnie C.; Foss, Catherine A.; Pomper, Martin G.

    2014-01-01

    Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Positron emission tomography/computed tomography (PET/CT) with emerging radiopharmaceuticals promises accurate staging of primary disease, restaging of recurrent disease, detection of metastatic lesions and, ultimately, for predicting the aggressiveness of disease. Prostate-specific membrane antigen (PSMA) is a well-characterized imaging biomarker of PCa. Because PSMA levels are directly related to androgen independence, metastasis and progression, PSMA could prove an important target for the development of new radiopharmaceuticals for PET. Preclinical data for new PSMA-based radiotracers are discussed and include new 89Zr- and 64Cu-labeled anti-PSMA antibodies and antibody fragments, 64Cu-labeled aptamers, and 11C-, 18F-, 68Ga-, 64Cu-, and 86Y-labeled low molecular weight inhibitors of PSMA. Several of these agents, namely 68Ga-HBED-CC conjugate 15, 18F-DCFBC 8, and BAY1075553 are particularly promising, each having detected sites of PCa in initial clinical studies. These early clinical results suggest that PET/CT using PSMA-targeted agents, especially with compounds of low molecular weight, will make valuable contributions to the management of PCa. PMID:23590171

  15. Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants

    SciTech Connect

    Hale, M.L.; Greiner, D.L.; McCarthy, K.F.

    1987-01-01

    Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous of intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone-marrow cells that could resonstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition, this marrow-cell population was able to protect lethally irradiated Lewis rats (9.5 GY) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.

  16. Prostate-specific membrane antigen as a target for cancer imaging and therapy

    PubMed Central

    KIESS, A. P.; BANERJEE, S. R.; MEASE, R. C.; ROWE, S. P.; RAO, A.; FOSS, C. A.; CHEN, Y.; YANG, X.; CHO, S. Y.; NIMMAGADDA, S.; POMPER, M. G.

    2016-01-01

    The prostate-specific membrane antigen (PSMA) is a molecular target whose use has resulted in some of the most productive work toward imaging and treating prostate cancer over the past two decades. A wide variety of imaging agents extending from intact antibodies to low-molecular-weight compounds permeate the literature. In parallel there is a rapidly expanding pool of antibody-drug conjugates, radiopharmaceutical therapeutics, small-molecule drug conjugates, theranostics and nanomedicines targeting PSMA. Such productivity is motivated by the abundant expression of PSMA on the surface of prostate cancer cells and within the neovasculature of other solid tumors, with limited expression in most normal tissues. Animating the field is a variety of small-molecule scaffolds upon which the radionuclides, drugs, MR-detectable species and nanoparticles can be placed with relative ease. Among those, the urea-based agents have been most extensively leveraged, with expanding clinical use for detection and more recently for radiopharmaceutical therapy of prostate cancer, with surprisingly little toxicity. PSMA imaging of other cancers is also appearing in the clinical literature, and may overtake FDG for certain indications. Targeting PSMA may provide a viable alternative or first-line approach to managing prostate and other cancers. PMID:26213140

  17. Cloning and sequence analysis of the Schistosoma mansoni membrane glycoprotein antigen gene GP22.

    PubMed

    el-Sherbeini, M; Ramadan, N; Bostian, K A; Knopf, P M

    1991-11-01

    A family of Schistosoma mansoni proteins (18-22 kDa, pI 5.3-5.8) are biosynthesized in juvenile worms and immunoprecipitated by antibodies uniquely present in protective Fischer rat antiserum. A cDNA clone, lambda gt11-40, expressing epitopes common to this protein family was used to obtain a genomic DNA clone, by hybridization with a lambda gt11-40 oligonucleotide probe. In the 1.37 kb of genomic DNA sequenced, an open reading frame of 182 amino acids was identified on the strand corresponding to lambda gt11-40 coding sequences, and those of identical independently isolated cDNA clones defining a 25-kDa surface membrane glycoprotein. The new S. mansoni gene is termed GP22. There are two candidate promoters, confirmed by primer extension studies with worm RNA. Promoter 1 (P1) is preceded by a G + C-rich region and potential CAAT sequences, and is to the 5'-side of P2. Transcription from P1 is initiated at 2 different sites, apparently producing mRNAs with different translation start sites (ATG). Decoding these mRNAs yields protein products of 182 (P1), 175 (P1), 140 (P2) and 136 (P2) amino acids. The polypeptides share the following features: a hydrophobic segment near the carboxy terminus sufficient to span a lipid bilayer, with a consensus sequence for thio-esterification by a fatty acid; an external domain containing 2 potential N-linked glycosylation sites; and a candidate leucine-zipper motif, suggesting the protein may exist as a dimer on the worm surface. While sharing these common features in their carboxy terminal regions, the three proteins differ in the length and properties of their amino termini. The 140-amino acid protein has a short hydrophobic amino terminus, while the 175- and 182-amino acid proteins have more extensive hydrophobic sequences, each preceded by a hydrophilic amino terminal sequence. The heterogeneity observed in 2-dimensional gels of the antigen may be explained in part by the size and charge differences among the proteins deduced

  18. No facilitator required for membrane transport of hydrogen sulfide

    PubMed Central

    Mathai, John C.; Missner, Andreas; Kügler, Philipp; Saparov, Sapar M.; Zeidel, Mark L.; Lee, John K.; Pohl, Peter

    2009-01-01

    Hydrogen sulfide (H2S) has emerged as a new and important member in the group of gaseous signaling molecules. However, the molecular transport mechanism has not yet been identified. Because of structural similarities with H2O, it was hypothesized that aquaporins may facilitate H2S transport across cell membranes. We tested this hypothesis by reconstituting the archeal aquaporin AfAQP from sulfide reducing bacteria Archaeoglobus fulgidus into planar membranes and by monitoring the resulting facilitation of osmotic water flow and H2S flux. To measure H2O and H2S fluxes, respectively, sodium ion dilution and buffer acidification by proton release (H2S ⇆ H+ + HS−) were recorded in the immediate membrane vicinity. Both sodium ion concentration and pH were measured by scanning ion-selective microelectrodes. A lower limit of lipid bilayer permeability to H2S, PM,H2S ≥ 0.5 ± 0.4 cm/s was calculated by numerically solving the complete system of differential reaction diffusion equations and fitting the theoretical pH distribution to experimental pH profiles. Even though reconstitution of AfAQP significantly increased water permeability through planar lipid bilayers, PM,H2S remained unchanged. These results indicate that lipid membranes may well act as a barrier to water transport although they do not oppose a significant resistance to H2S diffusion. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into an H2S barrier indicates that H2S transport through epithelial barriers, endothelial barriers, and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels. PMID:19805349

  19. Bacterial membranes enhance the immunogenicity and protective capacity of the surface exposed tick Subolesin-Anaplasma marginale MSP1a chimeric antigen.

    PubMed

    Contreras, Marinela; Moreno-Cid, Juan A; Domingos, Ana; Canales, Mario; Díez-Delgado, Iratxe; Pérez de la Lastra, José M; Sánchez, Emilio; Merino, Octávio; Zavala, Rigoberto López; Ayllón, Nieves; Boadella, Mariana; Villar, Margarita; Gortázar, Christian; de la Fuente, José

    2015-09-01

    Ticks are vectors of diseases that affect humans and animals worldwide. Tick vaccines have been proposed as a cost-effective and environmentally sound alternative for tick control. Recently, the Rhipicephalus microplus Subolesin (SUB)-Anaplasma marginale MSP1a chimeric antigen was produced in Escherichia coli as membrane-bound and exposed protein and used to protect vaccinated cattle against tick infestations. In this research, lipidomics and proteomics characterization of the E. coli membrane-bound SUB-MSP1a antigen showed the presence of components with potential adjuvant effect. Furthermore, vaccination with membrane-free SUB-MSP1a and bacterial membranes containing SUB-MSP1a showed that bacterial membranes enhance the immunogenicity of the SUB-MSP1a antigen in animal models. R. microplus female ticks were capillary-fed with sera from pigs orally immunized with membrane-free SUB, membrane bound SUB-MSP1a and saline control. Ticks ingested antibodies added to the blood meal and the effect of these antibodies on reduction of tick weight was shown for membrane bound SUB-MSP1a but not SUB when compared to control. Using the simple and cost-effective process developed for the purification of membrane-bound SUB-MSP1a, endotoxin levels were within limits accepted for recombinant vaccines. These results provide further support for the development of tick vaccines using E. coli membranes exposing chimeric antigens such as SUB-MSP1a. PMID:26219233

  20. Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum

    PubMed Central

    Blanchard, Nicolas; Gonzalez, Federico; Schaeffer, Marie; Joncker, Nathalie T; Cheng, Tiffany; Shastri, Anjali J; Robey, Ellen A; Shastri, Nilabh

    2016-01-01

    The parasite Toxoplasma gondii replicates in a specialized intracellular vacuole and causes disease in many species. Protection from toxoplasmosis is mediated by CD8+ T cells, but the T. gondii antigens and host genes required for eliciting protective immunity are poorly defined. Here we identified GRA6, a polymorphic protein secreted in the parasitophorous vacuole, as the source of the immunodominant and protective decapeptide HF10 presented by the H-2Ld major histocompatibility complex class I molecule. Presentation of the HF10–H-2Ld ligand required proteolysis by ERAAP, the endoplasmic reticulum aminopeptidase associated with antigen processing. Consequently, expansion of protective CD8+ T cell populations was impaired in T. gondii–infected ERAAP-deficient mice, which were more susceptible to toxoplasmosis. Thus, endoplasmic reticulum proteolysis is critical for eliciting protective immunity to a vacuolar parasite. PMID:18587399

  1. Prostate-specific membrane antigen protein expression in tumor tissue and risk of lethal prostate cancer

    PubMed Central

    Kasperzyk, Julie L.; Finn, Stephen P.; Flavin, Richard; Fiorentino, Michelangelo; Lis, Rosina; Hendrickson, Whitney K.; Clinton, Steven K.; Sesso, Howard D.; Giovannucci, Edward L.; Stampfer, Meir J.; Loda, Massimo; Mucci, Lorelei A.

    2013-01-01

    Background Over-expression of prostate-specific membrane antigen (PSMA) in tumor tissue and serum has been linked to increased risk of biochemical recurrence in surgically treated prostate cancer patients, but no studies have assessed its association with disease-specific mortality. Methods We examined whether high PSMA protein expression in prostate tumor tissue was associated with lethal disease, and with tumor biomarkers of progression, among participants of two US-based cohorts (n=902, diagnosed 1983–2004). We used Cox proportional hazards regression to calculate multivariable hazard ratios (HR) and 95% confidence intervals (CI) of lethal prostate cancer, defined as disease-specific death or development of distant metastases (n=95). Partial Spearman rank correlation coefficients were used to correlate PSMA with tumor biomarkers. Results During an average 13 years of follow-up, higher PSMA expression at prostatectomy was significantly associated with lethal prostate cancer (age-adjusted HRQuartile(Q)4vs.Q1=2.42; p-trend<0.01). This association was attenuated and non-significant (multivariable-adjusted HRQ4vs.Q1=1.01; p-trend=0.52) after further adjusting for Gleason score and PSA at diagnosis. High PSMA expression was significantly (p<0.05) correlated with higher Gleason score and PSA at diagnosis, increased tumor angiogenesis, lower vitamin D receptor and androgen receptor expression, and absence of ERG expression. Conclusions High tumor PSMA expression was not an independent predictor of lethal prostate cancer in the current study. PSMA expression likely captures, in part, malignant features of Gleason grade and tumor angiogenesis. Impact PSMA is not a strong candidate biomarker for predicting prostate cancer-specific mortality in surgically treated patients. PMID:24130224

  2. In vitro antigen-induced antibody responses to hepatitis B surface antigen in man. Kinetic and cellular requirements.

    PubMed Central

    Cupps, T R; Gerin, J L; Purcell, R H; Goldsmith, P K; Fauci, A S

    1984-01-01

    In this report we define the parameters of the human immune response after immunization with hepatitis B vaccine. 2 wk after booster immunization, there is significant spontaneous secretion of antibody to hepatitis B surface antigen (anti-HBs IgG), which is not further augmented by stimulation with antigen or pokeweed mitogen (PWM). By 4 wk there is little spontaneous secretion of specific antibody, and low doses of antigen or PWM produce significant increases in the amount of anti-HBs IgG produced. By 8 wk the peripheral blood mononuclear cells are refractory to stimulation by antigen, but anti-HBs IgG is produced in response to PWM. 0.5 yr or more after the last immunization, some individuals will manifest an antigen-induced specific antibody response. This induction of anti-HBs IgG by hepatitis B surface antigen (HBsAg) is monocyte- and T cell-dependent. Note that there is a dichotomy in the T cell response to HBsAg. The specific antibody response is clearly T cell dependent, but no in vitro T cell proliferative response to HBsAG could be demonstrated in the immunized individuals. Although the precise reason for the absent proliferative response to HBsAg despite well-established humoral immunity has not been determined, there was no evidence to suggest nonspecific suppression by HBsAg or the presence of an adherent suppressor cell population. The ability to evaluate antigen-induced, antigen-specific responses to HBsAg will be useful in defining the unique interaction between the human immune response and this clinically important viral agent. PMID:6332826

  3. Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

    PubMed

    Pattanapanyasat, K; Webster, H K; Udomsangpetch, R; Wanachiwanawin, W; Yongvanitchit, K

    1992-01-01

    A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites. PMID:1372210

  4. Plasma membrane-associated antigens on tumor cells derived from transitional-cell carcinoma of the human urinary bladder. II. Identification at the molecular level of plasma membrane-associated antigens.

    PubMed

    Schneider, M U; Paulie, S; Troye, M; Perlmann, P

    1980-08-01

    The surface proteins of seven human cell lines (three bladder carcinomas (TCC), two normal urothelial lines, one colon carcinoma, and one malignant melanoma) were labelled with 125I by the glucose oxidase-lactoperoxidase technique. Plasma membranes of the cells were isolated and analysed by sodium dodecyl sulphate electrophoresis (SDS-PAGE). When analysed under reducing conditions by staining with protein stain, approximately 45 distinct membrane polypeptides were detected in all membrane preparations. Although the banding patterns for all cell lines were very similar, a 23 K and a 110 K band were only seen in the five unrothelial lines. When the same gels were analysed by autoradiography, between 13 and 17 bands were detected for each of the cell lines. However, in this case, analysis revealed individual and stable banding profiles for each. One 180 K band and one 100 K band were only seen in the autoradiographs of the two normal lines but not in those of the tumor membranes. Analysis under non-reducing conditions gave similar results. The antigenicity of these surface components was analysed by incubating detergent extracts of surface-iodinated cells with IgG from a rabbit anti-TCC serum, absorbed with fetal bovine serum and bound to protein A (from Staphylococcus aureus) on a matrix of Sepharose 4B. Analysis of the eluates by autoradiography after SDS-PAGE under reducing conditions showed that many of the labelled polypeptides were antigenic and shared by all seven cell lines. Analysis of eluates from IgG preparations, exhaustively absorbed with human spleen, revealed the presence of at least one antigenic 110 K polypeptide confined to the membrane of the urothelial cells. Preparation of a rabbit antiserum to this 110 K component, isolated from one of the TCC-lines and tested by ADCC, indicated that this polypeptide constitutes an important surface antigen, present on urothelial cells of both TCC- and normal origin but absent from the colon carcinoma and

  5. Tolerance induction in memory CD4 T cells requires two rounds of antigen-specific activation.

    PubMed

    David, Alexandria; Crawford, Frances; Garside, Paul; Kappler, John W; Marrack, Philippa; MacLeod, Megan

    2014-05-27

    A major goal for immunotherapy is to tolerize the immune cells that coordinate tissue damage in autoimmune and alloantigen responses. CD4 T cells play a central role in many of these conditions and improved antigen-specific regulation or removal of these cells could revolutionize current treatments. A confounding factor is that little is known about whether and how tolerance is induced in memory CD4 T cells. We used MHC class II tetramers to track and analyze a population of endogenous antigen-specific memory CD4 T cells exposed to soluble peptide in the absence of adjuvant. We found that such memory T cells proliferated and reentered the memory pool apparently unperturbed by the incomplete activation signals provided by the peptide. Upon further restimulation in vivo, CD4 memory T cells that had been previously exposed to peptide proliferated, provided help to primary responding B cells, and migrated to inflamed sites. However, these reactivated memory cells failed to survive. The reduction in T-cell number was marked by low expression of the antiapoptotic molecule B cell lymphoma 2 (Bcl2) and increased expression of activated caspase molecules. Consequently, these cells failed to sustain a delayed-type hypersensitivity response. Moreover, following two separate exposures to soluble antigen, no T-cell recall response and no helper activity for B cells could be detected. These results suggest that the induction of tolerance in memory CD4 T cells is possible but that deletion and permanent removal of the antigen-specific T cells requires reactivation following exposure to the tolerogenic antigen. PMID:24821788

  6. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    PubMed Central

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies. PMID:23023212

  7. Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

    PubMed Central

    Curto, M.-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M.-Henar

    2014-01-01

    The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell–cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell–cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell–cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  8. Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

    PubMed

    Curto, M-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M-Henar

    2014-04-01

    The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  9. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    PubMed

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  10. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine

    PubMed Central

    Dick Jr, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    ABSTRACT The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90–110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  11. Identification and characterization of the 2D6 and Mr 23,000 antigens on the plasma membrane of rat spermatozoa.

    PubMed Central

    Jones, R; Brown, C R

    1987-01-01

    Previous investigations [Jones, Brown, von Glos & Gaunt (1985) Exp. Cell Res. 156, 31-44] have demonstrated the appearance of a new antigenic determinant (recognized by monoclonal antibody 2D6) on the plasma membrane of rat spermatozoa during post-testicular maturation in the epididymis. Identification of the 2D6 antigen on Western blots from one-dimensional SDS/polyacrylamide gels revealed that it co-migrated with a membrane protein (designated Mr 23,000 antigen) present on testicular and immature germ cells, suggesting that one antigen might be a modified version of the other. In the present work, however, we demonstrate that, although they have similar Mr and are present in soluble and membrane-bound forms, the 2D6 and Mr 23,000 antigens are biochemically and immunologically distinct molecules. The properties of the antigens are described and compared. The Mr 23,000 antigen is present on both testicular and cauda epididymidal spermatozoa, has a pI of 6.1, contains no detectable carbohydrate, is not tissue-specific and is degraded by V8 protease. By contrast, the 2D6 antigen is glycosylated, has a broad pI from 4.5 to 6.1, is tissue- and species-specific and is resistant to digestion with V8 protease. Its role in sperm-egg recognition is discussed. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2439064

  12. CD1d-restricted antigen presentation by Vγ9Vδ2-T cells requires trogocytosis.

    PubMed

    Schneiders, Famke L; Prodöhl, Jan; Ruben, Jurjen M; O'Toole, Tom; Scheper, Rik J; Bonneville, Marc; Scotet, Emmanuel; Verheul, Henk M W; de Gruijl, Tanja D; van der Vliet, Hans J

    2014-08-01

    CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and play a dominant role in antitumor immunity. Clinical trials with α-GalCer-pulsed monocyte-derived dendritic cells (moDC) have shown anecdotal antitumor activity in advanced cancer. It was reported that phosphoantigen (pAg)-activated Vγ9Vδ2-T cells can acquire characteristics of professional antigen-presenting cells (APC). Considering the clinical immunotherapeutic applications, Vγ9Vδ2-T APC can offer important advantages over moDC, potentially constituting an attractive novel APC platform. Here, we demonstrate that Vγ9Vδ2-T APC can present antigens to iNKT. However, this does not result from de novo synthesis of CD1d by Vγ9Vδ2-T, but critically depends on trogocytosis of CD1d-containing membrane fragments from pAg-expressing cells. CD1d-expressing Vγ9Vδ2-T cells were able to activate iNKT in a CD1d-restricted and α-GalCer-dependent fashion. Although α-GalCer-loaded moDC outperformed Vγ9Vδ2-T APC on a per cell basis, Vγ9Vδ2-T APC possess unique features with respect to clinical immunotherapeutic application that make them an interesting platform for consideration in future clinical trials. PMID:24934445

  13. PET imaging of prostate-specific membrane antigen in prostate cancer: current state of the art and future challenges

    PubMed Central

    Rowe, SP; Gorin, MA; Allaf, ME; Pienta, KJ; Tran, PT; Pomper, MG; Ross, AE; Cho, SY

    2016-01-01

    BACKGROUND Prostate-specific membrane antigen (PSMA) is a cell surface enzyme that is highly expressed in prostate cancer (PCa) and is currently being extensively explored as a promising target for molecular imaging in a variety of clinical contexts. Novel antibody and small-molecule PSMA radiotracers labeled with a variety of radionuclides for positron emission tomography (PET) imaging applications have been developed and explored in recent studies. METHODS A great deal of progress has been made in defining the clinical utility of this class of PET agents through predominantly small and retrospective clinical studies. The most compelling data to date has been in the setting of biochemically recurrent PCa, where PSMA-targeted radiotracers have been found to be superior to conventional imaging and other molecular imaging agents for the detection of locally recurrent and metastatic PCa. RESULTS Early data, however, suggest that initial lymph node staging before definitive therapy in high-risk primary PCa patients may be limited, although intraoperative guidance may still hold promise. Other examples of potential promising applications for PSMA PET imaging include non-invasive characterization of primary PCa, staging and treatment planning for PSMA-targeted radiotherapeutics, and guidance of focal therapy for oligometastatic disease. CONCLUSIONS However, all of these indications and applications for PCa PSMA PET imaging are still lacking and require large, prospective, systematic clinical trials for validation. Such validation trials are needed and hopefully will be forthcoming as the fields of molecular imaging, urology, radiation oncology and medical oncology continue to define and refine the utility of PSMA-targeted PET imaging to improve the management of PCa patients. PMID:27136743

  14. Localization and physical mapping of the prostate-specific membrane antigen (PSM) gene to human chromosome 11

    SciTech Connect

    Rinker-Schaeffer, C.W.; Hawkins, A.L.; Griffin, C.A.; Isaacs, J.T.

    1995-11-01

    The prostate-specific membrane antigen (PSM) was identified by the monoclonal antibody 7E11-C5.3, which was raised against the human prostatic carcinoma cell line LNCaP. The PSM antigen is expressed by normal, neoplastic, and metastatic prostatic tissues. The 2.65-kb cDNA encoding the 100-kDa PSM glycoprotein was cloned from LNCaP cells. Studies have shown that the expression of PSM is tissue-specific. In the present study monochromosomal somatic cell hybrids were used to localize the PSM gene to human chromosome 11. Using this information, initial mapping studies identified two potential PSM gene loci at 11p11.1-p13 and 11q14. Further high-stringency analysis using cosmid probes identified the 11q14 region as the location of the PSM gene. 10 refs., 2 figs.

  15. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    PubMed

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system. PMID:26481477

  16. An Extended Surface Loop on Toxoplasma gondii Apical Membrane Antigen 1 (AMA1) Governs Ligand Binding Selectivity

    PubMed Central

    Parker, Michelle L.; Boulanger, Martin J.

    2015-01-01

    Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs) on the parasite surface and rhoptry neck 2 (RON2) proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop). Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2) peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON2 invasion

  17. Erythrocyte Membrane Antigen Frequencies in Patients with Type II Congenital Smell Loss

    PubMed Central

    Stateman, William A.; Henkin, Robert I.; Knöppel, Alexandra; Flegel, Willy A.

    2014-01-01

    OBJECTIVE The objective of this study was to determine whether there are genetic factors associated with Type II congenital smell loss. STUDY DESIGN The expression frequencies of 16 erythrocyte antigens among patients with Type II congenital smell loss were determined and compared to those of a large control group. METHODS Blood samples were obtained from 99 patients with Type II congenital smell loss. Presence of the erythrocyte surface antigens A, B, M, N, S, s, Fya, Fyb, D, C, c, E, e, K, Jka, and Jkb was analyzed by blood group serology. Comparisons of expression frequencies of these antigens were made between the patients and a large control group. RESULTS Patients tested for the Duffy b antigen (Fyb haplotype) exhibited a statistically significant 11% decrease in expression frequency compared to the controls. There were no significant differences between patients and controls in the expression frequencies for all other erythrocyte antigens (A, B, M, N, S, s, Fya, D, C, c, E, e, K, Jka, or Jkb). CONCLUSIONS These findings describe the presence of a previously unrevealed genetic tendency among patients with Type II congenital smell loss related to erythrocyte surface antigen expression. The deviation in expression rate of Duffy b suggests a target gene and chromosome region in which future research into this form of congenital smell loss may reveal a more specific genetic basis for Type II congenital smell loss. PMID:25456515

  18. Metastasis-associated 5T4 oncofoetal antigen is concentrated at microvillus projections of the plasma membrane.

    PubMed

    Carsberg, C J; Myers, K A; Evans, G S; Allen, T D; Stern, P L

    1995-08-01

    The 5T4 oncofoetal antigen is a 72 kDa glycoprotein defined by a monoclonal antibody raised against human placental trophoblast and is expressed in many different carcinomas but detected only at low levels in some normal epithelia. Immunohistochemical analysis of the patterns of expression in colorectal carcinomas has indicated a significant association between the presence of the antigen in tumour cells and metastatic spread. A cDNA encoding the 5T4 molecules has been isolated and the extracellular portion contains several leucine-rich repeats which have been implicated in cellular interactions. To study the cell biological role of 5T4 molecules, murine L cells (A9 derivative) were stably transfected with 5T4 cDNA under the control of the CMV immediate-early promoter. The 5T4-expressing cells exhibited a more spindle-shaped morphology compared to the vector alone transfected cells. Confocal immunofluorescence microscopy revealed a 'polkadot' pattern of 5T4 antigen expression, heterogeneous in intensity between cells, but distributed over the entire cell surface. Transmission and scanning electron microscopy showed that the 5T4 antigen is concentrated at microvillus projections of the plasma membrane both in the transfected A9 cells and in various carcinoma cell lines. Such projections express an array of surface molecules which function in cell adhesion and motility. This association of 5T4 antigen with microvillus projections was also observed in various carcinoma cell lines. 5T4 expression in A9 cells was also associated with an altered pattern of cell division, decreased cell-substratum adhesion and increased cellular motility. These results support the hypothesis that 5T4 molecules may have a direct function in trophoblast and tumour cell invasion processes. PMID:7593330

  19. Elevated 68Ga Prostate-Specific Membrane Antigen Activity in Metastatic Non-Small Cell Lung Cancer.

    PubMed

    Shetty, Deepa; Loh, Han; Bui, Chuong; Mansberg, Robert; Stevanovic, Amanda

    2016-05-01

    A 71-year-old man with a background of treated stage IIIB non-small cell lung cancer was referred for Ga prostate-specific membrane antigen (PSMA) PET/CT for staging of prostate cancer. In addition to the PSMA uptake in the known prostate malignancy, the study also demonstrated increased PSMA uptake in an enlarging left lower lobe lung mass with diffusely increased PSMA uptake in an enlarged thyroid gland and bilateral enlarged supraclavicular lymph nodes. Fine-needle aspiration biopsy of the thyroid gland and a left supraclavicular lymph node demonstrated metastatic adenocarcinoma from a primary lung cancer. PMID:26828144

  20. Metastatic superscan in prostate carcinoma on gallium-68-prostate-specific membrane antigen positron emission tomography/computed tomography scan

    PubMed Central

    Agarwal, Krishan Kant; Tripathi, Madhavi; Kumar, Rajeev; Bal, Chandrasekhar

    2016-01-01

    We describe the imaging features of a metastatic superscan on gallium-68 Glu-NH-CO-NH-Lys-(Ahx)-[Ga-68(HBED-CC)], abbreviated as gallium-68-prostate-specific membrane antigen (68Ga-PSMA) positron emission tomography/computed tomography (PET/CT) imaging. 68Ga-PSMA is novel radiotracer undergoing evaluation for PET/CT imaging of prostate carcinoma. This patient had a superscan of metastases on conventional bone scintigraphy and was referred for 68Ga-PSMA PET/CT to evaluate the feasibility of 177Lu-PSMA therapy. PMID:27095868

  1. Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45.

    PubMed

    Pritsch, Michael; Ben-Khaled, Najib; Chaloupka, Michael; Kobold, Sebastian; Berens-Riha, Nicole; Peter, Annabell; Liegl, Gabriele; Schubert, Sören; Hoelscher, Michael; Löscher, Thomas; Wieser, Andreas

    2016-01-01

    Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV) are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT) and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3) were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells) were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 10(5) could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases. PMID:27239480

  2. Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45

    PubMed Central

    Pritsch, Michael; Ben-Khaled, Najib; Chaloupka, Michael; Kobold, Sebastian; Berens-Riha, Nicole; Peter, Annabell; Liegl, Gabriele; Schubert, Sören; Hoelscher, Michael; Löscher, Thomas; Wieser, Andreas

    2016-01-01

    Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV) are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT) and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3) were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells) were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 105 could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases. PMID:27239480

  3. In vitro induction of non-responsiveness in cloned normal inducer T cells by antigen and purified Ia incorporated into planar membranes

    SciTech Connect

    Quill, H.; Fox, B.; Carlson, L.; Pardoll, D.; Schwartz, R.H.

    1986-03-05

    Incubation of cytochrome c-specific E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/-containing planar membranes and an antigenic peptide analogue of moth cytochrome c resulted in a specific increase in cell volume of 40-50% as measured by Coulter Counter analysis. No change in cell volume was seen in the absence of antigen, or when A/sub ..beta..//sup k/A/sub ..cap alpha..//sup k/-planar membranes were used. T cell proliferation was never detected at any time from one to eight days after incubation with E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/-membranes at a wide range of antigen concentrations. Furthermore, only trace amounts of IL-2 were detected and no increase in IL-2 receptor expression was seen. IL-3 production, however, could be detected. T cells pre-incubated for one day with E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/-membranes plus antigen became non-responsive to subsequent normal stimulation with antigen and APC. Incorporation of /sup 3/H-thymidine was reduced by more than 90% and the production of both IL-2 and IL-3 was inhibited. Non-responsiveness persisted for at least eight days after exposure to E/sub ..beta..///sup k/E/sub ..cap alpha..//sup k/-membranes plus antigen. In contrast, T cells pre-incubated under control conditions remained fully responsive. These results demonstrate the specific induction of non-responsiveness in inducer T cells by antigen and purified E/sub ..beta..//sup k/E/sub ..cap alpha..//sup k/ in planar membranes.

  4. Ultraviolet-irradiated vaccinia virus recombinants, exposing HIV-envelope on their outer membrane, induce antibodies against this antigen in rabbits.

    PubMed

    Loewinger, M; Katz, E

    2002-01-01

    The construction and isolation of recombinants of vaccinia virus (IHD-J strain), bearing on their outer membrane a chimeric protein consisting of the cytoplasmic and transmembrane domains of vaccinia B5R protein and the external domain of HIV envelope, has been previously described by us. The present study aimed to investigate the potential use of such recombinants as a vaccine, following inactivation of their infectivity by ultraviolet (UV) irradiation. The minimal dose of UV irradiation, required for the complete inactivation of the infectivity of these recombinants, was determined. Injections of rabbits with the irradiated noninfectious recombinant viruses successfully induced specific antibodies against the HIV envelope antigen, in addition to those against the poxvirus. PMID:12479396

  5. Nano-clustering of ligands on surrogate antigen presenting cells modulates T cell membrane adhesion and organization.

    PubMed

    Dillard, Pierre; Pi, Fuwei; Lellouch, Annemarie C; Limozin, Laurent; Sengupta, Kheya

    2016-03-14

    We investigate the adhesion and molecular organization of the plasma membrane of T lymphocytes interacting with a surrogate antigen presenting cell comprising glass supported ordered arrays of antibody (α-CD3) nano-dots dispersed in a non-adhesive matrix of polyethylene glycol (PEG). The local membrane adhesion and topography, as well as the distribution of the T cell receptors (TCRs) and the kinase ZAP-70, are influenced by dot-geometry, whereas the cell spreading area is determined by the overall average density of the ligands rather than specific characteristics of the dots. TCR clusters are recruited preferentially to the nano-dots and the TCR cluster size distribution has a weak dot-size dependence. On the patterns, the clusters are larger, more numerous, and more enriched in TCRs, as compared to the homogeneously distributed ligands at comparable concentrations. These observations support the idea that non-ligated TCRs residing in the non-adhered parts of the proximal membrane are able to diffuse and enrich the existing clusters at the ligand dots. However, long distance transport is impaired and cluster centralization in the form of a central supramolecular cluster (cSMAC) is not observed. Time-lapse imaging of early cell-surface contacts indicates that the ZAP-70 microclusters are directly recruited to the site of the antibody dots and this process is concomitant with membrane adhesion. These results together point to a complex interplay of adhesion, molecular organization and activation in response to spatially modulated stimulation. PMID:26887857

  6. Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

    PubMed

    Krishnarjuna, Bankala; Lim, San Sui; Devine, Shane M; Debono, Cael O; Lam, Raymond; Chandrashekaran, Indu R; Jaipuria, Garima; Yagi, Hiromasa; Atreya, Hanudatta S; Scanlon, Martin J; MacRaild, Christopher A; Scammells, Peter J; Norton, Raymond S

    2016-06-01

    Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26804042

  7. Partial Purification of Integral Membrane Antigenic Proteins from Trypanosoma evansi That Display Immunological Cross-Reactivity with Trypanosoma vivax

    PubMed Central

    Velásquez, Norma P.; Camargo, Rocío E.; Uzcanga, Graciela L.; Bubis, José

    2014-01-01

    Trypanosoma evansi and Trypanosoma vivax, which are the major causative agents of animal trypanosomosis in Venezuela, have shown a very high immunological cross-reactivity. Since the production of T. vivax antigens is a limiting factor as this parasite is difficult to propagate in experimental animal models, our goal has been to identify and isolate antigens from T. evansi that cross-react with T. vivax. Here, we used the Venezuelan T. evansi TEVA1 isolate to prepare the total parasite lysate and its corresponding cytosolic and membranous fractions. In order to extract the T. evansi integral membrane proteins, the particulate portion was further extracted first with Triton X-100, and then with sodium dodecyl sulfate. After discarding the cytosolic and Triton X-100 solubilized proteins, we employed sedimentation by centrifugation on linear sucrose gradients to partially purify the sodium dodecyl sulfate-solubilized proteins from the Triton X-100 resistant particulate fraction of T. evansi. We obtained enriched pools containing polypeptide bands with apparent molecular masses of 27 kDa, 31 kDa, and 53 kDa, which were recognized by anti-T. vivax antibodies from experimentally and naturally infected bovines. PMID:24757558

  8. Lipopolysaccharide Modifications of a Cholera Vaccine Candidate Based on Outer Membrane Vesicles Reduce Endotoxicity and Reveal the Major Protective Antigen

    PubMed Central

    Leitner, Deborah R.; Feichter, Sandra; Schild-Prüfert, Kristina; Rechberger, Gerald N.; Reidl, Joachim

    2013-01-01

    The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay. PMID:23630951

  9. Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum.

    PubMed

    Radolf, J D; Norgard, M V; Schulz, W W

    1989-03-01

    Freeze fracture and deep etching were used to investigate the ultrastructural basis for the observation that anti-treponemal antibodies bind poorly to the surface of virulent Treponema pallidum. Fractures of T. pallidum outer membranes contained scarce, uniformly sized intramembranous particles (IMPs). IMPs on the convex faces often appeared to form linear arrays that wound in spirals about the organism. In contrast to the outer membrane, IMPs of the cytoplasmic membrane were randomly distributed, numerous, and heterogeneous in size. In Escherichia coli and T. pallidum cofractures, IMPs of the E. coli outer membranes were densely packed within the concave fracture faces, while the T. pallidum fractures were identical to the experiments lacking the E. coli internal controls. Outer membranes of two representative nonpathogenic treponemes, Treponema phagedenis biotype Reiter and Treponema denticola, contained numerous IMPs, which segregated preferentially with the concave halves. Examination of apposed replicas and deep-etched specimens indicated that at least some of the IMPs extend through the T. pallidum outer membrane and are exposed on the surface of the organism. The outer membrane of intact T. pallidum appears to contain a paucity of integral membrane proteins that can serve as targets for specific antibodies. These findings appear to represent an unusual parasitic strategy for evasion of host humoral defenses. PMID:2648388

  10. A monoclonal antibody directed against a human cell membrane antigen prevents cell substrate adhesion and tumor invasion.

    PubMed Central

    De Potter, C. R.; Schelfhout, A. M.; De Smet, F. H.; Van Damme, S.; de Ridder, L.; Dhont, E.; van Emmelo, J.

    1994-01-01

    It was the aim of this study to design mouse monoclonal antibodies (MAbs) that can inhibit the invasion of breast cancer cells in the host tissue. Therefore, MAbs were raised against epitopes on the extracellular domain of SK-BR-3 human breast cancer cells, and biological assays were performed to test the capability of the MAbs to inhibit cell substrate adhesion. MAb 14C5 bound an extracellular plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells and inhibited the cell substrate adhesion of these cells in vitro. The MAb delayed the adhesion of MCF-7 and SK-BR-3 cells on precultured embryonic heart fragments (PHFS). It inhibited the destruction of the PHF by MCF-7 cells and the invasion of the PHF by SK-BR-3 cells. The MAb reacted with an epitope on the cell membrane of in situ and invasive ductal carcinomas of the breast in immunohistochemistry. Poorly differentiated, highly invasive ductal carcinomas show extensive staining of long plasma membrane extensions. Normal multilayered epithelia, normal connective tissue, and tumors derived from these tissues as well as normal breast tissue were negative. From both cell lines a protein complex consisting of two subunits with molecular weight of 50 and 90 kd, respectively, was immunoprecipitated. It is concluded that the 14C5 antigen plays a role in cell substrate adhesion and subsequently also in invasion of breast cancer cells. The 14C5 MAb was able to inhibit cell substrate adhesion and invasion in vitro of breast cancer cells. Images Figure 3 Figure 4 Figure 5 PMID:8291615

  11. The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

    PubMed

    Guo, D; Bowden, M G; Pershad, R; Kaplan, H B

    1996-03-01

    A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521. PMID:8626291

  12. The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

    PubMed Central

    Guo, D; Bowden, M G; Pershad, R; Kaplan, H B

    1996-01-01

    A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521. PMID:8626291

  13. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling

    PubMed Central

    Sena, Laura A.; Li, Sha; Jairaman, Amit; Prakriya, Murali; Ezponda, Teresa; Hildeman, David A.; Wang, Chyung-Ru; Schumacker, Paul T.; Licht, Jonathan D.; Perlman, Harris; Bryce, Paul J.; Chandel, Navdeep S.

    2013-01-01

    SUMMARY It is widely appreciated that T cells increase glycolytic flux during activation, however the role of mitochondrial flux is unclear. Here we have shown that mitochondrial metabolism, in the absence of glucose metabolism, was sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs−/− mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, however Uqcrfs1−/− T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1−/− T cells were not lacking bioenergetically, but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through production of complex III ROS. PMID:23415911

  14. Genetic Locus Required for Antigenic Maturation of Rhizobium etli CE3 Lipopolysaccharide

    PubMed Central

    Duelli, Dominik M.; Tobin, Andrea; Box, Jodie M.; Kolli, V. S. Kumar; Carlson, Russell W.; Noel, K. Dale

    2001-01-01

    Rhizobium etli modifies lipopolysaccharide (LPS) structure in response to environmental signals, such as low pH and anthocyanins. These LPS modifications result in the loss of reactivity with certain monoclonal antibodies. The same antibodies fail to recognize previously isolated R. etli mutant strain CE367, even in the absence of such environmental cues. Chemical analysis of the LPS in strain CE367 demonstrated that it lacked the terminal sugar of the wild-type O antigen, 2,3,4-tri-O-methylfucose. A 3-kb stretch of DNA, designated as lpe3, restored wild-type antigenicity when transferred into CE367. From the sequence of this DNA, five open reading frames were postulated. Site-directed mutagenesis and complementation analysis suggested that the genes were organized in at least two transcriptional units, both of which were required for the production of LPS reactive with the diagnostic antibodies. Growth in anthocyanins or at low pH did not alter the specific expression of gusA from the transposon insertion of mutant CE367, nor did the presence of multiple copies of lpe3 situated behind a strong, constitutive promoter prevent epitope changes induced by these environmental cues. Mutations of the lpe genes did not prevent normal nodule development on Phaseolus vulgaris and had very little effect on the occupation of nodules in competition with the wild-type strain. PMID:11567006

  15. Quantitative proteomic analysis of Shigella flexneri and Shigella sonnei Generalized Modules for Membrane Antigens (GMMA) reveals highly pure preparations

    PubMed Central

    Maggiore, Luana; Yu, Lu; Omasits, Ulrich; Rossi, Omar; Dougan, Gordon; Thomson, Nicholas R.; Saul, Allan; Choudhary, Jyoti S.; Gerke, Christiane

    2016-01-01

    Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517. PMID:26746581

  16. Surface membrane antigen expression changes induced in vitro by exogenous growth factors in chronic lymphocytic leukemia cells.

    PubMed

    Vilpo, J; Hulkkonen, J; Hurme, M; Vilpo, L

    2002-09-01

    The factors determining the growth and survival of cells in B chronic lymphocytic leukemia (CLL) have remained poorly understood. We investigated the effects of optimal mitogen combinations (OMCs) on the expression of 26 surface membrane antigens among 33 CLL patients. The seven OMCs used were selected after pre-testing 14 combinations of (1) S. aureus Cowan I (SAC), (2) interleukin-2 (IL-2), (3) tumor necrosis factor alpha (TNF-alpha) and (4) 12-O-tetradecanoylphorbol 13-acetate (TPA; also known as phorbol 12-myristate 13-acetate or PMA). In flow cytometry we revealed that OMCs induced statistically highly significant upregulation of the expression of CD5, CD11c, CD19, CD22, CD23, CD25, CD38, CD40, CD45, CD45RO, CD95, CD126, CD130 and FMC7, and downregulation of CD20 and CD124 expression. Interestingly, the expression of CD27, CD45RA, CD79b, CD80, CD122 and that of the immunoglobulin gene superfamily members CD21, Ig-kappa, Ig-lambda, Ig-delta and Ig-micro were not significantly affected under similar conditions. The expression of several antigens was co-regulated, suggesting common regulatory pathways. These antigens include CD11c/CD5, CD11c/CD22, CD11c/CD126, CD11c/FMC7 as well as CD27/CD45, CD27/CD45RA and CD27/CD79b. Upregulation of surface antigen expression, induced by OMCs, should be applicable in antibody therapy in vitro and in vivo, and in negative stem cell selection for autotransplantation. Furthermore, the current strategy to enhance cell surface antigen expression may be a versatile tool to raise humoral and cell-mediated host defense against CLL cells. Upregulation of proteins mediating positive growth signals (eg CD25, CD40) and negative signals or apoptosis (eg CD95) may be used to sensitize cells to chemotherapy and programmed cell death. PMID:12200683

  17. Cross-presentation through langerin and DC-SIGN targeting requires different formulations of glycan-modified antigens.

    PubMed

    Fehres, Cynthia M; Kalay, Hakan; Bruijns, Sven C M; Musaafir, Sara A M; Ambrosini, Martino; van Bloois, Louis; van Vliet, Sandra J; Storm, Gert; Garcia-Vallejo, Juan J; van Kooyk, Yvette

    2015-04-10

    Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based antigen targeting to both the human DC-specific C-type lectin receptor (CLR) DC-SIGN and the LC-specific CLR langerin. Since DC-SIGN and langerin are able to recognize the difucosylated oligosaccharide Lewis Y (Le(Y)), we prepared neoglycoconjugates bearing this glycan epitope to allow targeting of both lectins. Le(Y)-modified liposomes, with an approximate diameter of 200nm, were significantly endocytosed by DC-SIGN(+) DCs and mediated efficient antigen presentation to CD4(+) and CD8(+) T cells. Surprisingly, although langerin bound to Le(Y)-modified liposomes, LCs exposed to Le(Y)-modified liposomes could not endocytose liposomes nor mediate antigen presentation to T cells. However, LCs mediated an enhanced cross-presentation when antigen was delivered through langerin using Le(Y)-modified synthetic long peptides. In contrast, Le(Y)-modified synthetic long peptides were recognized by DC-SIGN, but did not trigger antigen internalization nor antigen cross-presentation. These data demonstrate that langerin and DC-SIGN have different size requirements for antigen uptake. Although using glycans remains an interesting option in the design of anti-cancer vaccines targeting multiple CLRs, aspects such as molecule size and conformation need to be taken in consideration. PMID:25656175

  18. Caspase-Activated DNase is Required to Maintain Tolerance to Lupus Nuclear AutoAntigens

    PubMed Central

    Jog, Neelakshi R.; Frisoni, Lorenza; Shi, Qin; Monestier, Marc; Hernandez, Sairy; Craft, Joe; Luning Prak, Eline T.; Caricchio, Roberto

    2011-01-01

    Objective Caspase Activated DNase (CAD) is an endonuclease that is activated by active caspase 3 during apoptosis and is responsible for degradation of chromatin into nucleosomal units. These nucleosomal units are then included in apoptotic bodies. The presence of apoptotic bodies is considered important for the generation of auto-antigens in autoimmune diseases such as lupus, which are characterized by the presence of anti-nuclear antibodies. Methods The present study was carried out to determine the role of CAD in Sle1, Sle123 and 3H9 spontaneous models of lupus, where autoimmunity is genetically pre-determined. We also determined the ability of lupus auto-antibodies to bind to CAD deficient or sufficient apoptotic cells. Results The deficiency of CAD resulted in higher anti-dsDNA antibody titers in lupus-prone mice. Surprisingly, the absence of CAD only exacerbated genetically pre-determined autoimmune responses. To further determine whether nuclear modifications are required to maintain tolerance to nuclear auto-antigens, we used the 3H9 mouse, an anti-DNA heavy chain knock-in. In this model, the autoreactive B cells are tolerized by anergy. In line with the Sle1 and Sle123 CAD mutant mice, CAD deficient 3H9 mice spontaneously generated anti-DNA antibodies. We finally show that auto-antibodies with specificities towards histone/DNA complexes bind more to CAD deficient apoptotic cells compared to CAD sufficient apoptotic cells. Conclusions We propose that in mice genetically predisposed to lupus, nuclear apoptotic modifications are required to maintain tolerance. In the absence of these modifications, apoptotic chromatin is abnormally exposed, facilitating the autoimmune response. PMID:22127758

  19. Thy-1 antigen: selective association in lymphoid organs with the vascular basement membrane involved in lymphocyte recirculation.

    PubMed Central

    Ritter, M A; Morris, R J

    1980-01-01

    The cell surface differentiation antigen, Thy-1, was demonstrated by immunofluorescence to be associated with collagen-based connective tissue (mainly basement membrane) around some blood vessels in rat lymphoid organs. This association is highly selective: only certain types of blood vessel within a given lymphoid organ were found to be Thy-1+; and different lymphoid organs (thymus, bone marrow, lymph node and spleen) had characteristic differences in the types of blood vessel that bear Thy-1. In lymph node and spleen the vessels that were Thy-1+ were those involved in lymphocyte recirculation and homing (post-capillary venules and arterioles of white pulp); the possibility that Thy-1 may function in mediating selective adhesion of small lymphocytes to extracellular substrates during recirculation is discussed. Images Figure 2 Figure 3 Figure 1 Figure 4 PMID:6991398

  20. In Vivo Visualization of Prostate-Specific Membrane Antigen in Adenoid Cystic Carcinoma of the Salivary Gland.

    PubMed

    Lütje, Susanne; Sauerwein, Wolfgang; Lauenstein, Thomas; Bockisch, Andreas; Poeppel, Thorsten D

    2016-06-01

    As the prostate-specific membrane antigen (PSMA) is overexpressed in the neovasculature of several malignancies, it might serve as a target in oncology. Ga-PSMA PET/CT and PET/MRI were performed in a female who developed pulmonary metastases from an adenoid cystic carcinoma of the right sublingual salivary gland after incomplete resection of the primary tumor and radiotherapy. Uptake of Ga-PSMA in tumors was observed, indicating PSMA expression. Moreover, a new cerebral metastasis was detected. Potentially, Ga-PSMA PET might be used for noninvasive assessment of adenoid cystic carcinoma to evaluate whether patients apply for PSMA-based radiotherapy when no further treatment options are available. PMID:27055136

  1. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

    PubMed

    Hoehn, G T; Clark, V L

    1990-12-01

    The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus. PMID:2123827

  2. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

    PubMed Central

    Hoehn, G T; Clark, V L

    1990-01-01

    The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus. Images PMID:2123827

  3. A promising new ELISA diagnostic test for cattle babesiosis based on Babesia bigemina Apical Membrane Antigen-1.

    PubMed

    Torina, Alessandra; Cordaro, Antonio; Blanda, Valeria; D'Agostino, Rosalia; Scimeca, Salvatore; Scariano, Maria E; Sireci, Guido; Lelli, Rossella

    2016-01-01

    Babesiosis due to Babesia bigemina is a relevant tick-borne disease, affecting cattle worldwide. Many surface proteins of the pathogen including the Apical Membrane Antigen 1 (AMA-1) - have been analysed for vaccine and diagnostic purposes. This study focused on B. bigemina AMA-1 and on its use for the assessment of diagnostic tests. After bioinformatic analyses, AMA-1 codifying region was amplified and cloned into an expression vector used to induce protein synthesis in Escherichia coli cells. AMA-1 was purified by affinity chromatography and used to set up the best condition for an ELISA protocol. Bovine field sera positive to B. bigemina were used to evaluate the presence of anti-AMA-1 antibodies. In order to verify the assay specificity, sera positive to Babesia bovis or to the piroplasm Theileria annulata were also included. Significant differences were obtained between sera negative to both B. bigemina and B. bovis and samples positive to B. bigemina, to B. bovis or to both pathogens. No significant reaction was observed with T. annulata positive sera. The results showed that AMA-1 protein is suitable to be used as antigen in diagnostic assays for babesiosis diagnosis in cattle, as it does not show any cross reaction with anti-T. annulata antibodies. PMID:27033532

  4. Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

    PubMed

    Wingender, Gerhard; Birkholz, Alysia M; Sag, Duygu; Farber, Elisa; Chitale, Sampada; Howell, Amy R; Kronenberg, Mitchell

    2015-10-15

    Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness. PMID:26355152

  5. Subdominant antigens in bacterial vaccines: Am779 is subdominant in the anaplasma marginale outer membrane vaccine but does not associate with protective immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Identification of specific antigens responsible for the ability of complex immunogens to induce protection is a major goal in development of bacterial vaccines. Much of the investigation has focused on highly abundant and highly immunodominant outer membrane proteins. Recently however, genomic and p...

  6. Brucella outer membrane lipoprotein shares antigenic determinants with Escherichia coli Braun lipoprotein and is exposed on the cell surface.

    PubMed Central

    Gómez-Miguel, M J; Moriyón, I; López, J

    1987-01-01

    In an enzyme-linked immunosorbent assay (ELISA), purified Brucella abortus and Escherichia coli peptidoglycan-linked lipoproteins gave a strong cross-reaction with sera from rabbits hyperimmunized with the heterologous lipoprotein. When smooth E. coli cells were used as ELISA antigens, the immunological cross-reaction was not observed unless the cells were treated to remove lipopolysaccharide and other outer membrane components. In contrast, intact cells from smooth strains of B. abortus and Brucella melitensis bound anti-lipoprotein immunoglobulin G, and the controls performed by ELISA showed that this reaction was not due to antibodies to the lipopolysaccharide, group 3 outer membrane proteins, or porins. Electron microscopy of cells labeled with antilipoprotein serum and protein A-colloidal gold showed specific labeling of smooth cells from both B. abortus and B. melitensis, even though unspecific labeling by nonimmune serum was observed with rough B. abortus. These results confirm the close similarity between E. coli and Brucella peptidoglycan-linked lipoproteins and show that, in contrast to E. coli, the lipoprotein of B. abortus and B. melitensis is partially exposed on the surface of smooth cells. Images PMID:2432014

  7. A Remote Arene-Binding Site on Prostate Specific Membrane Antigen Revealed by Antibody-Recruiting Small Molecules

    SciTech Connect

    Zhang, Andrew X.; Murelli, Ryan P.; Barinka, Cyril; Michel, Julien; Cocleaza, Alexandra; Jorgensen, William L.; Lubkowski, Jacek; Spiegel, David A.

    2010-09-27

    Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar arene-binding sites throughout the proteome could prove widely enabling in the optimization of small molecule-protein interactions.

  8. Protective effect of intranasal immunization with Neospora caninum membrane antigens against murine neosporosis established through the gastrointestinal tract

    PubMed Central

    Ferreirinha, Pedro; Dias, Joana; Correia, Alexandra; Pérez-Cabezas, Begoña; Santos, Carlos; Teixeira, Luzia; Ribeiro, Adília; Rocha, António; Vilanova, Manuel

    2014-01-01

    Neospora caninum is an Apicomplexa parasite that in the last two decades was acknowledged as the main pathogenic agent responsible for economic losses in the cattle industry. In the present study, the effectiveness of intranasal immunization with N. caninum membrane antigens plus CpG adjuvant was assessed in a murine model of intragastrically established neosporosis. Immunized mice presented a lower parasitic burden in the brain on infection with 5 × 107 tachyzoites, showing that significant protection was achieved by this immunization strategy. Intestinal IgA antibodies raised by immunization markedly agglutinated live N. caninum tachyzoites whereas previous opsonization with IgG antibodies purified from immunized mice sera reduced parasite survival within macrophage cells. Although an IgG1 : IgG2a ratio < 1 was detected in the immunized mice before and after infection, indicative of a predominant T helper type 1 immune response, no increased production of interferon-γ was detected in the spleen or mesenteric lymph nodes of the immunized mice. Altogether, these results show that mucosal immunization with N. caninum membrane proteins plus CpG adjuvant protect against intragastrically established neosporosis and indicate that parasite-specific mucosal and circulating antibodies have a protective role against this parasitic infection. PMID:24128071

  9. Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS.

    PubMed

    Janovská, Sylva; Pávková, Ivona; Hubálek, Martin; Lenco, Juraj; Macela, Ales; Stulík, Jirí

    2007-02-15

    Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins. PMID:17241671

  10. Co-administration of non-carrier nanoparticles boosts antigen immune response without requiring protein conjugation.

    PubMed

    Wibowo, Nani; Chuan, Yap P; Seth, Arjun; Cordoba, Yoann; Lua, Linda H L; Middelberg, Anton P J

    2014-06-17

    Nanotechnology promises a revolution in medicine including through new vaccine approaches. The use of nanoparticles in vaccination has, to date, focused on attaching antigen directly to or within nanoparticle structures to enhance antigen uptake by immune cells. Here we question whether antigen incorporation with the nanoparticle is actually necessary to boost vaccine effectiveness. We show that the immunogenicity of a sub-unit protein antigen was significantly boosted by formulation with silica nanoparticles even without specific conjugation of antigen to the nanoparticle. We further show that this effect was observed only for virus-sized nanoparticles (50 nm) but not for larger (1,000 nm) particles, demonstrating a pronounced effect of nanoparticle size. This non-attachment approach has potential to radically simplify the development and application of nanoparticle-based formulations, leading to safer and simpler nanoparticle applications in vaccine development. PMID:24793947

  11. Requirement of phosphatidylglycerol for photosynthetic function in thylakoid membranes

    PubMed Central

    Sato, Norihiro; Hagio, Miki; Wada, Hajime; Tsuzuki, Mikio

    2000-01-01

    To investigate the role of phosphatidylglycerol (PG) in photosynthesis, we constructed a mutant defective in the CDP-diacylglycerol synthase gene from a cyanobacterium, Synechocystis sp. PCC6803. The mutant, designated as SNC1, required PG supplementation for growth. Growth was repressed in PG-free medium concomitantly with the decrease in cellular content of PG. These results indicate that PG is essential, and that SNC1 is defective in PG synthesis. Decrease in PG content was accompanied by a reduction in the cellular content of chlorophyll, but with little effect on the contents of phycobilisome pigments, which showed that levels of chlorophyll–protein complexes decreased without alteration of those of phycobilisomes. Regardless of the decrease in the PG content, CO2-dependent photosynthesis by SNC1 was similar to that by the wild type on a chlorophyll basis, but consequently became lower on a cell basis. Simultaneously, the ratio of oxygen evolution of photosystem II (PSII) measured with p-benzoquinone to that of CO2-dependent photosynthesis, which ranged between 1.3 and 1.7 in the wild type. However, it was decreased in SNC1 from 1.3 to 0.4 during the early growth phase where chlorophyll content and CO2-dependent photosynthesis were little affected, and then finally to 0.1, suggesting that PSII first lost its ability to reduce p-benzoquinone and then decreased in its level and actual activity. These results indicate that PG contributes to the accumulation of chlorophyll–protein complexes in thylakoid membranes, and also to normal functioning of PSII. PMID:10984546

  12. Electrochemical detection of the binding of Bacillus anthracis protective antigen (PA) to the membrane receptor on macrophages through release of nitric oxide.

    PubMed

    Trouillon, Raphaël; Williamson, E Diane; Saint, Richard J; O'Hare, Danny

    2012-01-01

    Anthrax is a serious bacterial disease of man and animals whose pathogenesis involves the secretion of lethal toxins in the host. The intracellular delivery of toxic complexes involves a complex structural rearrangement of sub-domains of the exotoxin protective antigen (PA). We have used a biocompatible microelectrode array, coated with J774 mouse macrophages, to detect PA binding and intracellular signaling resulting in nitric oxide (NO) release. We have found that exposure of macrophages to PA in vitro activates the inducible isoform of NO synthase (iNOS), thus increasing the extracellular concentration of NO and nitrite, in a dose- and time-dependent manner. However, the cell-binding domain 4 of PA (PA4) could substitute for full-length PA to achieve equivalent NO release, suggesting that the heptamerisation of PA, ultimately required to deliver toxic complexes into the cell, is not a requirement for the activation of an intracellular cascade through the ERK 1/2 and the PI-3K/ Akt kinase pathways and that these events could be triggered by the binding of PA4 alone to its cell membrane receptor. Further, we have found that pre-incubation of the cells with azidothymidine, a pro-oxidant drug, significantly improves the limit of detection of rPA-induced NO release thus offering a sensitive tool for the analysis of the kinetics of anthrax intoxication and ultimately drug discovery. PMID:22672762

  13. Low Levels of Polymorphisms and No Evidence for Diversifying Selection on the Plasmodium knowlesi Apical Membrane Antigen 1 Gene

    PubMed Central

    Faber, Bart W.; Abdul Kadir, Khamisah; Rodriguez-Garcia, Roberto; Remarque, Edmond J; Saul, Frederick A.; Vulliez-Le Normand, Brigitte; Bentley, Graham A.; Kocken, Clemens H. M.; Singh, Balbir

    2015-01-01

    Infection with Plasmodium knowlesi, a zoonotic primate malaria, is a growing human health problem in Southeast Asia. P. knowlesi is being used in malaria vaccine studies, and a number of proteins are being considered as candidate malaria vaccine antigens, including the Apical Membrane Antigen 1 (AMA1). In order to determine genetic diversity of the ama1 gene and to identify epitopes of AMA1 under strongest immune selection, the ama1 gene of 52 P. knowlesi isolates derived from human infections was sequenced. Sequence analysis of isolates from two geographically isolated regions in Sarawak showed that polymorphism in the protein is low compared to that of AMA1 of the major human malaria parasites, P. falciparum and P. vivax. Although the number of haplotypes was 27, the frequency of mutations at the majority of the polymorphic positions was low, and only six positions had a variance frequency higher than 10%. Only two positions had more than one alternative amino acid. Interestingly, three of the high-frequency polymorphic sites correspond to invariant sites in PfAMA1 or PvAMA1. Statistically significant differences in the quantity of three of the six high frequency mutations were observed between the two regions. These analyses suggest that the pkama1 gene is not under balancing selection, as observed for pfama1 and pvama1, and that the PkAMA1 protein is not a primary target for protective humoral immune responses in their reservoir macaque hosts, unlike PfAMA1 and PvAMA1 in humans. The low level of polymorphism justifies the development of a single allele PkAMA1-based vaccine. PMID:25881166

  14. Neisseria meningitidis Native Outer Membrane Vesicles Containing Different Lipopolysaccharide Glycoforms as Adjuvants for Meningococcal and Nonmeningococcal Antigens

    PubMed Central

    Nagaputra, Jerry C.; Sadarangani, Manish; Hoe, J. Claire; Mehta, Ojas Hrakesh; Norheim, Gunnstein; Saleem, Muhammad; Chan, Hannah; Derrick, Jeremy P.; Feavers, Ian; Pollard, Andrew J.; Moxon, E. Richard

    2014-01-01

    We evaluated the adjuvant effect of a modified glycoform of lipopolysaccharide (LPS) (LgtB-LpxL1) compared to that of the nonmodified glycoform Lpxl1 serogroup B meningococcal H44/76 native outer membrane vesicles (nOMVs) on immune responses to vaccination with the recombinant meningococcal protein, rPorA, tetanus toxoid, or meningococcal serogroup C capsular polysaccharide. We used LgtB-LpxL1 LPS because the disruption of the lgtB gene, which results in the exposure of N-acetylglucosamine-galactose-glucose residues in the LPS outer core, has been shown to enhance the activation of human dendritic cells in vitro. The responses were compared to those of a monophosphoryl lipid A (MPL)-based adjuvant and to an aluminum hydroxide suspension. The nOMVs induced blood serum IgG responses against each of the three antigens comparable to those obtained with MPL or aluminum salt. However, nOMVs elicited (i) a lower IgG1/IgG2a ratio against rPorA and (ii) serum bactericidal antibody titers superior to those achieved with aluminum salt, reaching similar titers to those obtained with MPL. Similarly, bactericidal antibody titers induced by immunization with meningococcal serogroup C polysaccharide and nOMVs were similar to those obtained using MPL but were better than those with aluminum salt. Immunization with tetanus toxoid and nOMVs resulted in tetanus toxoid-specific IgG responses similar to those obtained when adjuvanted with aluminum salt. These results highlight the potential utility of meningococcal LpxL1 LPS-containing nOMVs as an adjuvant for recombinant meningococcal protein vaccines and suggest their possible use with a variety of other antigens. PMID:24351756

  15. CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance.

    PubMed

    Fowler, Sanna; Powrie, Fiona

    2002-10-01

    CD4(+) T cells play a vital role in mediating the tolerance induced at mucosal sites following exposure to non-pathogenic stimuli, and further understanding of the precise mechanisms by which these cells prevent aberrant responses is required. We have developed a model using transfer of DO11.10 TCR-transgenic bone marrow into irradiated recipients in which it has been possible to track antigen-specific CD4(+) cells in mesenteric lymph nodes (mLN), Peyer's patches (PP) and lamina propria following primary exposure to antigen. Using this model we have demonstrated initial activation in all three gut-associated lymphoid tissue compartments characterized by increases in the frequency of transgenic cells expressing CD69 and CD25. These cells subsequently enter a state of hyporesponsiveness both locally in the mLN and PP and in the periphery following feeding and challenge. Investigating the role of CTLA-4 either using anti-CTLA-4 mAb or by generating chimeras using DO11.10xCTLA-4(-/-) mice as donors we have clearly shown that antigen-specific cells require the expression of this regulatory molecule for oral tolerance. In contrast, oral tolerance was intact in chimeras generated using DO11.10xIL-10(-/-) cells, indicating that secretion of this cytokine by antigen-specific cells is not required. PMID:12355454

  16. Descending Vasa Recta Endothelial Membrane Potential Response Requires Pericyte Communication

    PubMed Central

    Zhang, Zhong; Payne, Kristie; Pallone, Thomas L.

    2016-01-01

    Using dual-cell electrophysiological recording, we examined the routes for equilibration of membrane potential between the pericytes and endothelia that comprise the descending vasa recta (DVR) wall. We measured equilibration between pericytes in intact vessels, between pericytes and endothelium in intact vessels and between pericytes physically separated from the endothelium. Dual pericyte recording on the abluminal surface of DVR showed that both resting potential and subsequent time-dependent voltage fluctuations after vasoconstrictor stimulation remained closely equilibrated, regardless of the agonist employed (angiotensin II, vasopressin or endothelin 1). When pericytes where removed from the vessel wall but retained physical contact with one another, membrane potential responses were also highly coordinated. In contrast, responses of pericytes varied independently when they were isolated from both the endothelium and from contact with one another. When pericytes and endothelium were in contact, their resting potentials were similar and their temporal responses to stimulation were highly coordinated. After completely isolating pericytes from the endothelium, their mean resting potentials became discordant. Finally, complete endothelial isolation eliminated all membrane potential responses to angiotensin II. We conclude that cell-to-cell transmission through the endothelium is not needed for pericytes to equilibrate their membrane potentials. AngII dependent responses of DVR endothelia may originate from gap junction coupling to pericytes rather than via receptor dependent signaling in the endothelium, per se. PMID:27171211

  17. LABORATORY STUDIES OF SOIL BEDDING REQUIREMENTS FOR FLEXIBLE MEMBRANE LINERS

    EPA Science Inventory

    The initial objective of this study was to investigate the performance of membrane liners during construction of hazardous waste landfills and develop a means for protecting the liners from damage. This objective included the development of laboratory tests that could be used to ...

  18. Molecular Insights into the Interaction between Plasmodium falciparum Apical Membrane Antigen 1 and an Invasion-Inhibitory Peptide

    PubMed Central

    Wang, Geqing; MacRaild, Christopher A.; Mohanty, Biswaranjan; Mobli, Mehdi; Cowieson, Nathan P.; Anders, Robin F.; Simpson, Jamie S.; McGowan, Sheena; Norton, Raymond S.; Scanlon, Martin J.

    2014-01-01

    Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1∶1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction. PMID:25343578

  19. Immunization with Apical Membrane Antigen 1 Confers Sterile Infection-Blocking Immunity against Plasmodium Sporozoite Challenge in a Rodent Model

    PubMed Central

    Schussek, Sophie; Trieu, Angela; Apte, Simon H.; Sidney, John; Sette, Alessandro

    2013-01-01

    Apical membrane antigen 1 (AMA-1) is a leading blood-stage malaria vaccine candidate. Consistent with a key role in erythrocytic invasion, AMA-1-specific antibodies have been implicated in AMA-1-induced protective immunity. AMA-1 is also expressed in sporozoites and in mature liver schizonts where it may be a target of protective cell-mediated immunity. Here, we demonstrate for the first time that immunization with AMA-1 can induce sterile infection-blocking immunity against Plasmodium sporozoite challenge in 80% of immunized mice. Significantly higher levels of gamma interferon (IFN-γ)/interleukin-2 (IL-2)/tumor necrosis factor (TNF) multifunctional T cells were noted in immunized mice than in control mice. We also report the first identification of minimal CD8+ and CD4+ T cell epitopes on Plasmodium yoelii AMA-1. These data establish AMA-1 as a target of both preerythrocytic- and erythrocytic-stage protective immune responses and validate vaccine approaches designed to induce both cellular and humoral immunity. PMID:23836827

  20. Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project.

    PubMed

    Escalante, A A; Grebert, H M; Chaiyaroj, S C; Magris, M; Biswas, S; Nahlen, B L; Lal, A A

    2001-04-01

    We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum. PMID:11295182

  1. Biodistribution and Radiation Dosimetry for a Probe Targeting Prostate-Specific Membrane Antigen for Imaging and Therapy

    PubMed Central

    Herrmann, Ken; Bluemel, Christina; Weineisen, Martina; Schottelius, Margret; Wester, Hans-Jürgen; Czernin, Johannes; Eberlein, Uta; Beykan, Seval; Lapa, Constantin; Riedmiller, Hubertus; Krebs, Markus; Kropf, Saskia; Schirbel, Andreas; Buck, Andreas K.; Lassmann, Michael

    2016-01-01

    Prostate-specific membrane antigen (PSMA) is a promising target for diagnosis and treatment of prostate cancer. EuK-Subkff-68Ga-DOTAGA (68Ga-PSMA Imaging & Therapy [PSMA I&T]) is a recently introduced PET tracer for imaging PSMA expression in vivo. Whole-body distribution and radiation dosimetry of this new probe were evaluated. Methods Five patients with a history of prostate cancer were injected intravenously with 91–148 MBq of 68Ga-PSMA I&T (mean ± SD, 128 ± 23 MBq). After an initial series of rapid whole-body scans, 3 static whole-body scans were acquired at 1, 2, and 4 h after tracer injection. Time-dependent changes of the injected activity per organ were determined. Mean organ-absorbed doses and effective doses were calculated using OLINDA/EXM. Results Injection of 150 MBq of 68Ga-PSMA I&T resulted in an effective dose of 3.0 mSv. The kidneys were the critical organ (33 mGy), followed by the urinary bladder wall and spleen (10 mGy each), salivary glands (9 mGy each), and liver (7 mGy). Conclusion 68Ga-PSMA I&T exhibits a favorable dosimetry, delivering organ doses that are comparable to (kidneys) or lower than those delivered by 18F-FDG. PMID:25883128

  2. Sequence Requirements for the Assembly of Simian Virus 40 T Antigen and the T-Antigen Origin Binding Domain on the Viral Core Origin of Replication

    PubMed Central

    Kim, Henry Y.; Barbaro, Brett A.; Joo, Woo S.; Prack, Andrea E.; Sreekumar, K. R.; Bullock, Peter A.

    1999-01-01

    The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd131–260) have been determined. Binding of the purified T-ag-obd131–260 is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers. PMID:10438844

  3. A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

    PubMed Central

    Mahdavi, Jafar; Pirinccioglu, Necmettin; Oldfield, Neil J.; Carlsohn, Elisabet; Stoof, Jeroen; Aslam, Akhmed; Self, Tim; Cawthraw, Shaun A.; Petrovska, Liljana; Colborne, Natalie; Sihlbom, Carina; Borén, Thomas; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

    2014-01-01

    Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis. PMID:24451549

  4. Influence of the B-band O-antigen chain in the Structure and Electrostatics of the Lipopolysaccharide Membrane of Pseudomonas aeruginosa

    SciTech Connect

    Soares, Thereza A.; Straatsma, TP; Lins, Roberto D.

    2008-02-01

    Classical molecular dynamics simulations of A-B+ LPS membrane model of Pseudomonas aeruginosa were carried out for 12+ ns. The B-band presents a remarkable flexibility remaining fully solvated and does not interact with the sugar units from the LPS core surface residues, in agreement atomic force microscopy experiments. Comparison with previous simulations of the rough LPS membrane suggests that the presence of the B-band promotes membrane expansion. In addition, this O-antigen chain dramatically alters the electrostatic potential and surface charge of the LPS membrane. This is illustrated by the resulting electrostatic surface potential. Comparison with the same property in rough LPS aids to explain the increased ability of B-band expressing microorganisms to adhere to cell surfaces and the necessity of these organisms to loose the O side chain for the development of acute infections.

  5. Proteomic Approach for Characterization of Immunodominant Membrane-Associated 30- to 36-Kilodalton Fraction Antigens of Leishmania infantum Promastigotes, Reacting with Sera from Mediterranean Visceral Leishmaniasis Patients

    PubMed Central

    Kamoun-Essghaier, Sayda; Guizani, Ikram; Strub, Jean Marc; Van Dorsselaer, Alain; Mabrouk, Kamel; Ouelhazi, Lazhar; Dellagi, Koussay

    2005-01-01

    The aim of the present study was to identify and characterize proteins of a 30- to 36-kDa fraction of Leishmania infantum promastigote membranes previously shown to be an immunodominant antigen(s) in Mediterranean visceral leishmaniasis (MVL) and a consistent and reliable serological marker of this disease. By the first approach, Coomassie-stained protein bands (32- and 33-kDa fractions) that specifically reacted by immunoblotting with sera from MVL patients were excised from the gel and submitted to enzymatic digestion to generate peptides. Four peptides were sequenced, three of which were shown to be definitely associated with MVL-reactive antigens and ascribed to a mitochondrial integral ADP-ATP carrier protein from L. major, a putative NADH cytochrome b5 reductase, and a putative mitochondrial carrier protein, respectively. The second approach combined two-dimensional gel electrophoresis of membrane antigens and mass spectrometry (liquid chromatography-mass spectrometry/mass spectrometry) by using a quadrupole time-of-flight analysis. Six immunoreactive spots that resolved within a molecular mass range of 30 to 36 kDa and a pH range of 6.7 to 7.4 corresponded to four Leishmania products. The sequences derived from two spots were ascribed to a beta subunit-like guanine nucleotide binding protein, known as the activated protein kinase C receptor homolog antigen LACK, and to a probable member of the aldehyde reductase family. One spot was identified as a probable ubiquinol-cytochrome c reductase (EC 1.10.2.2) Rieske iron-sulfur protein precursor. The remaining three spots were identified as truncated forms of elongation factor 1α. These antigens correspond to conserved proteins ubiquitously expressed in eukaryotic cells and represent potential candidates for the design of a reliable tool for the diagnosis of this disease. PMID:15699427

  6. Prostate cancer nodal oligometastasis accurately assessed using prostate-specific membrane antigen positron emission tomography-computed tomography and confirmed histologically following robotic-assisted lymph node dissection

    PubMed Central

    O’Kane, Dermot B.; Lawrentschuk, Nathan; Bolton, Damien M.

    2016-01-01

    We herein present a case of a 76-year-old gentleman, where prostate-specific membrane antigen positron emission tomography-computed tomography (PSMA PET-CT) was used to accurately detect prostate cancer (PCa), pelvic lymph node (LN) metastasis in the setting of biochemical recurrence following definitive treatment for PCa. The positive PSMA PET-CT result was confirmed with histological examination of the involved pelvic LNs following pelvic LN dissection. PMID:27141207

  7. Current Status of Prostate-Specific Membrane Antigen Targeting in Nuclear Medicine: Clinical Translation of Chelator Containing Prostate-Specific Membrane Antigen Ligands Into Diagnostics and Therapy for Prostate Cancer.

    PubMed

    Kratochwil, Clemens; Afshar-Oromieh, Ali; Kopka, Klaus; Haberkorn, Uwe; Giesel, Frederik L

    2016-09-01

    The prostate-specific membrane antigen (PSMA) is expressed by approximately 90% of prostate carcinomas. The expression correlates with unfavorable prognostic factors, such as a high Gleason score, infiltrative growth, metastasis, and hormone-independence. The high specificity, especially in the undifferentiated stage, makes it an excellent target for diagnosis and therapy. Therefore, antibodies and small molecule inhibitors have been developed for imaging and therapy. In 2011 PSMA-11, a ligand that consists of the Glu-urea-motif and the chelator HBED-CC, which can be exclusively radiolabeled with (68)Ga for PET imaging, presented the clinical breakthrough for prostate cancer diagnostics. In two large diagnostic studies (n = 319 and n = 248) PET/CT with PSMA-11 successfully localized the recurrent tumor in approximately 90% of patients with biochemical relapse. Integrating PSMA-PET/CT into the planning phase of radiotherapy, the treatment concept is changed in 30%-50% of the patients. The combination of the Glu-urea-motif with DOTA, which can be labeled with several diagnostic and therapeutic radionuclides, opened new avenues for therapeutic usage of the small-molecule PSMA ligands. In the beginning of 2016, there are four confirmative reports (n = 19, n = 24, n = 30, and n = 56) from four different centers reporting a PSA response in approximately 70% of patients treated with (177)Lu-labeled PSMA ligands. In conclusion, the data available up to now indicate a widespread use of PSMA ligands for diagnostic applications with respect to staging, detection of recurrence, or metastases in patients with rising tumor markers and for therapy in case of failure of guideline-compliant treatment. PMID:27553466

  8. Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen.

    PubMed

    Frigerio, Barbara; Benigni, Fabio; Luison, Elena; Seregni, Ettore; Pascali, Claudio; Fracasso, Giulio; Morlino, Sara; Valdagni, Riccardo; Mezzanzanica, Delia; Canevari, Silvana; Figini, Mariangela

    2015-11-01

    Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognised by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. (111)In radiolabelling was performed via the chelator Bz-NOTA, and (131)I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for (111)In-scFvD2B at 3h after injection (45%ID/g) and it was maintained up to 24h (26%ID/g). By contrast, kidney accumulation of (131)I-scFvD2B was only marginally (0.3%ID/g at 24h). At the optimal time point defined between 15h and 24h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with (131)I-scFvD2B yielding a significantly better target/background ratio compared to (111)In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution. PMID:26404855

  9. MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

    PubMed

    Aranda, Juan F; Reglero-Real, Natalia; Kremer, Leonor; Marcos-Ramiro, Beatriz; Ruiz-Sáenz, Ana; Calvo, María; Enrich, Carlos; Correas, Isabel; Millán, Jaime; Alonso, Miguel A

    2011-04-15

    Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes. PMID:21325632

  10. In Vitro Studies with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 (AMA1): Production and Activity of an AMA1 Vaccine and Generation of a Multiallelic Response

    PubMed Central

    Kennedy, Michael C.; Wang, Jin; Zhang, Yanling; Miles, Aaron P.; Chitsaz, Farideh; Saul, Allan; Long, Carole A.; Miller, Louis H.; Stowers, Anthony W.

    2002-01-01

    Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage. PMID:12438374

  11. Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins

    PubMed Central

    Li, Ming; Koshi, Tatsuhiro

    2015-01-01

    Cells must regulate the abundance and activity of numerous nutrient transporters in different organelle membranes to achieve nutrient homeostasis. As the recycling center and major storage organelle, lysosomes are essential for maintaining nutrient homeostasis. However, very little is known about mechanisms that govern the regulation of its membrane proteins. In this study, we demonstrated that changes of Zn2+ levels trigger the downregulation of vacuolar Zn2+ transporters. Low Zn2+ levels cause the degradation of the influx transporter Cot1, whereas high Zn2+ levels trigger the degradation of the efflux channel Zrt3. The degradation process depends on the vacuole membrane recycling and degradation pathway. Unexpectedly, we identified a RING domain–containing E3 ligase Tul1 and its interacting proteins in the Dsc complex that are important for the ubiquitination of Cot1 and partial ubiquitination of Zrt3. Our study demonstrated that the Dsc complex can function at the vacuole to regulate the composition and lifetime of vacuolar membrane proteins. PMID:26527740

  12. Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

    PubMed Central

    Edwards, W. Barry

    2013-01-01

    The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. PMID:23935860

  13. In Vitro Targeted Photodynamic Therapy with a Pyropheophorbide-a Conjugated Inhibitor of Prostate Specific Membrane Antigen

    PubMed Central

    Liu, Tiancheng; Wu, Lisa Y.; Choi, Joseph K.; Berkman, Clifford E.

    2009-01-01

    BACKROUND The lack of specific delivery of photosensitizers (PSs), represents a significant limitation of photodynamic therapy (PDT) of cancer. The biomarker prostate-specific membrane antigen (PSMA) has attracted considerable attention as a target for imaging and therapeutic applications for prostate cancer. Although recent efforts have been made to conjugate inhibitors of PSMA with imaging agents, there have been no reports on photosensitizer-conjugated PSMA inhibitors for targeted PDT of prostate cancer. The present study focuses on the use of a PSMA inhibitor-conjugate of pyropheophorbide-a (Ppa-conjugate 2) for targeted PDT to achieve apoptosis in PSMA+ LNCaP cells. METHODS Confocal laser scanning microscopy with a combination of nuclear staining and immunofluorescence methods were employed to monitor the specific imaging and PDT-mediated apoptotic effects on PSMA-positive LNCaP and PSMA-negative (PC-3) cells. RESULTS Our results demonstrated that PDT-mediated effects by Ppa-conjugate 2 were specific to LNCaP cells, but not PC-3 cells. Cell permeability was detected as early as 2 h by HOE33342/PI double-staining, becoming more intense by 4 h. Evidence for the apoptotic caspase cascade being activated was based on the appearance of PARP p85 fragment. TUNEL assay detected DNA fragmentation 16 h post-PDT, confirming apoptotic events. CONCLUSIONS Cell permeability by HOE33342/PI double-staining as well as PARP p85 fragment and TUNEL assays confirm cellular apoptosis in PSMA+ cells when treated with PS-inhibitor conjugate 2 and subsequently irradiated. It is expected that the PSMA targeting small-molecule of this conjugate can serve as a delivery vehicle for PDT and other therapeutic applications for prostate cancer. PMID:19142895

  14. Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates.

    PubMed

    Al-Qahtani, Ahmed A; Abdel-Muhsin, Abdel-Muhsin A; Bin Dajem, Saad M; AlSheikh, Adel Ali H; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Putaporntip, Chaturong; Jongwutiwes, Somchai

    2016-04-01

    The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries. PMID:26867816

  15. Crystal Structure of Plasmodium knowlesi Apical Membrane Antigen 1 and Its Complex with an Invasion-Inhibitory Monoclonal Antibody

    PubMed Central

    van der Eijk, Marjolein; Thomas, Alan W.; Singh, Balbir; Kocken, Clemens H. M.

    2015-01-01

    The malaria parasite Plasmodium knowlesi, previously associated only with infection of macaques, is now known to infect humans as well and has become a significant public health problem in Southeast Asia. This species should therefore be targeted in vaccine and therapeutic strategies against human malaria. Apical Membrane Antigen 1 (AMA1), which plays a role in Plasmodium merozoite invasion of the erythrocyte, is currently being pursued in human vaccine trials against P. falciparum. Recent vaccine trials in macaques using the P. knowlesi orthologue PkAMA1 have shown that it protects against infection by this parasite species and thus should be developed for human vaccination as well. Here, we present the crystal structure of Domains 1 and 2 of the PkAMA1 ectodomain, and of its complex with the invasion-inhibitory monoclonal antibody R31C2. The Domain 2 (D2) loop, which is displaced upon binding the Rhoptry Neck Protein 2 (RON2) receptor, makes significant contacts with the antibody. R31C2 inhibits binding of the Rhoptry Neck Protein 2 (RON2) receptor by steric blocking of the hydrophobic groove and by preventing the displacement of the D2 loop which is essential for exposing the complete binding site on AMA1. R31C2 recognizes a non-polymorphic epitope and should thus be cross-strain reactive. PkAMA1 is much less polymorphic than the P. falciparum and P. vivax orthologues. Unlike these two latter species, there are no polymorphic sites close to the RON2-binding site of PkAMA1, suggesting that P. knowlesi has not developed a mechanism of immune escape from the host’s humoral response to AMA1. PMID:25886591

  16. Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

    NASA Astrophysics Data System (ADS)

    Luo, Zhang; Liu, Zhixin; Fu, Jianping; Zhang, Qiusheng; Huang, Bei; Nie, Pin

    2016-02-01

    Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes (IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon (IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.

  17. Genetic detection of Babesia bigemina from Mongolian cattle using apical membrane antigen-1 gene-based PCR assay.

    PubMed

    Sivakumar, Thillaiampalam; Altangerel, Khukhuu; Battsetseg, Badgar; Battur, Banzragch; Aboulaila, Mahmoud; Munkhjargal, Tserendorj; Yoshinari, Takeshi; Yokoyama, Naoaki; Igarashi, Ikuo

    2012-06-01

    We developed a new nested PCR (nPCR) assay based on the Babesia bigemina apical membrane antigen-1 (AMA-1) gene sequence for parasite-specific detection. The primers were designed to amplify 738-bp and 211-bp fragments of the AMA-1 gene by primary and nested PCRs, respectively. The assay was proven to be specific for the B. bigemina, whereas the previously established SpeI-AvaI nPCR assay amplified not only the target fragment of B. bigemina but also a homologous one from Babesia ovata. The AMA-1 nPCR assay was also evaluated using field DNA samples extracted from 266 bovine blood samples collected from Mongolia in 2010. In a comparative evaluation, 90 (33.8%) and 25 (9.4%) of the blood samples showed positive reactions for B. bigemina by the SpeI-AvaI nPCR and AMA-1 nPCR assays, respectively. The sequencing analysis of the nPCR products confirmed that the AMA-1 nPCR method had specifically detected the target B. bigemina DNA. However, 4 different kinds of sequences were determined among the SpeI-AvaI nPCR amplicons. Two of them were derived from B. bigemina and B. ovata, while the origins of the others were unknown. In the current study, the presence of B. bigemina was clearly demonstrated among Mongolian cattle populations by the current nPCR assay for the first time. Furthermore, our findings also indicate that the AMA-1 nPCR assay may be a useful diagnostic tool for the specific detection of B. bigemina. PMID:22284301

  18. Timely Closure of the Prospore Membrane Requires SPS1 and SPO77 in Saccharomyces cerevisiae.

    PubMed

    Paulissen, Scott M; Slubowski, Christian J; Roesner, Joseph M; Huang, Linda S

    2016-07-01

    During sporulation in Saccharomyces cerevisiae, a double lipid bilayer called the prospore membrane is formed de novo, growing around each meiotic nucleus and ultimately closing to create four new cells within the mother cell. Here we show that SPS1, which encodes a kinase belonging to the germinal center kinase III family, is involved in prospore membrane development and is required for prospore membrane closure. We find that SPS1 genetically interacts with SPO77 and see that loss of either gene disrupts prospore membrane closure in a similar fashion. Specifically, cells lacking SPS1 and SPO77 produce hyperelongated prospore membranes from which the leading edge protein complex is not removed from the prospore membrane in a timely fashion. The SPS1/SPO77 pathway is required for the proper phosphorylation and stability of Ssp1, a member of the leading edge protein complex that is removed and degraded when the prospore membrane closes. Genetic dissection of prospore membrane closure finds SPS1 and SPO77 act in parallel to a previously described pathway of prospore membrane closure that involves AMA1, an activator of the meiotic anaphase promoting complex. PMID:27182947

  19. Protective efficacy of a 62-kilodalton antigen, HIS-62, from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

    PubMed Central

    Gomez, F J; Gomez, A M; Deepe, G S

    1991-01-01

    We reported previously that a detergent extract of the cell wall and cell membrane of Histoplasma capsulatum yeast cells contains antigens recognized by T cells. In T-cell immunoblot analysis, a region encompassing 62 kDa was stimulatory for an H. capsulatum-reactive T-cell line and T-cell clones derived from C57BL/6 mice. In this study, we isolated a 62-kDa band, termed HIS-62, from electrophoresed cell wall and cell membrane of H. capsulatum yeast cells and examined its antigenicity and immunogenicity. C57BL/6, BALB/c, and CBA/J mice that were immunized with viable H. capsulatum yeast cells mounted a delayed-type hypersensitivity response to HIS-62 that was stronger than that of normal controls. Spleen cells from each strain of mouse immunized with viable yeast cells proliferated vigorously in response to HIS-62; conversely, splenocytes from control animals did not recognize this antigen. A T-cell line and 5 of 5 T-cell clones from C57BL/6 mice, 10 of 15 BALB/c T-cell hybridomas, and 8 of 12 CBA/J T-cell hybridomas recognized HIS-62. A cutaneous delayed-type hypersensitivity response to the antigen was apparent in each strain of mouse that was injected with 80 micrograms of HIS-62 mixed with Freund adjuvant. In addition, spleen cells from HIS-62-immunized mice proliferated in vitro in response to this antigen. Vaccination of each strain of mouse with 80 micrograms of HIS-62 conferred protection against a lethal intravenous challenge with H. capsulatum yeast cells. Thus, HIS-62 appears to be an important target of the cellular immune response to H. capsulatum and induces a protective immune response in mice. Images PMID:1937804

  20. Highly Efficient Capture and Enumeration of Low Abundance Prostate Cancer Cells Using Prostate-Specific Membrane Antigen Aptamers Immobilized to a Polymeric Microfluidic Device

    PubMed Central

    Dharmasiri, Udara; Balamurugan, Subramanian; Adams, André A.; Okagbare, Paul I.; Obubuafo, Annie; Soper, Steven A.

    2010-01-01

    Prostate tumor cells over-express a prostate specific membrane antigen (PSMA) that can be used as a marker to select these cells from highly heterogeneous clinical samples, even when found in low abundance. Antibodies and aptamers have been developed that specifically bind to PSMA. In this study, anti-PSMA aptamers were immobilized onto the surface of a capture bed poised within a poly(methyl methacrylate), PMMA, microchip, which was fabricated into a high throughput micro-sampling unit (HTMSU) used for the selective isolation of rare circulating prostate tumor cells resident in a peripheral blood matrix. The HTMSU capture bed consisted of 51 ultra-high aspect ratio parallel curvilinear channels with a width similar to the prostate cancer cell dimensions. The surface density of the PSMA-specific aptamers on a UV-modified PMMA microfluidic capture bed surface was determined to be 8.4 × 1012 molecules/cm2. Using a linear velocity for optimal cell capture in the aptamer-tethered HTMSU (2.5 mm/s), a recovery of 90% of LNCaP cells (prostate cancer cell line; used as a model in this example) was found. Due to the low abundance of these cells, the input volume required was 1 mL and this could be processed in approximately 29 min using an optimized linear flow rate of 2.5 mm/s. Captured cells were subsequently released intact from the affinity surface using 0.25% (w/v) trypsin followed by counting individual cells using a contact conductivity sensor integrated into the HTMSU that provided high detection and sampling efficiency (~100%) and did not require staining of the cells for enumeration. PMID:19722212

  1. Tyrosine motifs are required for prestin basolateral membrane targeting

    PubMed Central

    Zhang, Yifan; Moeini-Naghani, Iman; Bai, JunPing; Santos-Sacchi, Joseph; Navaratnam, Dhasakumar S.

    2015-01-01

    ABSTRACT Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that prestin targeting to the basolateral surface is dependent on AP1B (μ1B), and that prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (μ1B) in OHCs, and parallels between prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure. PMID:25596279

  2. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    PubMed Central

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-01-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity. PMID:27596047

  3. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs.

    PubMed

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-01-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity. PMID:27596047

  4. Epstein-Barr Virus Nuclear Antigen 3C Activates the Latent Membrane Protein 1 Promoter in the Presence of Epstein-Barr Virus Nuclear Antigen 2 through Sequences Encompassing an Spi-1/Spi-B Binding Site

    PubMed Central

    Zhao, Bo; Sample, Clare E.

    2000-01-01

    The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein Jκ, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. Jκ DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind Jκ activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of Jκ. Furthermore, activation of the LMP-1 promoter is a unique function of EBNA-3C, not shared by EBNA-3A and EBNA-3B. The DNA element through which EBNA-3C activates the LMP-1 promoter includes a Spi-1/Spi-B binding site, previously characterized as an important EBNA-2 response element. Although this element has considerable homology to mouse immunoglobulin light chain promoter sequences to which the mouse homologue of Spi-1 binds with its dimerization partner IRF4, we demonstrate that the IRF4-like binding sites in the LMP-1 promoter do not play a role in EBNA-3C-mediated activation. Both EBNA-2 and EBNA-3C were required for transcription mediated through a 41-bp region of the LMP-1 promoter encompassing the Spi binding site. However, EBNA-3C had no effect on transcription mediated in conjunction with the EBNA-2 activation domain fused to the GAL4 DNA-binding domain, suggesting that it does not function as an adapter between EBNA-2 and the cellular transcriptional machinery. Like EBNA-2, EBNA-3C bound directly to both Spi-1 and Spi-B in vitro. This interaction was mediated by a region of EBNA-3C encompassing a likely basic leucine zipper (bZIP) domain and the ets domain of Spi-1 or Spi-B, reminiscent of

  5. Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

    PubMed Central

    Venkatesan, M M; Buysse, J M; Oaks, E V

    1992-01-01

    An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases

  6. c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

    PubMed Central

    Polack, A; Hörtnagel, K; Pajic, A; Christoph, B; Baier, B; Falk, M; Mautner, J; Geltinger, C; Bornkamm, G W; Kempkes, B

    1996-01-01

    Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8816814

  7. Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis

    PubMed Central

    Rossi, Omar; Caboni, Mariaelena; Negrea, Aurel; Necchi, Francesca; Alfini, Renzo; Micoli, Francesca; Saul, Allan; MacLennan, Calman A.

    2016-01-01

    Invasive nontyphoidal Salmonella (iNTS) disease is a neglected disease with high mortality in children and HIV-positive individuals in sub-Saharan Africa, caused primarily by Africa-specific strains of Salmonella enterica serovars Typhimurium and Enteritidis. A vaccine using GMMA (generalized modules for membrane antigens) from S. Typhimurium and S. Enteritidis containing lipid A modifications to reduce potential in vivo reactogenicity is under development. GMMA with penta-acylated lipid A showed the greatest reduction in the level of cytokine release from human peripheral blood monocytes from that for GMMA with wild-type lipid A. Deletion of the lipid A modification genes msbB and pagP was required to achieve pure penta-acylation. Interestingly, ΔmsbB ΔpagP GMMA from S. Enteritidis had a slightly higher stimulatory potential than those from S. Typhimurium, a finding consistent with the higher lipopolysaccharide (LPS) content and Toll-like receptor 2 (TLR2) stimulatory potential of the former. Also, TLR5 ligand flagellin was found in Salmonella GMMA. No relevant contribution to the stimulatory potential of GMMA was detected even when the flagellin protein FliC from S. Typhimurium was added at a concentration as high as 10% of total protein, suggesting that flagellin impurities are not a major factor for GMMA-mediated immune stimulation. Overall, the stimulatory potential of S. Typhimurium and S. Enteritidis ΔmsbB ΔpagP GMMA was close to that of Shigella sonnei GMMA, which are currently in phase I clinical trials. PMID:26865597

  8. Synthesis and SAR of ⁹⁹mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates.

    PubMed

    Lu, Genliang; Maresca, Kevin P; Hillier, Shawn M; Zimmerman, Craig N; Eckelman, William C; Joyal, John L; Babich, John W

    2013-03-01

    Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel (99m)Tc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC(50) values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC(50)=4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({(99m)Tc(CO)(3)}(+)) to afford the {(99m)Tc(CO)(3)}(+) complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {(99m)Tc(CO)(3)}(+) radiolabeled PSMA inhibitors. PMID:23333070

  9. 68Ga-Labeled Anti-Prostate-Specific Membrane Antigen Peptide as Marker for Androgen Deprivation Therapy Response in Prostate Cancer.

    PubMed

    Schlenkhoff, Carl Diedrich; Gaertner, Florian; Essler, Markus; Hauser, Stefan; Ahmadzadehfar, Hojjat

    2016-05-01

    Prostate cancer was diagnosed in a 71-year-old man with an elevated prostate-specific antigen. The CT of the abdomen showed multiple para-aortal lymph nodes, and thus, a Ga anti-prostate-specific membrane antigen (PSMA-11) PET/CT was initiated, which showed, aside from the prostate cancer and multiple iliacal and para-aortal lymph node metastases, an increased tracer uptake in a lymph node left cervical. According to this advanced disease, a palliative therapy with GnRH agonist was initiated. A second PSMA-11 PET/CT was performed 4 months later, which showed a very good response; thus, additional radiation of the pelvis and the draining lymphatic system was performed. PMID:26859213

  10. Generation of antigen-specific Foxp3+ regulatory T-cells in vivo following administration of diabetes-reversing tolerogenic microspheres does not require provision of antigen in the formulation.

    PubMed

    Engman, Carl; Wen, Yi; Meng, Wilson S; Bottino, Rita; Trucco, Massimo; Giannoukakis, Nick

    2015-09-01

    We have developed novel antisense oligonucleotide-formulated microspheres that can reverse hyperglycemia in newly-onset diabetic mice. Dendritic cells taking up the microspheres adopt a restrained co-stimulation ability and migrate to the pancreatic lymph nodes when injected into an abdominal region that is drained by those lymph nodes. Furthermore, we demonstrate that the absolute numbers of antigen-specific Foxp3+ T regulatory cells are increased only in the lymph nodes draining the site of administration and that these T-cells proliferate independently of antigen supply in the microspheres. Taken together, our data add to the emerging model where antigen supply may not be a requirement in "vaccines" for autoimmune disease, but the site of administration - subserved by lymph nodes draining the target organ - is in fact critical to foster the generation of antigen-specific regulatory cells. The implications of these observations on "vaccine" design for autoimmunity are discussed and summarized. PMID:25773782

  11. Expression, purification and identification of an immunogenic fragment in the ectodomain of prostate-specific membrane antigen

    PubMed Central

    TAO, RONG; NI, ZHENHUA; LIU, CHONG; ZHU, MIN; JI, XIAOWEN; CHEN, XUEMIN; SHEN, JIANGFAN; TU, SHAOHUA

    2016-01-01

    The present study aimed to identify, express and purify an immunogenic fragment in the ectodomain of prostate-specific membrane antigen (PSMA) within a fusion protein. The PSMA amino acid sequence published in National Center for Biotechnology Information GenBank was used to determine sequence homology and immunogenic index analyses, additionally using BLASTN, Protean and ExPASy software to predict the polypeptide sequences of immunogenic epitopes. The gene sequence encoding the ectodomain of the polypeptide immunogenic fragments, containing the identified immunogenic epitopes, was generated using whole-gene synthesis. Prokaryotic expression vector pET-32a-r-ectodomain-PSMA was constructed and the recombinant plasmids were transformed into competent BL21 (DE3) Escherichia coli, which was followed by induction of recombinant protein expression using isopropyl-β-D-thiogalactopyranoside. Fusion proteins were isolated and purified using affinity chromatography and their immune activity was subsequently investigated using western blot analysis. Purified protein was used to immunize BALB/c mice in order to generate polyclonal antibodies, and the binding of polyclonal antibodies to prostate cancer cell lines in vitro was evaluated using flow cytometry. A total of 3 polypeptide fragments with high specificity were identified following analysis using numerous software packages, and the gene sequences encoding regions containing the 2 most immunogenic fragments were synthesized and successfully inserted into the prokaryotic expression vector pET-32a-r-ectodomain-PSMA. The recombinant PSMA protein fragment had a molecular weight of ~50 kDa and 95% purity. Western blot analysis revealed that the r-ectodomain-PSMA fusion protein specifically bound to the anti-PSMA ectodomain monoclonal antibody. Flow cytometry demonstrated that polyclonal antibodies raised against these recombinant proteins could specifically bind to PSMA-positive LNCaP cells, but not to PSMA-negative PC-3

  12. Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI.

    PubMed

    Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J

    2016-07-01

    Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd(3+) contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd(3+) binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 ± 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 ± 0.1 × 10(-22) M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM(-1) s(-1) and r2 of 37.9 mM(-1) s(-1) per Gd (55.2 and 75.8 mM(-1) s(-1) per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM(-1) s(-1) per Gd (188.0 mM(-1) s(-1) per molecule) and r1 of 18.6 mM(-1) s(-1) per Gd (37.2 mM(-1) s(-1) per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI. PMID:26961235

  13. Elevated CTLA-4 expression on CD4 T cells from periodontitis patients stimulated with Porphyromonas gingivalis outer membrane antigen

    PubMed Central

    Aoyagi, T; Yamazaki, K; Kabasawa-Katoh, Y; Nakajima, T; Yamashita, N; Yoshie, H; Hara, K

    2000-01-01

    To characterize the T cell response to Porphyromonas gingivalis, we examined the expression of costimulatory molecules on T cells derived from adult periodontitis patients with high serum antibody titre to P. gingivalis. The expression of CD28, CTLA-4, CD40 ligand (CD40L) on CD4+ T cells was analysed by flow cytometry. IL-10 and transforming growth factor-beta (TGF-β) mRNA expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent image analysis. Peripheral blood mononuclear cells (PBMC) derived from periodontitis patients showed higher proliferative responses to P. gingivalis outer membrane (OM) than those from healthy controls (P < 0.05). The percentage of CTLA-4+ cells within CD4+ T cells of patients was significantly higher than that of healthy controls after P. gingivalis OM stimulation (33.0% versus 11.9%, P < 0.01). There was no significant difference in the percentages of CD28+ cells and CD40L+ cells, and the percentage of CD40L+ cells was low in both groups even after stimulation. Stimulation of PBMC with P. gingivalis OM induced significantly higher IL-10 mRNA expression in periodontitis patients than in healthy controls (P < 0.05). The level of TGF-β mRNA expression of patients tended to be higher than that of healthy controls, but there was no significant difference. To elucidate the functional role of CTLA-4, we further investigated the secondary proliferative response to P. gingivalis OM. Interestingly, P. gingivalis OM stimulation did not enhance antigen-specific secondary response. Anti-CTLA-4 MoAb had no effect on proliferation in the presence of P. gingivalis OM. CTLA-4Ig suppressed the proliferative response significantly (P < 0.01). These results suggest that T cell responses to P. gingivalis OM may be regulated by CTLA-4 that is expressed at the late phase of T cell activation, and, in part, immunosuppressive cytokines. Taken together, CTLA-4 may play a crucial role in the pathogenesis of chronic

  14. Preclinical Evaluation of 86Y-Labeled Inhibitors of Prostate-Specific Membrane Antigen for Dosimetry Estimates

    PubMed Central

    Banerjee, Sangeeta Ray; Foss, Catherine A.; Pullambhatla, Mrudula; Wang, Yuchuan; Srinivasan, Senthamizhchelvan; Hobbs, Robert F.; Baidoo, Kwamena E.; Brechbiel, Martin W.; Nimmagadda, Sridhar; Mease, Ronnie C.; Sgouros, George; Pomper, Martin G.

    2016-01-01

    86Y (half-life = 14.74 h, 33% β+) is within an emerging class of positron-emitting isotopes with relatively long physical half-lives that enables extended imaging of biologic processes. We report the synthesis and evaluation of 3 low-molecular-weight compounds labeled with 86Y for imaging the prostate-specific membrane antigen (PSMA) using PET. Impetus for the study derives from the need to perform dosimetry estimates for the corresponding 90Y-labeled radiotherapeutics. Methods Multistep syntheses were used in preparing 86Y-4–6. PSMA inhibition constants were evaluated by competitive binding assay. In vivo characterization using tumor-bearing male mice was performed by PET/CT for 86Y-4–6 and by biodistribution studies of 86Y-4 and 86Y-6 out to 24 h after injection. Quantitative whole-body PET scans were recorded to measure the kinetics for 14 organs in a male baboon using 86Y-6. Results Compounds 86Y-4–6 were obtained in high radiochemical yield and purity, with specific radioactivities of more than 83.92 GBq/µmol. PET imaging and biodistribution studies using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor-bearing mice revealed that 86Y-4–6 had high site-specific uptake in PSMA-positive PC-3 PIP tumor starting at 20 min after injection and remained high at 24 h. Compound 86Y-6 demonstrated the highest tumor uptake and retention, with 32.17 ± 7.99 and 15.79 ± 6.44 percentage injected dose per gram (%ID/g) at 5 and 24 h, respectively. Low activity concentrations were associated with blood and normal organs, except for the kidneys, a PSMA-expressing tissue. PET imaging in baboons reveals that all organs have a 2-phase (rapid and slow) clearance, with the highest uptake (8 %ID/g) in the kidneys at 25 min. The individual absolute uptake kinetics were used to calculate radiation doses using the OLINDA/EXM software. The highest mean absorbed dose was received by the renal cortex, with 1.9 mGy per MBq of 86Y-6. Conclusion Compound 86Y-6 is a promising

  15. A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

    PubMed Central

    Kushner, Nicholas; Zhang, Dong; Touzjian, Neal; Essex, Max; Lieberman, Judy; Lu, Yichen

    2003-01-01

    Anthrax protective antigen (PA) is a 735-aa polypeptide that facilitates the exit of anthrax lethal factor (LF) from the endosome to the cytosol where the toxin acts. We recently found, however, that a fusion protein of the detoxified N-terminal domain of lethal factor (LFn) with a foreign peptide could induce CD8 T cell immune responses in the absence of PA. Because CD8 T cells recognize peptides derived from proteins degraded in the cytosol, this result suggests that lethal factor may be capable of entering the cytosol independently of PA. To investigate this further, the intracellular trafficking of an LFn-enhanced green fluorescent protein fusion protein (LFn-GFP) in the presence or absence of PA was examined by using confocal microscopy. LFn-GFP is able to enter the cytosol without PA. Moreover, it efficiently colocalizes with the proteosome 20s subunit, which degrades proteins into peptides for presentation to CD8 T cells by the MHC class I pathway. We further demonstrate that in the presence of an immune adjuvant LFn fusion protein without PA is able to effectively elicit anti-HIV cytotoxic T lymphocyte in inbred mice. These results indicate that LFn may be used without PA in a protein vaccine as a carrier to deliver antigens into the cytosol for efficient induction of T lymphocyte responses. Furthermore, these results enable us to propose a modified molecular mechanism of anthrax lethal toxin. PMID:12740437

  16. Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

    PubMed Central

    Fogg, Mark H.; Teterina, Natalya L.; Ehrenfeld, Ellie

    2003-01-01

    Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation. PMID:14557626

  17. Antigen identification using skin deficient in basement-membrane protein: a novel tool for the diagnosis of subepidermal immunobullous diseases.

    PubMed

    Rao, R; Bhogal, B; Groves, R

    2013-04-01

    Common unifying features of the subepidermal blistering diseases are the presence of tense blisters clinically and demonstration by immunofluorescence of linear deposition of immunoreactants along the dermoepidermal junction. Further characterization of subtype is possible by identification of the target antigen by immunoblotting. However, immunoblotting is time-consuming and may not be practical for routine use in the laboratory. In this report, we describe a simple technique to identify the target antigen by indirect immunofluorescence, using epidermolysis bullosa skin as substrate. PMID:23517360

  18. Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

    NASA Astrophysics Data System (ADS)

    Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

    2016-06-01

    Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high

  19. Development of Prostate Specific Membrane Antigen (PSMA) Inhibitors Coupled to 99mTc(CO)3+ with Enhanced Specific Activity for SPECT Imaging

    SciTech Connect

    Paul; D.; Benny,; Clifford; Berkman,; Jeffery; Bryan

    2011-12-20

    The overall objectives of the project were two fold: 1) the development of new facile reactions for coupling radioactive complexes with biomolecules and 2) the development of a novel molecular imaging targeting vector for Prostate Specific Membrane Antigen (PSMA) for prostate cancer. The didactic approach allowed the synergistic exploration of new technologies for coupling reactions of radioactive complexes that can be applied to a novel targeting moiety. As part of the project, a number of students (undergraduate, graduate and post-doctoral) were trained in radiochemical techniques for preparing and characterizing radiometal complexes. Results from the experiments within the project have generated several presentations and publications.

  20. Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

    PubMed

    Espósito, Danillo L A; Nguyen, Jennifer B; DeWitt, David C; Rhoades, Elizabeth; Modis, Yorgo

    2015-07-01

    Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process. PMID:25740960

  1. Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles

    PubMed Central

    Espósito, Danillo L. A.; Nguyen, Jennifer B.; DeWitt, David C.; Rhoades, Elizabeth

    2015-01-01

    Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7–20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process. PMID:25740960

  2. Methane to syngas conversion. Part I. Equilibrium conditions and stability requirements of membrane materials

    NASA Astrophysics Data System (ADS)

    Frade, J. R.; Kharton, V. V.; Yaremchenko, A.; Naumovich, E.

    Thermodynamic data have been used to predict the dependence of methane conversion on temperature and oxygen partial pressure in mixed conducting membrane reactors, and the corresponding fractions of water vapor, H 2, CO and CO 2. The relations between methane conversion, gas composition and oxygen partial pressure were also used to formulate the oxygen balance in mixed conducting membrane reactors, with tubular reactor and continuous stirred tank reactor (CSTR) configurations. A single dimensionless parameter accounts for the combined effects of geometric parameters of the membrane reactor, the permeability of the membrane material, and flow rate at the entry of the reactor. Selected examples were calculated to illustrate the effects of steam to methane and inert to methane ratios in the gas entering the reactor. The values of oxygen partial pressure required to attain the highest yield of CO and H 2 were also used to estimate the stability requirements to be met by mixed conducting membrane materials. Suitable membrane designs might be needed to bridge the difference between the conditions inside the reactors and the stability limits of known mixed conductors.

  3. The two neutrophil plasma membrane markers alkaline phosphatase and HLA class I antigen localize differently in granule-deficient cytoplasts. An ideal plasma membrane marker in human neutrophils is still lacking.

    PubMed

    Pellmé, Sara; Dahlgren, Claes; Karlsson, Anna

    2007-08-31

    Neutrophil function relies largely on the ability of the cell to mobilize its different granules and vesicles to the cell surface and thereby expose and/or release effector molecules to the surrounding tissue. To properly identify these subcellular compartments is thus a prerequisite for studies of neutrophil physiology. A range of specific markers for the classical granules is available, but finding optimal markers for the secretory vesicles and plasma membrane has historically been more challenging. Latent and non-latent alkaline phosphatase activities are often used to distinguish these two light membrane structures, but the outcome using this technique depends on the level of cellular activation. Therefore, HLA-I was introduced some years ago as a specific, stimulation-independent marker for the plasma membrane. In this study we however report that detailed fractionation studies of neutrophil cytoplasts, lacking secretory vesicles, granules and other dense organelles, reveal that the HLA-I antigen is not only co-localizing with the plasma membrane marker ALP, but is also present in other, more dense organelles. Further, we found the mixed enzyme-linked immunosorbent assay (MELISA), detecting the beta(2)-microglobulin/HLA-I complex, to be negatively influenced by uncomplexed beta(2)-microglobulin present in the specific granules and secretory vesicles, making it difficult to use HLA-I as a plasma membrane marker during maturation of for example phagolysosomes. PMID:17673253

  4. Association of a Protective Monoclonal IgA with the O Antigen of Salmonella enterica Serovar Typhimurium Impacts Type 3 Secretion and Outer Membrane Integrity

    PubMed Central

    Forbes, Stephen J.; Martinelli, Daniel; Hsieh, Chyongere; Ault, Jeffrey G.; Marko, Michael; Mannella, Carmen A.

    2012-01-01

    Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is an energetically demanding process, involving the transfer of effector proteins from invading bacteria into host cells via a specialized organelle known as the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion system (T3SS). By a mechanism that remains poorly understood, entry of S. Typhimurium into epithelial cells is inhibited by Sal4, a monoclonal, polymeric IgA antibody that binds an immunodominant epitope within the O-antigen (O-Ag) component of lipopolysaccharide. In this study, we investigated how the binding of Sal4 to the surface of S. Typhimurium influences T3SS activity, bacterial energetics, and outer membrane integrity. We found that Sal4 treatment impaired T3SS-mediated translocon formation and attenuated the delivery of tagged effector proteins into epithelial cells. Sal4 treatment coincided with a partial reduction in membrane energetics and intracellular ATP levels, possibly explaining the impairment in T3SS activity. Sal4's effects on bacterial secretion and energetics occurred concurrently with an increase in O-Ag levels in culture supernatants, alterations in outer membrane permeability, and changes in surface ultrastructure, as revealed by transmission electron microscopy and cryo-electron microscopy. We propose that Sal4, by virtue of its ability to bind and cross-link the O-Ag, induces a form of outer membrane stress that compromises the integrity of the S. Typhimurium cell envelope and temporarily renders the bacterium avirulent. PMID:22473607

  5. VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes

    PubMed Central

    Glover, Lucy; Hutchinson, Sebastian; Horn, David

    2016-01-01

    Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I–transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I–transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I–transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a “winner-takes-all” mechanism of allelic exclusion. PMID:27226299

  6. VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes.

    PubMed

    Glover, Lucy; Hutchinson, Sebastian; Alsford, Sam; Horn, David

    2016-06-28

    Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I-transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I-transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I-transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a "winner-takes-all" mechanism of allelic exclusion. PMID:27226299

  7. Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

    PubMed Central

    Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

    2012-01-01

    The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

  8. Identification of the critical attribute(s) of EBV gp350 antigen required for elicitation of a neutralizing antibody response in vivo.

    PubMed

    Servat, Esteban; Ro, Bodrey W; Cayatte, Corinne; Gemmell, Lorraine; Barton, Chris; Rao, Eileen; Lin, Rui; Zuo, Fengrong; Woo, Jennifer C; Hayes, Gregory M

    2015-11-27

    Vaccine prophylaxis with EBV glycoprotein 350 (gp350) subunit plus adjuvant has been demonstrated clinically to protect individuals against infectious mononucleosis (IM), but the specifications of the antigen required to elicit this protection has remained largely theoretical. Previous studies have shown that antibodies to gp350 comprise the principle component of EBV-neutralizing sera. Further, a murine monoclonal antibody against gp350 (clone 72A1) is able to prevent infection by the virus both in vitro and in vivo. In the present study, we identify the 72A1 epitope on recombinant gp350 antigen as the site required for binding to CD21 on human B cells. We also identify the need for conformational-dependence of the antigen to generate EBV-neutralizing antibodies in vivo. Further, we have characterized the glycosylation status and antigenicity profiles of both native and denatured CHO-produced soluble gp350 as well as non-glycosylated protein produced in Escherichia coli. Collectively our in vitro and in vivo data demonstrate the requirement for a conformationally accessible 72A1 epitope on gp350 to elicit EBV-neutralizing responses, and establish this as a critical attribute of this vaccine antigen. These data provide direction for commercial vaccine development, as the absence of this epitope on either E. coli-expressed or denatured gp350, may limit production and purification options for the antigen. PMID:26485517

  9. 5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt's lymphoma line rael.

    PubMed Central

    Masucci, M G; Contreras-Salazar, B; Ragnar, E; Falk, K; Minarovits, J; Ernberg, I; Klein, G

    1989-01-01

    Nonproductive infection of B lymphocytes by Epstein-Barr virus (EBV) is associated with a highly restricted expression of viral genes. In growth-transformed lymphoblastoid cell lines, the products of these genes include a complex of at least six EBV nuclear antigens (EBNAs) (EBNA-1 through EBNA-6) and one membrane protein (latent membrane protein [LMP]). EBV-carrying Burkitt's lymphoma (BL) biopsies and derived cell lines that have retained a representative phenotype (group I BL lines) express only EBNA-1 (M. Rowe, D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, and A. B. Rickinson, EMBO J. 6:2743-2751, 1987). We have found that EBNA-2 through EBNA-6 and LMP can be up regulated by treating the group I BL line Rael with the DNA-demethylating agent 5-azacytidine (5-AzaC). The drug acted in a time- and dose-dependent manner. EBNA-2-positive cells were detected by anti-complement immunofluorescence staining just 12 h after addition of 4 microM 5-AzaC and reached a maximum number at 72 h, when up to 75% of the population was positive. EBNA-2, EBNA-3, EBNA-4, EBNA-4, EBNA-6, and LMP were demonstrated immunoblots starting at 48 h. The EBV-encoded early antigens and viral capsid antigens were also induced but at a lower level. EBNA-2 and the lytic cycle-associated antigens appeared with a different time course and in largely nonoverlapping cell subpopulations, as demonstrated by double fluorescence staining. Thus, EBNA-2 expression was not restricted to lytically infected cells, nor was EBNA-2 required for entry into the lytic cycle. The coding and regulatory sequences of EBNA-2 and LMP were found to be highly methylated in Rael cells and were, as expected, demethylated after 5-AzaC treatment. These findings suggest that DNA methylation may participate in the regulation of growth transformation-associated viral genes in BL cells. Images PMID:2470924

  10. Chimerically fused antigen rich of overlapped epitopes from latent membrane protein 2 (LMP2) of Epstein–Barr virus as a potential vaccine and diagnostic agent

    PubMed Central

    Lin, Xiaoyun; Chen, Shao; Xue, Xiangyang; Lu, Lijun; Zhu, Shanli; Li, Wenshu; Chen, Xiangmin; Zhong, Xiaozhi; Jiang, Pengfei; Sename, Torsoo Sophia; Zheng, Yi; Zhang, Lifang

    2016-01-01

    Epstein–Barr virus (EBV) is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 (LMP2) of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte (CTL), helper T cell (Th) and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195∼232 and LMP2aa419∼436. The EBV-LMP2m protein was expressed in E. coli BL21 (DE3) after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma (NPC) patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA (60.53% sensitivity and 76.86% specificity). The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients. PMID:25864917

  11. Chimerically fused antigen rich of overlapped epitopes from latent membrane protein 2 (LMP2) of Epstein-Barr virus as a potential vaccine and diagnostic agent.

    PubMed

    Lin, Xiaoyun; Chen, Shao; Xue, Xiangyang; Lu, Lijun; Zhu, Shanli; Li, Wenshu; Chen, Xiangmin; Zhong, Xiaozhi; Jiang, Pengfei; Sename, Torsoo Sophia; Zheng, Yi; Zhang, Lifang

    2016-07-01

    Epstein-Barr virus (EBV) is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 (LMP2) of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte (CTL), helper T cell (Th) and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195∼232 and LMP2aa419∼436. The EBV-LMP2m protein was expressed in E. coli BL21 (DE3) after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma (NPC) patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA (60.53% sensitivity and 76.86% specificity). The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients. PMID:25864917

  12. Quality control of mitochondrial protein synthesis is required for membrane integrity and cell fitness

    PubMed Central

    Richter, Uwe; Lahtinen, Taina; Marttinen, Paula; Suomi, Fumi

    2015-01-01

    Mitochondrial ribosomes synthesize a subset of hydrophobic proteins required for assembly of the oxidative phosphorylation complexes. This process requires temporal and spatial coordination and regulation, so quality control of mitochondrial protein synthesis is paramount to maintain proteostasis. We show how impaired turnover of de novo mitochondrial proteins leads to aberrant protein accumulation in the mitochondrial inner membrane. This creates a stress in the inner membrane that progressively dissipates the mitochondrial membrane potential, which in turn stalls mitochondrial protein synthesis and fragments the mitochondrial network. The mitochondrial m-AAA protease subunit AFG3L2 is critical to this surveillance mechanism that we propose acts as a sensor to couple the synthesis of mitochondrial proteins with organelle fitness, thus ensuring coordinated assembly of the oxidative phosphorylation complexes from two sets of ribosomes. PMID:26504172

  13. The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds

    PubMed Central

    Fernandes, Maria Cecilia; Corrotte, Matthias; Miguel, Danilo C.; Tam, Christina; Andrews, Norma W.

    2015-01-01

    ABSTRACT The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca2+-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca2+-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targeted membrane delivery. PMID:25380822

  14. Membrane repair of human skeletal muscle cells requires Annexin-A5.

    PubMed

    Carmeille, Romain; Bouvet, Flora; Tan, Sisareuth; Croissant, Coralie; Gounou, Céline; Mamchaoui, Kamel; Mouly, Vincent; Brisson, Alain R; Bouter, Anthony

    2016-09-01

    Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin. PMID:27286750

  15. Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly.

    PubMed

    Lee, Il-Hyung; Kai, Hiroyuki; Carlson, Lars-Anders; Groves, Jay T; Hurley, James H

    2015-12-29

    The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding. PMID:26668364

  16. The requirement of membrane lymphotoxin for the presence of dendritic cells in lymphoid tissues.

    PubMed

    Wu, Q; Wang, Y; Wang, J; Hedgeman, E O; Browning, J L; Fu, Y X

    1999-09-01

    Although several cytokines, including tumor necrosis factor (TNF), can promote the growth of dendritic cells (DCs) in vitro, the cytokines that naturally regulate DC development and function in vivo have not been well defined. Here, we report that membrane lymphotoxin (LT), instead of TNF, regulates the migration of DCs in the spleen. LTalpha(-/-) mice, lacking membrane LTalpha/beta and LTalpha(3), show markedly reduced numbers of DCs in the spleen. Unlike wild-type mice and TNF(-/-) mice that have densely clustered DCs in the T cell zone and around the marginal zone, splenic DCs in LTalpha(-/-) mice are randomly distributed. The reduced number of DCs in lymphoid tissues of LTalpha(-/-) mice is associated with an increased number of DCs in nonlymphoid tissues. The number of splenic DCs in LTalpha(-/-) mice is restored when additional LT-expressing cells are provided. Blocking membrane LTalpha/beta in wild-type mice markedly diminishes the accumulation of DCs in lymphoid tissues. These data suggest that membrane LT is an essential ligand for the presence of DCs in the spleen. Mice deficient in TNF receptor, which is the receptor for both soluble LTalpha(3) and TNF-alpha(3) trimers, have normal numbers of DCs. However, LTbetaR(-/-) mice show reduced numbers of DCs, similar to the mice lacking membrane LT alpha/beta. Taken together, these results support the notion that the signaling via LTbetaR by membrane LTalpha/beta is required for the presence of DCs in lymphoid tissues. PMID:10477548

  17. A conserved amphipathic helix is required for membrane tubule formation by Yop1p

    PubMed Central

    Brady, Jacob P.; Claridge, Jolyon K.; Smith, Peter G.; Schnell, Jason R.

    2015-01-01

    The integral membrane proteins of the DP1 (deleted in polyposis) and reticulon families are responsible for maintaining the high membrane curvature required for both smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, and mutations in these proteins lead to motor neuron diseases, such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain reticulon homology domains (RHDs) that have unusually long hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilize membrane curvature. We have characterized the secondary structure and dynamics of the DP1 family protein produced from the YOP1 gene (Yop1p) and identified a C-terminal conserved amphipathic helix (APH) that, on its own, interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming APHs. Together, these results indicate that APHs play a previously unrecognized role in RHD membrane curvature stabilization. PMID:25646439

  18. CD8+ T Cell Fate and Function Influenced by Antigen-Specific Virus-Like Nanoparticles Co-Expressing Membrane Tethered IL-2

    PubMed Central

    Wojta-Stremayr, Daniela; Neunkirchner, Alina; Srinivasan, Bharani; Trapin, Doris; Schmetterer, Klaus G.; Pickl, Winfried F.

    2015-01-01

    A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here determined whether systemically toxic cytokines such as IL-2 can be restricted to the site of antigen presentation and used as ‘natural adjuvants’. Therefore, we devised antigen-presenting virus-like nanoparticles (VNP) co-expressing IL-2 attached to different membrane-anchors and assessed their potency to modulate CD8+ T cell responses in vitro and in vivo. Efficient targeting of IL-2 to lipid rafts and ultimately VNP was achieved by fusing IL-2 at its C-terminus to a minimal glycosylphosphatidylinositol (GPI)-anchor acceptor sequence. To identify optimal membrane-anchor dimensions we inserted one (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b between IL-2 and the minimal GPI-anchor acceptor sequence of CD16b (GPI). We found that the 2IgGPI version was superior to all other evaluated IL-2 variants (IL-2v) in terms of its i) degree of targeting to lipid rafts and to the VNP surface, ii) biological activity, iii) co-stimulation of cognate T cells in the absence of bystander activation and iv) potency to induce differentiation and acquisition of CD8+ T cell effector functions in vitro and in vivo. In contrast, the GPI version rather favored memory precursor cell formation. These results exemplify novel beneficial features of membrane-bound IL-2, which in addition to its mere T cell stimulatory capacity include the induction of differential effector and memory functions in CD8+ T lymphocytes. PMID:25946103

  19. Design and preclinical evaluation of a 99mTc-labelled diabody of mAb J591 for SPECT imaging of prostate-specific membrane antigen (PSMA)

    PubMed Central

    2014-01-01

    Background Sensitive and specific detection of nodal status, sites of metastases and low-volume recurrent disease could greatly improve management of patients with advanced prostate cancer. Prostate-specific membrane antigen (PSMA) is a well-established marker for prostate carcinoma with increased levels of expression in high-grade, hormone-refractory and metastatic disease. The monoclonal antibody (mAb) J591 is directed against an extracellular epitope of PSMA and has been shown to efficiently target disseminated disease including metastases in lymph nodes and bone. Its use as a diagnostic imaging agent however is limited due to its slow pharmacokinetics. In this study a diabody derived from mAb J591 was developed as a single photon emission computed tomography (SPECT) tracer with improved pharmacokinetics for the detection of PSMA expression in prostate cancer. Methods A diabody in VH-VL orientation and with a C-terminal cysteine was expressed in HEK293T cells and purified by a combination of metal ion affinity and size exclusion chromatography. Specificity and affinity were determined in cell binding studies. For SPECT imaging, the diabody was site-specifically labelled with [99mTc(CO)3]+ via the C-terminal His tag and evaluated in a subcutaneous DU145/DU145-PSMA prostate carcinoma xenograft model. Results J591C diabody binds to PSMA-expressing cells with low nanomolar affinity (3.3 ± 0.2 nM). SPECT studies allowed imaging of tumour xenografts with high contrast from 4 h post injection (p.i.). Ex vivo biodistribution studies showed peak tumour uptake of the tracer of 12.1% ± 1.7% injected dose (ID)/g at 8 h p.i. with a tumour to blood ratio of 8.0. Uptake in PSMA-negative tumours was significantly lower with 6.3% ± 0.5% at 8 h p.i. (p < 0.001). Conclusion The presented diabody has favourable properties required to warrant its further development for antibody-based imaging of PSMA expression in prostate cancer, including PSMA-specific uptake

  20. Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

    PubMed Central

    Blanco, D R; Champion, C I; Exner, M M; Shang, E S; Skare, J T; Hancock, R E; Miller, J N; Lovett, M A

    1996-01-01

    We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure. PMID:8955283

  1. Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific Ia-restricted, but not allospecific, stimulation of T lymphocytes.

    PubMed Central

    Falo, L D; Benacerraf, B; Rock, K L

    1986-01-01

    The corecognition of antigen and class II major histocompatibility complex (MHC) molecules (Ia molecules) by the T-cell receptor is a cell surface event. Before antigen is recognized, it must be taken up, processed, and displayed on the surface of an Ia-bearing accessory cell (antigen-presenting cell, APC). The exact nature of antigen processing and the subsequent associations of antigen with the APC plasma membrane, Ia molecules, and/or the T-cell receptor are not well defined. To further analyze these events, we have characterized the processing and presentation of the soluble polypeptide antigen bovine insulin. We found that this antigen requires APC-dependent processing, as evidenced by the inability of metabolically inactivated APCs to present native antigen to antigen plus Ia-specific T-T hybridomas. The ability of the same APCs to present antigen after uptake and processing showed that this antigen subsequently becomes stably associated with the APC plasma membrane. To characterize the basis for this association, we analyzed its sensitivity to enzymatic digestion. APCs exposed to antigen, treated with phospholipase A2, and then immediately fixed lost the ability to stimulate bovine insulin plus I-Ad-specific hybridomas. In contrast, the ability of these same APCs to stimulate I-Ad allospecific hybridomas was unaffected. This effect of phospholipase is not mimicked by the broadly active protease Pronase, nor is there evidence for contaminating proteases in the phospholipase preparation. These results suggest that one consequence of antigen processing may be an antigen-lipid association that contributes to the anchoring of antigen to the APC membrane. The implications of this model are discussed. PMID:3529095

  2. Targeting bacterial membranes: NMR spectroscopy characterization of substrate recognition and binding requirements of D-arabinose-5-phosphate isomerase.

    PubMed

    Airoldi, Cristina; Sommaruga, Silvia; Merlo, Silvia; Sperandeo, Paola; Cipolla, Laura; Polissi, Alessandra; Nicotra, Francesco

    2010-02-01

    Lipopolysaccharide (LPS) is an essential component of the outer membrane of gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide, and the O-antigen. The inner-core region is highly conserved and contains at least one residue of 3-deoxy-D-manno-octulosonate (Kdo). Arabinose-5-phosphate isomerase (API) is an aldo-keto isomerase catalyzing the reversible isomerization of D-ribulose-5-phosphate (Ru5P) to D-arabinose-5-phosphate (A5P), the first step of Kdo biosynthesis. By exploiting saturation transfer difference (STD) NMR spectroscopy, the structural requirements necessary for API substrate recognition and binding were identified, with the aim of designing new API inhibitors. In addition, simple experimental conditions for the STD experiments to perform a fast, robust, and efficient screening of small libraries of potential API inhibitors, allowing the identification of new potential leads, were set up. Due to the essential role of API enzymes in LPS biosynthesis and gram-negative bacteria survival, by exploiting these data, a new generation of potent antibacterial drugs could be developed. PMID:20039350

  3. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains.

    PubMed Central

    Verstreate, D R; Winter, A J

    1984-01-01

    Outer membrane proteins were solubilized from 49 strains of Brucella abortus by sequential extraction of physically disrupted cells with N-lauroylsarcosinate and a dipolar ionic detergent (Verstreate et al., Infect. Immun. 35:979-989, 1982). The strains tested included standard agglutination test strain 1119, virulent strain 2308, and eight reference strains representing each of the biotypes; the remainder were isolates from cattle in North America with natural infections and included biotypes 1, 2, and 4. Three principal protein groups with apparent molecular weights of 88,000 to 94,000 (group 1), 35,000 to 40,000 (group 2, now established as porins [Douglas et al., Infect. Immun. 44:16-21, 1984]), and 25,000 to 30,000 (group 3) were observed in every strain. Some variability in banding patterns occurred among strains, but intrastrain variation was sufficient to preclude the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of outer membrane proteins for differentiating among strains of B. abortus. One antigen ([b]) was shared among the porin proteins, and three others ([c], ([d], and ([ e]) were shared among the group 3 proteins of all of the strains tested, indicating that these relationships are probably species wide. These results suggest that it may be possible to use outer membrane proteins from a representative strain of B. abortus in a vaccine for species-wide immunization. Images PMID:6434426

  4. Membrane-localized estrogen receptor α is required for normal organ development and function.

    PubMed

    Pedram, Ali; Razandi, Mahnaz; Lewis, Michael; Hammes, Stephen; Levin, Ellis R

    2014-05-27

    Steroid receptors are found in discrete cellular locations, but it is unknown whether extranuclear pools are necessary for normal organ development. To assess this, we developed a point mutant estrogen receptor α (ERα) knockin mouse (C451A) that precludes palmitoylation and membrane trafficking of the steroid receptor in all organs. Homozygous knockin female mice (nuclear-only ERα [NOER]) show loss of rapid signaling that occurs from membrane ERα in wild-type mice. Multiple developmental abnormalities were found, including infertility, relatively hypoplastic uteri, abnormal ovaries, stunted mammary gland ductal development, and abnormal pituitary hormone regulation in NOER mice. These abnormalities were rescued in heterozygous NOER mice that were comparable to wild-type mice. mRNAs implicated in organ development were often poorly stimulated by estrogen only in homozygous NOER mice. We conclude that many organs require membrane ERα and resulting signal transduction to collaborate with nuclear ERα for normal development and function. PMID:24871949

  5. Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes.

    PubMed

    Pae, Janely; Liivamägi, Laura; Lubenets, Dmitri; Arukuusk, Piret; Langel, Ülo; Pooga, Margus

    2016-08-01

    Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides. PMID:27117133

  6. Triggered Ca2+ influx is required for extended synaptotagmin 1-induced ER-plasma membrane tethering

    PubMed Central

    Idevall-Hagren, Olof; Lü, Alice; Xie, Beichen; De Camilli, Pietro

    2015-01-01

    The extended synaptotagmins (E-Syts) are ER proteins that act as Ca2+-regulated tethers between the ER and the plasma membrane (PM) and have a putative role in lipid transport between the two membranes. Ca2+ regulation of their tethering function, as well as the interplay of their different domains in such function, remains poorly understood. By exposing semi-intact cells to buffers of variable Ca2+ concentrations, we found that binding of E-Syt1 to the PI(4,5)P2-rich PM critically requires its C2C and C2E domains and that the EC50 of such binding is in the low micromolar Ca2+ range. Accordingly, E-Syt1 accumulation at ER-PM contact sites occurred only upon experimental manipulations known to achieve these levels of Ca2+ via its influx from the extracellular medium, such as store-operated Ca2+ entry in fibroblasts and membrane depolarization in β-cells. We also show that in spite of their very different physiological functions, membrane tethering by E-Syt1 (ER to PM) and by synaptotagmin (secretory vesicles to PM) undergo a similar regulation by plasma membrane lipids and cytosolic Ca2+. PMID:26202220

  7. Crystal Structure of TDP-Fucosamine Acetyl Transferase (WECD) from Escherichia Coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

    SciTech Connect

    Hung,M.; Rangarajan, E.; Munger, C.; Nadeau, G.; Sulea, T.; Matte, A.

    2006-01-01

    Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence {yields}(3)-{alpha}-D-Fuc4NAc-(1{yields}4)-{beta}-D-ManNAcA-(1{yields}4)-{alpha}-D-GlcNAc-(1{yields}). While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-D-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Angstroms resolution and bound to acetyl-CoA at a 1.66-Angstroms resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.

  8. trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1.

    PubMed Central

    Casaz, P; Sundseth, R; Hansen, U

    1991-01-01

    Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein. Images PMID:1658359

  9. Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors.

    PubMed

    Benešová, Martina; Bauder-Wüst, Ulrike; Schäfer, Martin; Klika, Karel D; Mier, Walter; Haberkorn, Uwe; Kopka, Klaus; Eder, Matthias

    2016-03-10

    Since prostate-specific membrane antigen (PSMA) is up-regulated in nearly all stages of prostate cancer (PCa), PSMA can be considered as a viable diagnostic biomarker and treatment target in PCa. This project is focused on the development and evaluation of a series of compounds directed against PSMA. The modifications to the linker are designed to improve the binding potential and pharmacokinetics for theranostic application. In addition, the results help to further elucidate the structure-activity relationships (SAR) of the resulting PSMA inhibitors. Both in vitro and in vivo experiments of 18 synthesized PSMA inhibitor variants showed that systematic chemical modification of the linker has a significant impact on the tumor-targeting and pharmacokinetic properties. This approach can lead to an improved management of patients suffering from recurrent prostate cancer by the use of one radiolabeling precursor, which can be radiolabeled either with (68)Ga for diagnosis or with (177)Lu or (225)Ac for therapy. PMID:26878194

  10. Case with Brunsting-Perry-like localized subepidermal blister formations and immunoglobulin G antibodies against unidentified basement membrane zone antigen.

    PubMed

    Sato-Shibuya, Mami; Dainichi, Teruki; Egawa, Gyohei; Honda, Tetsuya; Otsuka, Atsushi; Ishii, Norito; Hashimoto, Takashi; Miyachi, Yoshiki; Kabashima, Kenji

    2016-04-01

    Brunsting-Perry type bullous pemphigoid is defined by the blister formation limited to the head and neck, and autoantibodies to type VII collagen are detected in several cases. However, the pathomechanisms and autoantigens in this condition remain unknown. We report a 20-year-old female patient with a more than 2-year history of recurrent tense blisters localized on the face with no distinct atrophic scar formation. The patient had neither extensive sun exposure nor a history suggestive of contact dermatitis. Oral betamethasone was effective on the skin lesions. Histopathology revealed subepidermal blister formation with dermal infiltrates of neutrophils. Although direct and indirect immunofluorescence tests detected immunoglobulin G antibodies to the basement membrane zone (BMZ), no known dermal or epidermal autoantigens were detected in immunoblot analyses. Therefore, this case may be a rare variant of Brunsting-Perry type localized bullous pemphigoid with autoantibodies to an undetermined BMZ antigen. PMID:26362108

  11. Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

    SciTech Connect

    Jhun, E.; Jhun, B.H.; Jones, L.R.; Jung, C.Y. )

    1991-05-25

    The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100{degree}C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein.

  12. Babesia divergens apical membrane antigen-1 (BdAMA-1): A poorly polymorphic protein that induces a weak and late immune response.

    PubMed

    Moreau, E; Bonsergent, C; Al Dybiat, I; Gonzalez, L M; Lobo, C A; Montero, E; Malandrin, L

    2015-08-01

    Babesiosis is an important veterinary and zoonotic tick borne disease caused by the hemoprotozoan Babesia spp. which infects red blood cell of its vertebrate host. In order to control the infection, vaccination that targets molecules involved in the invasion process of red blood cells could provide a good alternative to chemotherapy. Among these molecules, Apical Membrane Antigen-1 (AMA-1) has been described as an excellent vaccine candidate in Plasmodium spp. In this paper, we have investigated AMA-1 of Babesia divergens (BdAMA-1) as vaccine candidate by evaluating its polymorphism and by studying the humoral response against BdAMA-1 of sheep experimentally infected with B. divergens. Polymorphism of BdAMA-1 was investigated by sequencing the corresponding gene of 9 B. divergens isolates from different geographical areas in France. Two Bdama-1 haplotypes (A and B) could be defined based on 2 non-synonymous point mutations. In silico prediction of linear epitopes revealed that the antigenicity of the 2 haplotypes is very similar. Antibody production against the extracellular domain of BdAMA-1 is weak and late, between 1 and 5 months after the inoculation of parasites. Both IgG1 and IgG2 are components of the anti-BdAMA-1 response. These results indicate that while BdAMA-1 may not be an immuno-dominant antigen, it could induce a mixed type 1 and type 2 immune response. In light of these results, the potential of BdAMA-1 as vaccine candidate is discussed. PMID:25956948

  13. Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies*

    PubMed Central

    Kammerer, Robert; Rüttiger, Lukas; Riesenberg, Rainer; Schäuble, Constanze; Krupar, Rosemarie; Kamp, Annegret; Sunami, Kishiko; Eisenried, Andreas; Hennenberg, Martin; Grunert, Fritz; Bress, Andreas; Battaglia, Sebastiano; Schrewe, Heinrich; Knipper, Marlies; Schneider, Marlon R.; Zimmermann, Wolfgang

    2012-01-01

    The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16−/− mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16−/− mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range. PMID:22544735

  14. The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

    PubMed Central

    Galetto, Luciana; Bosco, Domenico; Balestrini, Raffaella; Genre, Andrea; Fletcher, Jacqueline; Marzachì, Cristina

    2011-01-01

    Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity. PMID

  15. [Influence of different products of platelet membrane glycoprotein monoclonal antibodies used internationally on tests for monoclonal antibody-specific immobilization of platelet antigens].

    PubMed

    Tang, Qiu-Min; Shen, Wei-Dong; Zhong, Zhou-Lin; Zhou, Yan; Wu, Guo-Guang

    2009-08-01

    This study was aimed to investigate the influence of different platelet membrane glycoprotein monoclonal antibodies (McAb) which are common used in laboratories on the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technique according to the request of 14th International Society of Blood Transfusion Platelet Immunology Workshop. 30 participant laboratories were provided with 10 known human platelet antigen (HPA) antibodies, 1 normal serum, 9 different McAbs (against GPIIb/IIIa, GPIa/IIa, GPIb/IX and GPIV respectively), and the same protocol. Each participant laboratory carried out the test as the protocol to compare the results of different McAbs against the same glycoprotein and submitted the data to organizer. The results indicated that in McAbs against GPIIb/IIIa, AP2, Gi-5 and PL2-73 showed higher mean S/CO than that of others; in GPIa/IIa, MBC202.2 and 143.1 showed higher mean S/CO than that of others; in GPIb/IX, 142.11 and CLB-MB45 (CD42b) showed higher mean S/CO than that of others; as to GPIV, 131.4 showed higher mean S/CO. In conclusion, capture effects of various McAbs are different, so that different products of McAbs exert influences on the sensitivity of MAIPA. To use a panel of McAbs against the same glycoprotein may avoid the false negative results. PMID:19698264

  16. Metastasis in urothelial carcinoma mimicking prostate cancer metastasis in Ga-68 prostate-specific membrane antigen positron emission tomography-computed tomography in a case of synchronous malignancy.

    PubMed

    Gupta, Manoj; Choudhury, Partha Sarathi; Gupta, Gurudutt; Gandhi, Jatin

    2016-01-01

    Prostate cancer is the second most common cancer in man. It commonly presents with urinary symptoms, bone pain, or diagnosed with elevated prostate-specific antigen.(PSA) levels. Correct staging and early diagnosis of recurrence by a precise imaging tool are the keys for optimum management. Molecular imaging of prostate cancer with Ga-68 prostate-specific membrane antigen.(PSMA), positron emission tomography-computed tomography.(PET-CT) has recently received significant attention and frequently used with a signature to prostate cancer-specific remark. However, this case will highlight the more cautious use of it. A-72-year-old male treated earlier for synchronous double malignancy.(invasive papillary urothelial carcinoma right ureter and carcinoma prostate) presented with rising PSA.(0.51.ng/ml) and referred for Ga-68 PSMA PET-CT, which showed a positive enlarged left supraclavicular lymph node. Lymph node biopsy microscopic and immunohistochemistry examination revealed metastatic carcinoma favoring urothelial origin. Specificity of PSMA scan to prostate cancer has been seen to be compromised in a certain situation mostly due to neoangiogenesis, and false positives emerged in renal cell cancer, differentiated thyroid cancer, glioblastoma, breast cancer brain metastasis, and paravertebral schwannomas. Understanding the causes of false positive will further enhance the confidence of interpretating PSMA scans. PMID:27385897

  17. Metastasis in urothelial carcinoma mimicking prostate cancer metastasis in Ga-68 prostate-specific membrane antigen positron emission tomography-computed tomography in a case of synchronous malignancy

    PubMed Central

    Gupta, Manoj; Choudhury, Partha Sarathi; Gupta, Gurudutt; Gandhi, Jatin

    2016-01-01

    Prostate cancer is the second most common cancer in man. It commonly presents with urinary symptoms, bone pain, or diagnosed with elevated prostate-specific antigen.(PSA) levels. Correct staging and early diagnosis of recurrence by a precise imaging tool are the keys for optimum management. Molecular imaging of prostate cancer with Ga-68 prostate-specific membrane antigen.(PSMA), positron emission tomography-computed tomography.(PET-CT) has recently received significant attention and frequently used with a signature to prostate cancer-specific remark. However, this case will highlight the more cautious use of it. A-72-year-old male treated earlier for synchronous double malignancy.(invasive papillary urothelial carcinoma right ureter and carcinoma prostate) presented with rising PSA.(0.51.ng/ml) and referred for Ga-68 PSMA PET-CT, which showed a positive enlarged left supraclavicular lymph node. Lymph node biopsy microscopic and immunohistochemistry examination revealed metastatic carcinoma favoring urothelial origin. Specificity of PSMA scan to prostate cancer has been seen to be compromised in a certain situation mostly due to neoangiogenesis, and false positives emerged in renal cell cancer, differentiated thyroid cancer, glioblastoma, breast cancer brain metastasis, and paravertebral schwannomas. Understanding the causes of false positive will further enhance the confidence of interpretating PSMA scans. PMID:27385897

  18. Bone Marrow Recovery and Subsequent Chemotherapy Following Radiolabeled Anti-Prostate-Specific Membrane Antigen Monoclonal Antibody J591 in Men with Metastatic Castration-Resistant Prostate Cancer

    PubMed Central

    Tagawa, Scott T.; Akhtar, Naveed H.; Nikolopoulou, Anastasia; Kaur, Gurveen; Robinson, Brian; Kahn, Renee; Vallabhajosula, Shankar; Goldsmith, Stanley J.; Nanus, David M.; Bander, Neil H.

    2013-01-01

    Radioimmunotherapy (RIT) has demonstrated efficacy with acceptable toxicity leading to approval in non-Hodgkin’s lymphoma, but has been slower to develop for the treatment of advanced solid tumors. Prostate cancer (PC) represents a good candidate for RIT based upon high exposure to circulating antibodies at common disease sites with a specific, highly expressed cell-surface antigen of prostate-specific membrane antigen. Four phase I and II trials utilizing 177Lu- or 90Y-J591 have been reported. Long-term toxicity and chemotherapy administration was analyzed. As expected, the only serious toxicity observed was myelosuppression. Grade 4 thrombocytopenia occurred in 33.3% without significant hemorrhage and grade 4 neutropenia occurred in 17.3% with 0.07% febrile neutropenia. Nearly all subjects (97.3%) recovered to grade 0 or 1 platelets and all had complete neutrophil recovery. The majority (81.3%) received chemotherapy at any time, with 61.3% receiving chemotherapy following RIT. Ten subjects underwent bone marrow biopsies at some point in their disease course following RIT for low counts; all had diffuse PC infiltration without evidence of myelodysplasia or leukemia. As expected, myelosuppression occurs following therapeutic doses of RIT for men with metastatic castration-resistant PC. However, toxicity is predictable and self-limited, with the majority of patients who do not refuse able to receive cytotoxic chemotherapy following RIT. PMID:23986881

  19. Radioimmunotherapy of Metastatic Prostate Cancer with ¹⁷⁷Lu-DOTAhuJ591 Anti Prostate Specific Membrane Antigen Specific Monoclonal Antibody.

    PubMed

    Vallabhajosula, Shankar; Nikolopoulou, Anastasia; Jhanwar, Yuliya S; Kaur, Gurveen; Tagawa, Scott T; Nanus, David M; Bander, Neil H; Goldsmith, Stanley J

    2016-01-01

    Prostate specific membrane antigen (PSMA) is the single most well-validated prostate cancer (PCa)-specific cell membrane antigen known. It is present in high levels in 95% of PCa, and is an ideal target to develop radiopharmaceuticals for imaging studies and radionuclide therapy. Humanized J591 monoclonal antibody (mAb) binds specifically with nanomolar affinity to the extracellular domain of PSMA. After binding, the PSMA-antibody complex is rapidly internalized, increasing the potential utility of PSMA as a target for the delivery of mAb-conjugated radionuclides or cytotoxins. J591 mAb was labeled with 177Lu at a high specific activity (10-30 mCi/mg) using DOTA as the bifunctional chelate. The preclinical data in PSMA positive xenografts, strongly suggested that 177;Lu-J591 mAb is an ideal radiopharmaceutical for RIT of metastatic PCa. Since October 2000, five clinical studies (phase I and II) were performed in subjects with metastatic castration-resistant prostate cancer (CRPC) using 177Lu-J591. The methodology and the results of these clinical studies are briefly reviewed in this article. The maximum tolerated dose (MTD) as a single dose was 70 mCi2. Based on dose fractionation (DF), MTD was 90 mCi/m2(2 doses of 45 mCi/m2, 2 wks apart). Phase II study in patients with progressive metastatic CRPC, at a dose of 65- 70 mCi/m2 resulted in significant PSA declines in 60% of the patients. While myelosuppression was the dose limiting toxicity, DF alone or in combination with docetaxel also resulted in significant PSA declines with much less toxicity. 177Lu imaging studies demonstrated accurate targeting of known metastatic sites in >90% of patients and those with stronger PSMA expression by semi-quantitative imaging had more PSA declines. These clinical studies clearly documented the potential therapeutic value of radioimmunotherapy (RIT) in metastatic PCa. PMID:25771365

  20. Polarized granzyme release is required for antigen-driven transendothelial migration of human effector memory CD4 T cells

    PubMed Central

    Manes, Thomas D.; Pober, Jordan S.

    2014-01-01

    Human effector memory (EM) CD4 T cells may transmigrate across endothelial cell (EC) monolayers either in response to inflammatory chemokines or in response to TCR recognition of antigen presented on the surface of the EC. The kinetics, morphologic manifestations, and molecular requirements of chemokine- and TCR-driven transendothelial migration (TEM) differ significantly. Here we report that while the MTOC and cytosolic granules follow the nucleus across the endothelium in a uropod during chemokine-driven TEM, MTOC reorientation to the contact region between the T cell and the EC, accompanied by dynein-driven transport of granzyme-containing granules to and exocytosis at the contact region, are early events in TCR-driven but not chemokine-driven TEM. Inhibitors of either granule function or of granzyme proteolytic activity can arrest TCR-driven TEM, implying a requirement for granule discharge in the process. In the final stages of TCR-driven TEM, the MTOC precedes, rather than follows, the nucleus across the endothelium. Thus TCR-driven TEM of EM CD4 T cells appears to be a novel process that more closely resembles immune synapse formation than it does conventional chemotaxis. PMID:25367116

  1. Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer.

    PubMed

    Pintscher, Sebastian; Kuleta, Patryk; Cieluch, Ewelina; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

    2016-03-25

    In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemesb The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on hemebligand mutants of cytochromebc1in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functionalin vivo This reveals that cytochromebc1can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemesbin this cytochrome and in other membranous cytochromesbact as electronic connectors for the catalytic sites with no fine tuning in ΔEm_b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential. PMID:26858251

  2. Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer*

    PubMed Central

    Pintscher, Sebastian; Kuleta, Patryk; Cieluch, Ewelina; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur

    2016-01-01

    In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc1 in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc1 can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔEm_b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential. PMID:26858251

  3. Phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular transport

    PubMed Central

    Xu, Peng; Baldridge, Ryan D.; Chi, Richard J.; Burd, Christopher G.

    2013-01-01

    Vesicle-mediated protein transport between organelles of the secretory and endocytic pathways is strongly influenced by the composition and organization of membrane lipids. In budding yeast, protein transport between the trans-Golgi network (TGN) and early endosome (EE) requires Drs2, a phospholipid translocase in the type IV P-type ATPase family. However, downstream effectors of Drs2 and specific phospholipid substrate requirements for protein transport in this pathway are unknown. Here, we show that the Arf GTPase-activating protein (ArfGAP) Gcs1 is a Drs2 effector that requires a variant of the ArfGAP lipid packing sensor (+ALPS) motif for localization to TGN/EE membranes. Drs2 increases membrane curvature and anionic phospholipid composition of the cytosolic leaflet, both of which are sensed by the +ALPS motif. Using mutant forms of Drs2 and the related protein Dnf1, which alter their ability to recognize phosphatidylserine, we show that translocation of this substrate to the cytosolic leaflet is essential for +ALPS binding and vesicular transport between the EE and the TGN. PMID:24019533

  4. A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

    NASA Astrophysics Data System (ADS)

    Kalra, Rajkumar S.; Wadhwa, Renu

    2015-02-01

    Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing & transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

  5. A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

    SciTech Connect

    Kalra, Rajkumar S. Wadhwa, Renu

    2015-02-27

    Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

  6. Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid RequirementsD⃞

    PubMed Central

    Naslavsky, Naava; Weigert, Roberto; Donaldson, Julie G.

    2004-01-01

    Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin. PMID:15146059

  7. Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

    SciTech Connect

    Lo, Yuan-Hung; Ho, Po-Chun; Chen, Min-Shan; Hugo, Eric; Ben-Jonathan, Nira; Wang, Shao-Chun

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT

  8. Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1

    PubMed Central

    Povelones, Megan L.; Tiengwe, Calvin; Gluenz, Eva; Gull, Keith; Englund, Paul T.; Jensen, Robert E.

    2016-01-01

    Summary In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knockdown of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential, and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes. PMID:23336702

  9. Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis

    PubMed Central

    Nawaratna, Sujeevi S. K.; Gobert, Geoffrey N.; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P.; Jones, Malcolm K.

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  10. Lysosome-associated membrane glycoprotein (LAMP)--preliminary study on a hidden antigen target for vaccination against schistosomiasis.

    PubMed

    Nawaratna, Sujeevi S K; Gobert, Geoffrey N; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P; Jones, Malcolm K

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16-25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  11. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection

    PubMed Central

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-01-01

    Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed. PMID:27525325

  12. A Prospective Pilot Study of 89Zr-J591/Prostate Specific Membrane Antigen Positron Emission Tomography in Men with Localized Prostate Cancer Undergoing Radical Prostatectomy

    PubMed Central

    Osborne, Joseph R.; Green, David A.; Spratt, Daniel E.; Lyashchenko, Serge; Fareedy, Shoaib B.; Robinson, Brian D.; Beattie, Bradley J.; Jain, Manu; Lewis, Jason S.; Christos, Paul; Larson, Steven M.; Bander, Neil H.; Scherr, Douglas S.

    2015-01-01

    Purpose In this pilot study we explored the feasibility of 89Zr labeled J591 monoclonal antibody positron emission tomography of localized prostate cancer. Materials and Methods Before scheduled radical prostatectomy 11 patients were injected intravenously with 89Zr-J591, followed 6 days later by whole body positron emission tomography. Patients underwent surgery the day after imaging. Specimens were imaged by ex vivo micro positron emission tomography and a custom 3 Tesla magnetic resonance scanner coil. Positron emission tomography images and histopathology were correlated. Results Median patient age was 61 years (range 47 to 68), median prostate specific antigen was 5.2 ng/ml (range 3.5 to 12.0) and median biopsy Gleason score of the 11 index lesions was 7 (range 7 to 9). On histopathology 22 lesions were identified. Median lesion size was 5.5 mm (range 2 to 21) and median Gleason score after radical prostatectomy was 7 (range 6 to 9). Eight of 11 index lesions (72.7%) were identified by in vivo positron emission tomography. Lesion identification improved with increasing lesion size for in vivo and ex vivo positron emission tomography (each p <0.0001), and increasing Gleason score (p = 0.14 and 0.01, respectively). Standardized uptake values appeared to correlate with increased Gleason score but not significantly (p = 0.19). Conclusions To our knowledge this is the first report of 89Zr-J591/prostate specific membrane antigen positron emission tomography in localized prostate cancer cases. In this setting 89Zr-J591 bound to tumor foci in situ and positron emission tomography identified primarily Gleason score 7 or greater and larger tumors, likely corresponding to clinically significant disease warranting definitive therapy. A future, larger clinical validation trial is planned to better define the usefulness of 89Zr-J591 positron emission tomography for localized prostate cancer. PMID:24135437

  13. An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DR alpha promoter and activation by SV40 T-antigen.

    PubMed Central

    Cox, P M; Goding, C R

    1992-01-01

    Constitutive expression of major histocompatibility complex class II (MHC II) antigens normally occurs in B-lymphocytes and antigen presenting cells of the monocyte/macrophage lineage. However, many malignant tumours and transformed cells express these proteins aberrantly. We demonstrate here that the MHC II DR alpha promoter is constitutively active both in the SV40 large T antigen transformed cell line, COS, and in CV1 cells from which they are derived. As an approach to understanding the molecular mechanisms underlying aberrant DR alpha expression we have examined the cis- and trans-acting requirements for DR alpha transcription in these cell types. Electrophoretic mobility shift assays showed that the region immediately 3' to the X-box was bound by a member of the ATF/CREB family of transcription factors. Using deletions and point mutations in the DR alpha promoter we demonstrate that, in contrast to B-cells, the octamer motif and conserved X- and Y-boxes make only a minor contribution to promoter function while single point mutations in the ATF/CREB motif reduced transcription up to 20-fold. In addition, we show that the DR alpha promoter is activated by SV40 large T-antigen and that activation requires an intact ATF/CREB motif. Similar data were obtained using B16 melanoma cells. These results suggest that the ATF/CREB motif may be a target for transcription deregulation in several transformed cell types. Images PMID:1329030

  14. Native bivalvular endocarditis by Gemella haemolysans requiring venovenous extracorporeal membrane oxygenation.

    PubMed

    Ando, Akika; Kagihara, Jaclyn; Chung, Heath; Bolger, Dennis Thomas

    2016-01-01

    A 24-year-old otherwise healthy man presented with a 3-week history of malaise, headache, fever and rigors after he was treated with oral clindamycin for left parotitis and Gemella haemolysans bacteraemia. He developed G. haemolysans infective endocarditis, septic emboli and heart failure due to progressive bivalvular disease. He underwent urgent mechanical aortic valve replacement and mitral valve repair, which required venovenous extracorporeal membrane oxygenation, to support severe respiratory failure. This is the first documented case of G. haemolysans infective endocarditis affecting native aortic and mitral valves in a healthy adult. PMID:27539135

  15. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae.

    PubMed

    Okumura, Yuuya; Nakamura, Tsuyoshi S; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong; Tachikawa, Hiroyuki

    2016-01-01

    Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation

  16. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae

    PubMed Central

    Okumura, Yuuya; Nakamura, Tsuyoshi S.; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong

    2015-01-01

    ABSTRACT Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall

  17. A Chemical Genetics Approach Reveals H,K-ATPase-Mediated Membrane Voltage is Required for Planarian Head Regeneration

    PubMed Central

    Beane, Wendy Scott; Morokuma, Junji; Adams, Dany Spencer; Levin, Michael

    2012-01-01

    SUMMARY Biophysical signaling is required for both embryonic polarity and regenerative outgrowth. Exploiting endogenous ion transport for regenerative therapies will require direct regulation of membrane voltage. Here, we develop a pharmacological method to target ion transporters, uncovering a novel role for membrane voltage as a key regulator of anterior polarity in regenerating planaria. Utilizing the highly specific inhibitor, SCH-28080, our data reveal that H+,K+-ATPase-mediated membrane depolarization is essential for anterior gene expression and brain induction. H+,K+-ATPase-independent manipulation of membrane potential with ivermectin confirms that depolarization drives head formation, even at posterior-facing wounds. Using this chemical genetics approach, we demonstrate that membrane voltage controls head-vs.-tail identity during planarian regeneration. Our data suggest well-characterized drugs (already approved for human use) might be exploited to control adult stem cell-driven pattern formation during the regeneration of complex structures. PMID:21276941

  18. Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp.

    PubMed Central

    Velasco, J; Díaz, R; Grilló, M J; Barberán, M; Marín, C; Blasco, J M; Moriyón, I

    1997-01-01

    Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B. melitensis brucellin. O. anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B. melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O. anthropi. No antibodies to O. anthropi cytosolic proteins were detected in the sera of Brucella-free hosts. Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O. anthropi proteins of similar molecular weight. No IgG to the O-specific polysaccharide of O. anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts. The sera of sheep, goats, and rabbits infected with B. melitensis contained IgG to O. anthropi rough lipopolysaccharide and lipid A, and B. ovis and O. anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B. ovis-infected rams. The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O. anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals. PMID:9144364

  19. RipA, a cytoplasmic membrane protein conserved among Francisella species, is required for intracellular survival.

    PubMed

    Fuller, James R; Craven, Robin R; Hall, Joshua D; Kijek, Todd M; Taft-Benz, Sharon; Kawula, Thomas H

    2008-11-01

    Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS Delta ripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS Delta ripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS Delta ripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence. PMID:18765722

  20. Transporter associated with antigen processing-like (ABCB9) stably expressed in Chinese hamster ovary-K1 cells is sorted to the microdomains of lysosomal membranes.

    PubMed

    Fujimoto, Yasuyuki; Kamakura, Aya; Motohashi, Yu; Ohashi-Kobayashi, Ayako; Maeda, Masatomo

    2011-01-01

    The carboxyl terminus of a human ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL), was tagged with green fluorescence protein (GFP), and the resulting fusion protein (TAPL-GFP) was stably expressed in Chinese hamster ovary (CHO)-K1 cells. The GFP signal was co-localized with that of LysoTracker but not that of MitoTracker, as visualized under a microscope. TAPL-GFP was co-sedimented with lysosomal marker cathepsin D on Percoll density gradient centrifugation. These results indicated that TAPL is a lysosomal ABC transporter but not a mitochondrial one. It was not solubilized completely with a non-ionic detergent under ice-cold conditions, and was co-sedimented with flotillin-1 on sucrose density gradient centrifugation. A similar result was obtained with high pH-treatment. Furthermore, treatment with methyl-β-cyclodextrin resulted in an altered distribution of TAPL-GFP. These results suggest that TAPL may be localized to the microdomains (lipid rafts) of lysosomal membranes enriched in cholesterol. PMID:21212514

  1. Computational Prediction and Identification of Epstein-Barr Virus Latent Membrane Protein 2A Antigen-Specific CD8+ T-Cell Epitopes

    PubMed Central

    Wang, Bing; Yao, Kun; Liu, Genyan; Xie, Fangyi; Zhou, Feng; Chen, Yun

    2009-01-01

    Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC) is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A (LMP2A) is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three (LMP2A264-272, LMP2A426-434 and LMP2A356-364) of six peptides respectively showed that the numbers of spots forming cells (SFC) ranged from 55.7 to 80.6 SFC/5 × 104 CD8+ T cells and the responding index (RI) ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2-expressing target cells. As a result, LMP2A264-272 (QLSPLLGAV), LMP2A426-434 (CLGGLLTMV) and LMP2A356-364 (FLYALALLL) were identified as LMP2A-specific CD8+ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC. PMID:19403058

  2. Structure-Activity Relationship of (18)F-Labeled Phosphoramidate Peptidomimetic Prostate-Specific Membrane Antigen (PSMA)-Targeted Inhibitor Analogues for PET Imaging of Prostate Cancer.

    PubMed

    Dannoon, Shorouk; Ganguly, Tanushree; Cahaya, Hendry; Geruntho, Jonathan J; Galliher, Matthew S; Beyer, Sophia K; Choy, Cindy J; Hopkins, Mark R; Regan, Melanie; Blecha, Joseph E; Skultetyova, Lubica; Drake, Christopher R; Jivan, Salma; Barinka, Cyril; Jones, Ella F; Berkman, Clifford E; VanBrocklin, Henry F

    2016-06-23

    A series of phosphoramidate-based prostate specific membrane antigen (PSMA) inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorine-18 analogs were evaluated for use as positron emission tomography (PET) imaging agents for prostate cancer. To gain insight into their modes of binding, they were also cocrystallized with the extracellular domain of PSMA. All analogs exhibited irreversible binding to PSMA with IC50 values ranging from 0.4 to 1.3 nM. In vitro assays showed binding and rapid internalization (80-95%, 2 h) of the radiolabeled ligands in PSMA(+) cells. In vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with tumor to blood ratios of 25.6:1, 63.6:1, and 69.6:1 for [(18)F]4, [(18)F]5, and [(18)F]6, respectively, at 2 h postinjection. Installation of aminohexanoic acid (AH) linkers in the phosphoramidate scaffold improved their PSMA binding and inhibition and was critical for achieving suitable in vivo imaging properties, positioning [(18)F]5 and [(18)F]6 as favorable candidates for future prostate cancer imaging clinical trials. PMID:27228467

  3. Initial Evaluation of [18F]DCFPyL for Prostate-Specific Membrane Antigen (PSMA)-Targeted PET Imaging of Prostate Cancer

    PubMed Central

    Szabo, Zsolt; Mena, Esther; Rowe, Steven P.; Plyku, Donika; Nidal, Rosa; Eisenberger, Mario A.; Antonarakis, Emmanuel S.; Fan, Hong; Dannals, Robert F.; Chen, Ying; Mease, Ronnie C.; Vranesic, Melin; Bhatnagar, Akrita; Sgouros, George; Cho, Steve Y.; Pomper, Martin G.

    2015-01-01

    Purpose Prostate-specific membrane antigen (PSMA) is a recognized target for imaging prostate cancer. Here we present initial safety, biodistribution, and radiation dosimetry results with [18F]DCFPyL, a second-generation fluorine-18-labeled small-molecule PSMA inhibitor, in patients with prostate cancer. Procedures Biodistribution was evaluated using sequential positron-emission tomography (PET) scans in nine patients with prostate cancer. Time-activity curves from the most avid tumor foci were determined. The radiation dose to selected organs was estimated using OLINDA/EXM. Results No major radiotracer-specific adverse events were observed. Physiologic accumulation was observed in known sites of PSMA expression. Accumulation in putative sites of prostate cancer was observed (SUVmax up to >100, and tumor-to-blood ratios up to >50). The effective radiation dose from [18F]DCFPyL was 0.0139 mGy/MBq or 5 mGy (0.5 rem) from an injected dose of 370 MBq (10 mCi). Conclusions [18F]DCFPyL is safe with biodistribution as expected, and its accumulation is high in presumed primary and metastatic foci. The radiation dose from [18F]DCFPyL is similar to that from other PET radiotracers. PMID:25896814

  4. Multilevel Precision-Based Rational Design of Chemical Inhibitors Targeting the Hydrophobic Cleft of Toxoplasma gondii Apical Membrane Antigen 1 (AMA1)

    PubMed Central

    Muralikumar, Shalini; Mahalakshmi, B; Lily Therese, K; Madhavan, HN; Alameen, Mohamed; Thirumudi, Indhuja

    2016-01-01

    Toxoplasma gondii is an intracellular Apicomplexan parasite and a causative agent of toxoplasmosis in human. It causes encephalitis, uveitis, chorioretinitis, and congenital infection. T. gondii invades the host cell by forming a moving junction (MJ) complex. This complex formation is initiated by intermolecular interactions between the two secretory parasitic proteins—namely, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) and is critically essential for the host invasion process. By this study, we propose two potential leads, NSC95522 and NSC179676 that can efficiently target the AMA1 hydrophobic cleft, which is a hotspot for targeting MJ complex formation. The proposed leads are the result of an exhaustive conformational search-based virtual screen with multilevel precision scoring of the docking affinities. These two compounds surpassed all the precision levels of docking and also the stringent post docking and cumulative molecular dynamics evaluations. Moreover, the backbone flexibility of hotspot residues in the hydrophobic cleft, which has been previously reported to be essential for accommodative binding of RON2 to AMA1, was also highly perturbed by these compounds. Furthermore, binding free energy calculations of these two compounds also revealed a significant affinity to AMA1. Machine learning approaches also predicted these two compounds to possess more relevant activities. Hence, these two leads, NSC95522 and NSC179676, may prove to be potential inhibitors targeting AMA1-RON2 complex formation towards combating toxoplasmosis. PMID:27445648

  5. Multilevel Precision-Based Rational Design of Chemical Inhibitors Targeting the Hydrophobic Cleft of Toxoplasma gondii Apical Membrane Antigen 1 (AMA1).

    PubMed

    Vetrivel, Umashankar; Muralikumar, Shalini; Mahalakshmi, B; Lily Therese, K; Madhavan, H N; Alameen, Mohamed; Thirumudi, Indhuja

    2016-06-01

    Toxoplasma gondii is an intracellular Apicomplexan parasite and a causative agent of toxoplasmosis in human. It causes encephalitis, uveitis, chorioretinitis, and congenital infection. T. gondii invades the host cell by forming a moving junction (MJ) complex. This complex formation is initiated by intermolecular interactions between the two secretory parasitic proteins-namely, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) and is critically essential for the host invasion process. By this study, we propose two potential leads, NSC95522 and NSC179676 that can efficiently target the AMA1 hydrophobic cleft, which is a hotspot for targeting MJ complex formation. The proposed leads are the result of an exhaustive conformational search-based virtual screen with multilevel precision scoring of the docking affinities. These two compounds surpassed all the precision levels of docking and also the stringent post docking and cumulative molecular dynamics evaluations. Moreover, the backbone flexibility of hotspot residues in the hydrophobic cleft, which has been previously reported to be essential for accommodative binding of RON2 to AMA1, was also highly perturbed by these compounds. Furthermore, binding free energy calculations of these two compounds also revealed a significant affinity to AMA1. Machine learning approaches also predicted these two compounds to possess more relevant activities. Hence, these two leads, NSC95522 and NSC179676, may prove to be potential inhibitors targeting AMA1-RON2 complex formation towards combating toxoplasmosis. PMID:27445648

  6. Development and screening of a series of antibody-conjugated and silica coated iron-oxide nanoparticles for targeting the Prostate Specific Membrane Antigen

    PubMed Central

    Mukherjee, Amarnath; Darlington, Thomas; Baldwin, Richard; Holz, Charles; Olson, Sage; Kulkarni, Prakash; DeWeese, Theodore L.; Getzenberg, Robert H.; Ivkov, Robert

    2014-01-01

    The Prostate Specific Membrane Antigen (PSMA) is an established target for the delivery of cancer therapeutic and imaging agents due to its high expression on the surface of prostate cancer cells and within the neovasculature of other solid tumors. Here we describe the synthesis and screening of antibody-conjugated silica-coated iron oxide nanoparticles for PSMA-specific cell targeting. The humanized anti-PSMA antibody, HuJ591, was conjugated to a series of nanoparticles with varying densities of polyethylene glycol and primary amine groups. Customized assays utilizing iron spectral absorbance and Enzyme-Linked Immunoassay (ELISA) were developed to screen microgram quantities of nanoparticle formulations for immunoreactivity and cell targeting ability. Antibody and PSMA-specific targeting of the optimized nanoparticle was evaluated using an isogenic PSMA-positive and PSMA-negative cell line pair. Specific nanoparticle targeting was confirmed by iron quantification with inductively coupled plasma mass spectrometry (ICP-MS). These methods and nanoparticles support the promise of targeted theranostic agents for future treatment of prostate and other cancers. PMID:24591351

  7. [(18)F]Fluorobenzoyllysinepentanedioic Acid Carbamates: New Scaffolds for Positron Emission Tomography (PET) Imaging of Prostate-Specific Membrane Antigen (PSMA).

    PubMed

    Yang, Xing; Mease, Ronnie C; Pullambhatla, Mrudula; Lisok, Ala; Chen, Ying; Foss, Catherine A; Wang, Yuchuan; Shallal, Hassan; Edelman, Hannah; Hoye, Adam T; Attardo, Giorgio; Nimmagadda, Sridhar; Pomper, Martin G

    2016-01-14

    Radiolabeled urea-based low-molecular weight inhibitors of the prostate-specific membrane antigen (PSMA) are under intense investigation as imaging and therapeutic agents for prostate and other cancers. In an effort to provide agents with less nontarget organ uptake than the ureas, we synthesized four (18)F-labeled inhibitors of PSMA based on carbamate scaffolds. 4-Bromo-2-[(18)F]fluorobenzoyllysineoxypentanedioic acid (OPA) carbamate [(18)F]23 and 4-iodo-2-[(18)F]fluorobenzoyllysine OPA carbamate [(18)F]24 in particular exhibited high target-selective uptake in PSMA+ PC3 PIP tumor xenografts, with tumor-to-kidney ratios of >1 by 4 h postinjection, an important benchmark. Because of its high tumor uptake (90% injected dose per gram of tissue at 2 h postinjection) and high tumor-to-organ ratios, [(18)F]23 is promising for clinical translation. Prolonged tumor-specific uptake demonstrated by [(18)F]24, which did not reach equilibrium during the 4 h study period, suggests carbamates as alternative scaffolds for mitigating dose to nontarget tissues. PMID:26629713

  8. Review of Salvage Therapy for Biochemically Recurrent Prostate Cancer: The Role of Imaging and Rationale for Systemic Salvage Targeted Anti-Prostate-Specific Membrane Antigen Radioimmunotherapy

    PubMed Central

    Kosuri, Satyajit; Akhtar, Naveed H.; Smith, Michael; Osborne, Joseph R.; Tagawa, Scott T.

    2012-01-01

    Despite local therapy with curative intent, approximately 30% of men suffer from biochemical relapse. Though some of these PSA relapses are not life threatening, many men eventually progress to metastatic disease and die of prostate cancer. Local therapy is an option for some men, but many have progression of disease following local salvage attempts. One significant issue in this setting is the lack of reliable imaging biomarkers to guide the use of local salvage therapy, as the likely reason for a low cure rate is the presence of undetected micrometastatic disease outside of the prostate/prostate bed. Androgen deprivation therapy is a cornerstone of therapy in the salvage setting. While subsets may benefit in terms of delay in time to metastatic disease and/or death, research is ongoing to improve salvage systemic therapy. Prostate-specific membrane antigen (PSMA) is highly overexpressed by the majority of prostate cancers. While initial methods of exploiting PSMA's high and selective expression were suboptimal, additional work in both imaging and therapeutics is progressing. Salvage therapy and imaging modalities in this setting are briefly reviewed, and the rationale for PSMA-based systemic salvage radioimmunotherapy is described. PMID:22693495

  9. Effect of Chelators on the Pharmacokinetics of 99mTc-Labeled Imaging Agents for the Prostate-Specific Membrane Antigen (PSMA)

    PubMed Central

    Banerjee, Sangeeta Ray; Pullambhatla, Mrudula; Foss, Catherine A.; Falk, Alexander; Byun, Youngjoo; Nimmagadda, Sridhar; Mease, Ronnie C.; Pomper, Martin G.

    2013-01-01

    Technetium-99m, the most commonly used radionuclide in nuclear medicine, can be attached to biologically important molecules through a variety of chelating agents, the choice of which depends upon the imaging application. The prostate-specific membrane antigen (PSMA) is increasingly recognized as an important target for imaging and therapy of prostate cancer (PCa). Three different 99mTc-labeling methods were employed to investigate the effect of the chelator on the biodistribution and PCa tumor uptake profiles of 12 new urea based PSMA-targeted radiotracers. This series includes hydrophilic ligands for radiolabeling with the [99mTc(CO)3]+ core (L8-10), traditional NxSy-based chelating agents with varying charge and polarity for the 99mTc-oxo core (L11-18), and a 99mTc-organohydrazine-labeled radioligand (L19). 99mTc(I)-Tricarbonyl-labeled [99mTc]L8 produced the highest PSMA+ PC3 PIP to PSMA− PC3 flu tumor ratios, and demonstrated the lowest retention in normal tissues including kidney after 2 h. These results suggest that choice of chelator is an important pharmacokinetic consideration in the development of 99mTc-labeled radiopharmaceuticals targeting PSMA. PMID:23799782

  10. Secretion of nuclease across the outer membrane of Serratia marcescens and its energy requirements.

    PubMed Central

    Suh, Y; Benedik, M J

    1997-01-01

    Extracellular secretion of Serratia marcescens nuclease occurs as a two-step process via a periplasmic intermediate. Unlike other extracellular proteins secreted by gram-negative bacteria by the general secretory pathway, nuclease accumulates in the periplasm in its active form for an unusually long time before its export into the growth medium. The energy requirements for extracellular secretion of nuclease from the periplasm were investigated. Our results suggest that the second step of secretion across the outer membrane is dependent upon the external pH; acidic pH effectively but reversibly blocks extracellular secretion. However, electrochemical proton gradient, and possibly ATP hydrolysis, are not required for this step. We suggest that nuclease uses a novel mechanism for the second step of secretion in S. marcescens. PMID:9006020

  11. Novel Mutants Define Genes Required for the Expression of Human Histocompatibility Leukocyte Antigen DM: Evidence for Loci on Human Chromosome 6p

    PubMed Central

    Fling, Steven P.; Rak, Jennifer; Muczynski, Kimberly A.; Arp, Benjamin; Pious, Donald

    1997-01-01

    We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II–associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II–peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM null mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II–restricted antigen processing. PMID:9348304

  12. Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes

    PubMed Central

    1996-01-01

    In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis- Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed. PMID:8636207

  13. Immunomodulatory Glycan Lacto-N-Fucopentaose III Requires Clathrin-Mediated Endocytosis To Induce Alternative Activation of Antigen-Presenting Cells

    PubMed Central

    Srivastava, Leena; Tundup, Smanla; Choi, Beak-San; Norberg, Thomas

    2014-01-01

    The mechanism of alternative activation of antigen-presenting cells (APCs) is largely unknown. Lacto-N-fucopentaose III (LNFPIII) is a biologically conserved pentasaccharide that contains the Lewisx trisaccharide. LNFPIII conjugates and schistosome egg antigens, which contain the Lewisx trisaccharide, drive alternative activation of APCs and induce anti-inflammatory responses in vivo, preventing inflammation-based diseases, including psoriasis, transplant organ rejection, and metabolic disease. In this study, we show that LNFPIII conjugates and schistosome egg antigens interact with APCs via a receptor-mediated process, requiring internalization of these molecules through a clathrin/dynamin-dependent but caveolus-independent endocytic pathway. Using inhibitors/small interfering RNA (siRNA) against dynamin and clathrin, we show for the first time that endocytosis of Lewisx-containing glycans is required to drive alternative maturation of antigen-presenting cells and Th2 immune responses. We identified mouse SIGNR-1 as a cell surface receptor for LNFPIII conjugates. Elimination of SIGNR-1 showed no effect on uptake of LNFPIII conjugates, suggesting that other receptors bind to and facilitate uptake of LNFPIII conjugates. We demonstrate that disruption of actin filaments partially prevented the entry of LNFPIII conjugates into APCs and that LNFPIII colocalizes with both early and late endosomal markers and follows the classical endosomal pathway leading to lysosome maturation. The results of this study show that the ability of LNFPIII to induce alternative activation utilizes a receptor-mediated process that requires a dynamin-dependent endocytosis. Thus, key steps have been defined in the previously unknown mechanism of alternative activation that ultimately leads to induction of anti-inflammatory responses. PMID:24566617

  14. Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.

    PubMed

    Chawla, Akhil; Alatrash, Gheath; Philips, Anne V; Qiao, Na; Sukhumalchandra, Pariya; Kerros, Celine; Diaconu, Iulia; Gall, Victor; Neal, Samantha; Peters, Haley L; Clise-Dwyer, Karen; Molldrem, Jeffrey J; Mittendorf, Elizabeth A

    2016-06-01

    Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response. PMID:27129972

  15. High-Level Expression, Purification and Characterization of A Recombinant Plasmodium vivax Apical Membrane Antigen 1: Implication for vivax Malaria Vaccine Development

    PubMed Central

    Salavatifar, Maryam; Zakeri, Sedigheh; Hayati Roodbari, Nasim; Djadid, Navid Dinparast

    2015-01-01

    Objective The apical membrane antigen-1 (AMA-1) is considered as a promising candidate for development of a malaria vaccine against Plasmodium parasites. The correct conformation of this protein appears to be necessary for the stimulation of parasite-inhibitory responses, and these responses, in turn, seem to be antibody-mediated. Therefore, in the present investigation, we expressed the Plasmodium vivax AMA-1 (PvAMA-1) ectodomain in Escherichia coli (E. coli), purified it using standard procedures and characterized it to determine its biological activities for it to be used as a potential target for developing a protective and safe vivax malaria vaccine. Materials and Methods In this experimental investigation, the ectodomain of PvAMA-1 antigen (GenBank accession no. JX624741) was expressed in the E. coli M15pQE30 expression system and purified with immobilized-metal affinity chromatography. The correct conformation of the recombinant protein was evaluated by Western blotting and indirect immunofluorescence antibody (IFA) test. In addition, the immunogenic properties of PvAMA-1 were evaluated in BALB/c mice with the purified protein emulsified in Freund’s adjuvant. Results In the present study, the PvAMA-1 ectodomain was expressed at a high-level (65 mg/L) using a bacterial system. Reduced and non-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as Western blot analysis confirmed the appropriate conformation and folding of PvAMA-1. The evaluation of immunogenic properties of PvAMA-1 showed that both T helper-1 and 2 cells (Th1 and Th2) responses were present in mice after three immunizations and persisted up to one year after the first immunization. Moreover, the antibodies raised against the recombinant PvAMA-1 in injected mice could recognize the native protein localized on P. vivax parasites. Conclusion We demonstrate that our recombinant protein had proper conformation and folding. Also, there were common epitopes in the

  16. A Multivalent Approach of Imaging Probe Design to Overcome an Endogenous Anion Binding Competition for Noninvasive Assessment of Prostate Specific Membrane Antigen

    PubMed Central

    Hao, Guiyang; Kumar, Amit; Dobin, Timothy; Öz, Orhan K.; Hsieh, Jer-Tsong; Sun, Xiankai

    2013-01-01

    2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid) (GPI) is a highly potent inhibitor of prostate specific membrane antigen (PSMA) with a rapid in vivo clearance profile from non-target organs including kidneys, but its use for imaging of PSMA is blocked by an endogenous anion (serum phosphate) competition, which compromises its specific binding to the antigen. Multi-presentation of a targeting molecule on a single entity has been recognized as a practical way for imaging sensitivity enhancement. Herein, we demonstrate a multivalent approach based on a 64Cu-specific bifunctional chelator scaffold to overcome the endogenous phosphate competition thus enabling the utility of GPI conjugates for in vivo detection of PSMA and imaging quantification. Both monomeric (H2CBT1G) and dimeric (H2CBT2G) conjugates were synthesized and labeled with 64Cu for in vitro and in vivo evaluations. A 4-fold enhancement of PSMA binding affinity was observed for H2CBT2G as compared to H2CBT1G from the PSMA competitive binding assays performed on LNCaP cells. In vivo PET imaging studies were conducted on mouse xenograft models established with a PSMA+ cell line, LNCaP, and PSMA− PC3 and H2009 cell lines. 64Cu-CBT2G showed significantly higher LNCaP tumor uptake than 64Cu-CBT1G at 1, 4, and 24 h post-injection (p.i.) (p < 0.05). In addition, tumor uptake of 64Cu-CBT2G remained steady out to 24 h p.i. (1.46 ± 0.54, 1.12 ± 0.56, and 1.00 ± 0.50 %ID/g at 1, 4 and 24 h p.i., respectively), while 64Cu-CBT1G showed a great decrease from 1 h to 4 h p.i. The PSMA imaging specificity of both H2CBT1G and H2CBT2G was demonstrated by their low uptake in PSMA− tumors (PC3 and H2009) and further confirmed by a significant signal reduction in PSMA+ LNCaP tumors in the blockade study. In addition, the LNCaP tumor uptake (%ID/g) of 64Cu-CBT2G was found to be in a positive linear correlation with the tumor size (R2 = 0.92, 0.94, and 0.93 for 1 h, 4 h, and 24 h p.i.). This

  17. Antigen and epitope specificity of anti-glomerular basement membrane antibodies in patients with goodpasture disease with or without anti-neutrophil cytoplasmic antibodies.

    PubMed

    Yang, Rui; Hellmark, Thomas; Zhao, Juan; Cui, Zhao; Segelmark, Marten; Zhao, Ming-Hui; Wang, Hai-Yan

    2007-04-01

    Goodpasture disease (GP) is defined by the presence of anti-glomerular basement membrane (anti-GBM) antibodies and rapidly progressive glomerulonephritis. Besides anti-GBM, many patients with GP produce anti-neutrophil cytoplasmic antibodies (ANCA). For elucidation of the pathophysiologic significance of ANCA in this setting, epitope and antigen specificity of the anti-GBM antibodies and antigen specificity of ANCA were studied. Bovine testis alpha(IV)NC1 (tNC1); recombinant human alpha1, alpha3, alpha4, and alpha5(IV)NC1 (ralpha1 through ralpha5); and three chimeric proteins that contain previously defined epitope regions designated E(A), E(B), and S2 were used to examine the anti-GBM antibodies by ELISA in 205 Chinese patients with GP with or without ANCA. In the 205 anti-GBM antibody-positive sera, 63 (30.7%) were also ANCA positive (61 myeloperoxidase-ANCA and six proteinase 3-ANCA, four being triple positive). All 205 sera recognized tNC1 and ralpha3(IV)NC1. In the double-positive group, 54.0, 66.7, 71.4% of the sera could recognize ralpha1, ralpha4, and ralpha5, respectively, compared with 49.3, 60.6, and 55.6% for patients with anti-GBM antibodies alone. The levels of the antibodies to ralpha3, tNC1, and the alpha3/alpha1 ratio were lower in the double-positive group than that in patients with anti-GBM antibody alone (P < 0.05). Most of the sera could recognize the epitope regions E(A), E(B), and S2, but the absorbance values to E(A), E(B), and S2 were lower in double-positive group (P < 0.05). Double-positive patients had a broader spectrum of anti-GBM antibodies and lower levels of antibodies against alpha3(IV)NC1 compared with that of patients with anti-GBM antibodies alone. PMID:17329569

  18. Physical Property Requirements of Ion-exchange Polymer Membranes for Acid-base Flow Batteries

    NASA Astrophysics Data System (ADS)

    Roddecha, Supacharee; Thayer, Peter; Jorne', Jacob; Anthamatten, Mitchell

    2013-03-01

    Flow batteries offer feasible solutions to grid-scale storage of intermittent power. We are developing a new type of flow battery that reversibly controls an acid-base neutralization reaction. The battery consists of two highly reversible hydrogen gas electrodes that are exposed to low and high pH process streams. A brine solution runs between the acid and base streams and is separated by cationic and anionic exchange membranes. For both charge and discharge phases, hydrogen gas is produced at one electrode and consumed at the other. During charging, an external potential is applied across the two electrodes to electrochemically produce acid and base from the fed brine solution. Discharge involves electrochemical neutralization of acid and base streams, resulting in current flow through an external load. Several charge and discharge cycles were performed to demonstrate proof of concept. Experiments were conducted to determine the physical property requirements of the ionic exchange polymer layers. Properties including ion conductivity, permselectivity, and membrane stability will be discussed.

  19. Properties required by extractants and diluents for the decontamination of liquid wastes using supported liquid membranes

    SciTech Connect

    Dozol, J.F.; Rouquette, H.; Eymard, S.; Tournois, B.

    1993-12-31

    Macrocyclic extractants are now being studied more and more often for the decontamination of radioactive liquid wastes: coronands (crown ethers, azacrown...) and cryptands. As these very sophisticated compounds are expensive, the best technique is supported liquid membranes which need a very low extractant inventory. This paper deals with the properties required by the extractant and the diluent in order to be used in an SLM device and to ensure a stable and efficient SLM: solubility of the extractant in organic compounds and in aqueous solutions; size of crown ether cavities; influence of the substituent groups on the selectivity of the crown ether; and influence of the properties of the diluent (polarity, transport of acidity) on the efficiency of the process and on the stability of the membrane (interfacial tension between the organic and aqueous phases, solubility in the aqueous phase). The influence of these parameters is illustrated by experiments performed in order to remove strontium and cesium from high sodium content liquid waste. The studies described in this paper are focused on the decategorization of evaporator concentrates arising from the reprocessing of spent fuel.

  20. BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

    PubMed Central

    Weber, Kathrin; Harper, Nicholas; Schwabe, John; Cohen, Gerald M.

    2013-01-01

    Summary BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models. PMID:24120870

  1. Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane.

    PubMed

    Diekert, K; de Kroon, A I; Ahting, U; Niggemeyer, B; Neupert, W; de Kruijff, B; Lill, R

    2001-10-15

    The import of proteins into the mitochondrial intermembrane space differs in various aspects from the classical import pathway into the matrix. Apocytochrome c defines one of several pathways known to reach the intermembrane space, yet the components and pathways involved in outer membrane translocation are poorly defined. Here, we report the reconstitution of the apocytochrome c import reaction using proteoliposomes harbouring purified components. Import specifically requires the protease-resistant part of the TOM complex and is driven by interactions of the apoprotein with internal parts of the complex (involving Tom40) and the 'trans-side receptor' cytochrome c haem lyase. Despite the necessity of TOM complex function, the translocation pathway of apocytochrome c does not overlap with that of presequence-containing preproteins. We conclude that the TOM complex is a universal preprotein translocase that mediates membrane passage of apocytochrome c and other preproteins along distinct pathways. Apocytochrome c may provide a paradigm for the import of other small proteins into the intermembrane space such as factors used in apoptosis and protection from stress. PMID:11598006

  2. Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation

    SciTech Connect

    Ji, Peng; Lodish, Harvey F.

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Ankyrin and band 3 are not required for erythroblasts enucleation. Black-Right-Pointing-Pointer Loss of ankyrin does not affect erythroid membrane glycoprotein expression. Black-Right-Pointing-Pointer Loss of band 3 influences erythroid membrane glycoprotein expression. -- Abstract: During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex.

  3. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    PubMed

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  4. Development of (99m)Tc-labeled asymmetric urea derivatives that target prostate-specific membrane antigen for single-photon emission computed tomography imaging.

    PubMed

    Kimura, Hiroyuki; Sampei, Sotaro; Matsuoka, Daiko; Harada, Naoya; Watanabe, Hiroyuki; Arimitsu, Kenji; Ono, Masahiro; Saji, Hideo

    2016-05-15

    Prostate-specific membrane antigen (PSMA) is expressed strongly in prostate cancers and is, therefore, an attractive diagnostic and radioimmunotherapeutic target. In contrast to previous reports of PMSA-targeting (99m)Tc-tricarbonyl complexes that are cationic or lack a charge, no anionic (99m)Tc-tricarbonyl complexes have been reported. Notably, the hydrophilicity conferred by both cationic and anionic charges leads to rapid hepatobiliary clearance, whereas an anionic charge might better enhance renal clearance relative to a cationic charge. Therefore, an improvement in rapid clearance would be expected with either cationic or anionic charges, particularly anionic charges. In this study, we designed and synthesized a novel anionic (99m)Tc-tricarbonyl complex ([(99m)Tc]TMCE) and evaluated its use as a single-photon emission computed tomography (SPECT) imaging probe for PSMA detection. Direct synthesis of [(99m)Tc]TMCE from dimethyl iminodiacetate, which contains both the asymmetric urea and succinimidyl moiety important for PSMA binding, was performed using our microwave-assisted one-pot procedure. The chelate formation was successfully achieved even though the precursor included a complicated bioactive moiety. The radiochemical yield of [(99m)Tc]TMCE was 12-17%, with a radiochemical purity greater than 98% after HPLC purification. [(99m)Tc]TMCE showed high affinity in vitro, with high accumulation in LNCaP tumors and low hepatic retention in biodistribution and SPECT/CT studies. These findings warrant further evaluation of [(99m)Tc]TMCE as an imaging agent and support the benefit of this strategy for the design of other PSMA imaging probes. PMID:27073053

  5. Production of Multimeric Prostate-Specific Membrane Antigen Small Molecule Radiotracers using a Solid-Phase 99mTc Pre-Loading Strategy

    PubMed Central

    Misra, Preeti; Humblet, Valerie; Pannier, Nadine; Maison, Wolfgang; Frangioni, John V.

    2008-01-01

    Small molecule ligands specific for prostate-specific membrane antigen (PSMA) have the potential to improve prostate cancer imaging. However, highly charged ligands are difficult to label with 99mTc and to purify. In this study, we present an adamantane-trimerized small molecule that has nanomolar binding to PSMA, but that also has 12 negative charges. Methods To convert this molecule into a clinically viable SPECT diagnostic, we have developed a simple, cartridge-based, solid-phase pre-labeling strategy that, within 25 minutes, converts readily available and inexpensive 99mTc-pertechnetate into a chemically pure complex, with a reactive N-hydroxysuccinimide (NHS) ester, in neat organic solvent. This stable intermediate can label any amine-containing small molecule or peptide with 99mTc in one step, with high specific activity, and without the need for HPLC. Results Solid-phase conversion of 99mTc-pertechnetate to 99mTc-MAS3-NHS could be completed in 25 minutes, with >99% radiochemical purity and with no co-ligands present. This intermediate was then conjugated to adamantane-trimerized GPI in one step with >95% yield, and no need for HPLC purification. The final molecule bound specifically to living human tumor cells expressing PSMA on their surface. Quantitative comparison was made among GPI monomer, GPI trimer, and their 99mTc derivatives. Conclusion Our study describes a simple cartridge-based conversion of 99mTc pertechnetate to a useful, pre-loaded NHS ester intermediate that takes only 25 minutes to prepare and results in >99% radiochemical purity. Using this chemistry, we produce a high specific activity, 99mTc-labeled, PSMA-targeted small molecule, and demonstrate gamma ray radioscintigraphic imaging of living human prostate cancer cells. PMID:17631555

  6. A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway.

    PubMed

    Zhang, Hailong; Liu, Xiaogang; Wu, Fengbo; Qin, Feifei; Feng, Ping; Xu, Ting; Li, Xiang; Yang, Li

    2016-01-01

    Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG's effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa. PMID:27196894

  7. A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway

    PubMed Central

    Zhang, Hailong; Liu, Xiaogang; Wu, Fengbo; Qin, Feifei; Feng, Ping; Xu, Ting; Li, Xiang; Yang, Li

    2016-01-01

    Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG’s effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa. PMID:27196894

  8. Towards Personalized Treatment of Prostate Cancer: PSMA I&T, a Promising Prostate-Specific Membrane Antigen-Targeted Theranostic Agent

    PubMed Central

    Chatalic, Kristell L.S.; Heskamp, Sandra; Konijnenberg, Mark; Molkenboer-Kuenen, Janneke D.M.; Franssen, Gerben M.; Clahsen-van Groningen, Marian C.; Schottelius, Margret; Wester, Hans-Jürgen; van Weerden, Wytske M.; Boerman, Otto C.; de Jong, Marion

    2016-01-01

    Prostate-specific membrane antigen (PSMA) is a well-established target for nuclear imaging and therapy of prostate cancer (PCa). Radiolabeled small-molecule PSMA inhibitors are excellent candidates for PCa theranostics—they rapidly and efficiently localize in tumor lesions. However, high tracer uptake in kidneys and salivary glands are major concerns for therapeutic applications. Here, we present the preclinical application of PSMA I&T, a DOTAGA-chelated urea-based PSMA inhibitor, for SPECT/CT imaging and radionuclide therapy of PCa. 111In-PSMA I&T showed dose-dependent uptake in PSMA-expressing tumors, kidneys, spleen, adrenals, lungs and salivary glands. Coadministration of 2-(phosphonomethyl)pentane-1,5-dioic acid (2-PMPA) efficiently reduced PSMA-mediated renal uptake of 111In-PSMA I&T, with the highest tumor/kidney radioactivity ratios being obtained using a dose of 50 nmol 2-PMPA. SPECT/CT clearly visualized subcutaneous tumors and sub-millimeter intraperitoneal metastases; however, high renal and spleen uptake in control mice (no 2-PMPA) interfered with visualization of metastases in the vicinity of those organs. Coadministration of 2-PMPA increased the tumor-to-kidney absorbed dose ratio during 177Lu-PSMA I&T radionuclide therapy. Hence, at equivalent absorbed dose to the tumor (36 Gy), coinjection of 2-PMPA decreased absorbed dose to the kidneys from 30 Gy to 12 Gy. Mice injected with 177Lu-PSMA I&T only, showed signs of nephrotoxicity at 3 months after therapy, whereas mice injected with 177Lu-PSMA I&T + 2-PMPA did not. These data indicate that PSMA I&T is a promising theranostic tool for PCa. PSMA-specific uptake in kidneys can be successfully tackled using blocking agents such as 2-PMPA. PMID:27162555

  9. The Prostate Specific Membrane Antigen Regulates the Expression of IL-6 and CCL5 in Prostate Tumour Cells by Activating the MAPK Pathways1

    PubMed Central

    Colombatti, Marco; Fracasso, Giulio; Scupoli, Maria Teresa; Cingarlini, Sara; Poffe, Ornella; Naim, Hassan Y.; Heine, Martin; Tridente, Giuseppe; Mainiero, Fabrizio; Ramarli, Dunia

    2009-01-01

    The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis. PMID:19242540

  10. A novel recombinant bivalent outer membrane protein of Vibrio vulnificus and Aeromonas hydrophila as a vaccine antigen of American eel (Anguilla rostrata).

    PubMed

    SongLin, Guo; PanPan, Lu; JianJun, Feng; JinPing, Zhao; Peng, Lin; LiHua, Duan

    2015-04-01

    The immogenicity of a novel vaccine antigen was evaluated after immunized American eels (Anguilla rostrata) with a recombinant bivalent expressed outer membrane protein (OMP) of Vibrio vulnificus and Aeromonas hydrophila. Three groups of eels were intraperitoneal (i.p) injected with phosphate-buffered saline (PBS group), formaline-killed-whole-cell (FKC) of A. hydrophila and V. vulnificus (FKC group) or the bivalent OMP (OMP group). On 14, 21, 28 and 42 days post-vaccination respectively, proliferation of the whole blood cells, titers of specific antibody and lysozyme activities of experimental eels were detected. On 28 day post-vaccination, eels from three groups were challenged by i.p injection of live A. hydrophila or V. vulnificus. The results showed that, compared with the PBS group, proliferation of whole blood cells in OMP group was significant enhanced on 28 days, and the serum titers of anti-A.hydrophila and anti-V. vulnificus antibody in eels of FKC and OMP group were significant increased on 14, 21 and 28d. Lysozyme Activities in serum, skin mucus, liver and kidney were significant changed between the three groups. Relative Percent Survival (RPS) after challenged A. hydrophila in KFC vs. PBS group and OMP vs. PBS group were 62.5% and 50% respectively, and the RPS challenged V. vulnificus in FKC and OMP vs. PBS group were 37.5% and 50% respectively. These results suggest that American eels immunized with the bivalent OMP would positively affect specific as well as non-specific immune parameters and protect against infection by the two pathogens in fresh water farming. PMID:25655329

  11. Phase II study of lutetium-177 labeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 for metastatic castration-resistant prostate cancer

    PubMed Central

    Tagawa, Scott T.; Milowsky, Matthew I.; Morris, Michael; Vallabhajosula, Shankar; Christos, Paul; Akhtar, Naveed H.; Osborne, Joseph; Goldsmith, Stanley J.; Larson, Steve; Taskar, Neeta Pandit; Scher, Howard I.; Bander, Neil H.; Nanus, David M.

    2013-01-01

    Purpose To assess the efficacy of a single infusion of radiolabeled anti-prostate specific membrane antigen monoclonal antibody J591 (177Lu-J591) by PSA decline, measurable disease response, and survival. Experimental Design In this dual-center phase II study, 2 cohorts with progressive metastatic castration-resistant prostate cancer received one dose of 177Lu-J591 (15 patients at 65 mCi/m2, 17 at 70 mCi/m2) with radionuclide imaging. Expansion cohort (n=15) received 70 mCi/m2 to verify response rate and examine biomarkers. Results 47 patients who progressed after hormonal therapies (55.3% also received prior chemotherapy) received 177Lu-J591. 10.6% experienced ≥ 50% decline in PSA, 36.2% experienced ≥ 30% decline, and 59.6% experienced any PSA decline following their single treatment. One of 12 with measurable disease experienced a partial radiographic response (8 with stable disease). Sites of prostate cancer metastases were targeted in 44 of 47 (93.6%) as determined by planar imaging. All experienced reversible hematologic toxicity with grade 4 thrombocytopenia occurring in 46.8% (29.8% received platelet transfusions) without significant hemorrhage. 25.5% experienced grade 4 neutropenia with 1 episode of febrile neutropenia. The phase I maximum tolerated dose (70 mCi/m2) resulted in more 30% PSA declines (46.9% vs 13.3%, p=0.048) and longer survival (21.8 vs 11.9 months, p=0.03), but also more grade 4 hematologic toxicity and platelet transfusions. No serious non-hematologic toxicity occurred. Those with poor PSMA imaging were less likely to respond. Conclusion A single dose of 177Lu-J591 was well-tolerated with reversible myelosuppression. Accurate tumor targeting and PSA responses were seen with evidence of dose-response. Imaging biomarkers appear promising. PMID:23714732

  12. The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway

    PubMed Central

    Roboti, Peristera; Sato, Keisuke; Lowe, Martin

    2015-01-01

    Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER–Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2). PMID:25717001

  13. The golgin GMAP-210 is required for efficient membrane trafficking in the early secretory pathway.

    PubMed

    Roboti, Peristera; Sato, Keisuke; Lowe, Martin

    2015-04-15

    Golgins are coiled-coil proteins that participate in membrane-tethering events at the Golgi complex. Golgin-mediated tethering is thought to be important for vesicular trafficking and Golgi organization. However, the degree to which individual golgins contribute to these processes is poorly defined, and it has been proposed that golgins act in a largely redundant manner. Previous studies on the golgin GMAP-210 (also known as TRIP11), which is mutated in the rare skeletal disorder achondrogenesis type 1A, have yielded conflicting results regarding its involvement in trafficking. Here, we re-investigated the trafficking role of GMAP-210, and found that it is indeed required for efficient trafficking in the secretory pathway. GMAP-210 acts at both the endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) and Golgi complex during anterograde trafficking, and is also required for retrograde trafficking to the ER. Using co-depletion experiments, we also found that GMAP-210 acts in a partially redundant manner with the golgin GM130 to ensure efficient anterograde cargo delivery to the cis-Golgi. In summary, our results indicate a role for GMAP-210 in several trafficking steps at the ER-Golgi interface, some of which are partially redundant with another golgin, namely GM130 (also known as GOLGA2). PMID:25717001

  14. The Membrane-Associated Sec1/Munc18 KEULE is Required for Phragmoplast Microtubule Reorganization During Cytokinesis in Arabidopsis.

    PubMed

    Steiner, Alexander; Müller, Lin; Rybak, Katarzyna; Vodermaier, Vera; Facher, Eva; Thellmann, Martha; Ravikumar, Raksha; Wanner, Gerhard; Hauser, Marie-Theres; Assaad, Farhah F

    2016-04-01

    Cytokinesis, the partitioning of the cytoplasm following nuclear division, requires extensive coordination between membrane trafficking and cytoskeletal dynamics. In plants, the onset of cytokinesis is characterized by the assembly of a bipolar microtubule array, the phragmoplast, and of a transient membrane compartment, the cell plate. Little is known about the coordination between membrane deposition at the cell plate and the dynamics of phragmoplast microtubules. In this study, we monitor the localization dynamics of microtubule and membrane markers throughout cytokinesis. Our spatiotemporal resolution is consistent with the general view that microtubule dynamics drive membrane movements. Nonetheless, we provide evidence for active sorting at the cell plate and show that this is, at least in part, mediated by the TRAPPII tethering complex. We also characterize phragmoplast microtubule organization and cell plate formation in a suite of cytokinesis-defective mutants. Of four mutant lines with defects in phragmoplast microtubule organization, only mor1 microtubule-associated mutants exhibited aberrant cell plates. Conversely, the mutants with the strongest impairment in phragmoplast microtubule reorganization are keule alleles, which have a primary defect in membrane fusion. Our findings identify the SEC1/Munc18 protein KEULE as a central regulatory node in the coordination of membrane and microtubule dynamics during plant cytokinesis. PMID:26700031

  15. Linking differences in membrane tension with the requirement for a contractile actomyosin scaffold during exocytosis in salivary glands

    PubMed Central

    Masedunskas, Andrius; Porat-Shliom, Natalie

    2012-01-01

    In all the major secretory organs regulated exocytosis is a fundamental process that is used for releasing molecules in the extracellular space. Molecules destined for secretion are packaged into secretory vesicles that fuse with the plasma membrane upon the appropriate stimulus. In exocrine glands, large secretory vesicles fuse with specialized domains of the plasma membrane, which form ductal structures that are in direct continuity with the external environment and exhibit various architectures and diameters. In a recent study, we used intravital microscopy to analyze in detail the dynamics of exocytic events in the salivary glands of live rodents under conditions that cannot be reproduced in in vitro or ex vivo model systems. We found that after the opening of the fusion pore large secretory vesicles gradually collapse with their limiting membranes being completely absorbed into the apical plasma membrane canaliculi within 40–60 sec. Moreover, we observed that this controlled collapse requires the contractile activity of actin and its motor myosin II, which are recruited onto the large secretory vesicles immediately after their fusion with the plasma membrane. Here we suggest that the actomyosin complex may be required to facilitate exocytosis in those systems, such as the salivary glands, in which the full collapse of the vesicles is not energetically favorable due to a difference in membrane tension between the large secretory vesicles and the canaliculi. PMID:22482019

  16. Membrane fusion by the GTPase atlastin requires a conserved C-terminal cytoplasmic tail and dimerization through the middle domain

    PubMed Central

    Moss, Tyler J.; Andreazza, Camilla; Verma, Avani; Daga, Andrea; McNew, James A.

    2011-01-01

    The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization. PMID:21690399

  17. Serum Concentrations of Antibodies against Outer Membrane Protein P6, Protein D, and T- and B-Cell Combined Antigenic Epitopes of Nontypeable Haemophilus influenzae in Children and Adults of Different Ages.

    PubMed

    Hua, Chun-Zhen; Hu, Wei-Lin; Shang, Shi-Qiang; Li, Jian-Ping; Hong, Li-Quan; Yan, Jie

    2016-02-01

    Nontypeable Haemophilus influenzae (NTHi) is one of the most common etiologies of acute otitis media, rhinosinusitis, and pneumonia. Outer membrane proteins (OMPs) are the main focus in new vaccine development against NTHi, as the H. influenzae type b (Hib) vaccine does not cover noncapsulated NTHi. The OMPs P6 and protein D are the most promising candidate antigens for an NTHi vaccine, and low antibody levels against them in serum may be correlated with infection caused by NTHi. In the current study, we measured the antibody titers against P6, protein D, and their T- and B-cell combined peptide epitopes in healthy individuals of different ages. We found that children <1 month old had the lowest antibody levels against NTHi P6, protein D, and their T- and B-cell combined antigenic epitopes. Antibody titers increased at ages 1 to 6 months, peaked at 7 months to 3 years, and remained high at 4 to 6 years. The antibody titers started to decrease after 6 years and were the lowest in the 21- to 30-year group. The geometric mean titers (GMTs) of T- and B-cell combined antigenic epitopes in P6 and protein D were positively correlated with those of the protein antigens. Among 12 peptides tested, P6-61, P6-123, and protein D-167 epitopes were better recognized than others in human serum. These findings might contribute to the development of an effective serotype-independent vaccine for H. influenzae. PMID:26677200

  18. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

    PubMed

    Schapire, Arnaldo L; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A

    2008-12-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  19. Plasma membrane H+-ATPase regulation is required for auxin gradient formation preceding phototropic growth

    PubMed Central

    Hohm, Tim; Demarsy, Emilie; Quan, Clément; Allenbach Petrolati, Laure; Preuten, Tobias; Vernoux, Teva; Bergmann, Sven; Fankhauser, Christian

    2014-01-01

    Phototropism is a growth response allowing plants to align their photosynthetic organs toward incoming light and thereby to optimize photosynthetic activity. Formation of a lateral gradient of the phytohormone auxin is a key step to trigger asymmetric growth of the shoot leading to phototropic reorientation. To identify important regulators of auxin gradient formation, we developed an auxin flux model that enabled us to test in silico the impact of different morphological and biophysical parameters on gradient formation, including the contribution of the extracellular space (cell wall) or apoplast. Our model indicates that cell size, cell distributions, and apoplast thickness are all important factors affecting gradient formation. Among all tested variables, regulation of apoplastic pH was the most important to enable the formation of a lateral auxin gradient. To test this prediction, we interfered with the activity of plasma membrane H+-ATPases that are required to control apoplastic pH. Our results show that H+-ATPases are indeed important for the establishment of a lateral auxin gradient and phototropism. Moreover, we show that during phototropism, H+-ATPase activity is regulated by the phototropin photoreceptors, providing a mechanism by which light influences apoplastic pH. PMID:25261457

  20. Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus

    PubMed Central

    Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

    2016-01-01

    Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses. PMID:27110810

  1. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

    PubMed

    Giansanti, Maria Grazia; Vanderleest, Timothy E; Jewett, Cayla E; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C; Brill, Julie A; Loerke, Dinah; Fuller, Margaret T; Blankenship, J Todd

    2015-11-01

    Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

  2. Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking

    PubMed Central

    VanRheenen, Susan M.; Cao, Xiaochun; Lupashin, Vladimir V.; Barlowe, Charles; Gerard Waters, M.

    1998-01-01

    SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex. PMID:9606204

  3. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila

    PubMed Central

    Giansanti, Maria Grazia; Vanderleest, Timothy E.; Jewett, Cayla E.; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C.; Brill, Julie A.; Loerke, Dinah; Fuller, Margaret T.; Blankenship, J. Todd

    2015-01-01

    Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

  4. Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sucrose synthase (SUS) cleaves sucrose to form UDP-glucose and fructose, and exists in soluble (s-SUS) and membrane-associated (m-SUS) forms, with the latter proposed to channel UDP-glucose to the cellulose synthase complex on the plasma membrane of plant cells during synthesis of cellulose. However...

  5. IgE-mediated enhancement of CD4+ T cell responses requires antigen presentation by CD8α− conventional dendritic cells

    PubMed Central

    Ding, Zhoujie; Dahlin, Joakim S.; Xu, Hui; Heyman, Birgitta

    2016-01-01

    IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4+ T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)+ B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c+ cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c+ cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α− conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4+ T cell proliferation ex vivo than were CD8α+ cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α− cDCs which induce proliferation of CD4+ T cells. PMID:27306570

  6. IgE-mediated enhancement of CD4(+) T cell responses requires antigen presentation by CD8α(-) conventional dendritic cells.

    PubMed

    Ding, Zhoujie; Dahlin, Joakim S; Xu, Hui; Heyman, Birgitta

    2016-01-01

    IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4(+) T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)(+) B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c(+) cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c(+) cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α(-) conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4(+) T cell proliferation ex vivo than were CD8α(+) cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α(-) cDCs which induce proliferation of CD4(+) T cells. PMID:27306570

  7. B-Cell Responses to Pregnancy-Restricted and -Unrestricted Plasmodium falciparum Erythrocyte Membrane Protein 1 Antigens in Ghanaian Women Naturally Exposed to Malaria Parasites

    PubMed Central

    Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F.; Barfod, Lea

    2014-01-01

    Protective immunity to Plasmodium falciparum malaria acquired after natural exposure is largely antibody mediated. IgG-specific P. falciparum EMP1 (PfEMP1) proteins on the infected erythrocyte surface are particularly important. The transient antibody responses and the slowly acquired protective immunity probably reflect the clonal antigenic variation and allelic polymorphism of PfEMP1. However, it is likely that other immune-evasive mechanisms are also involved, such as interference with formation and maintenance of immunological memory. We measured PfEMP1-specific antibody levels by enzyme-linked immunosorbent assay (ELISA) and memory B-cell frequencies by enzyme-linked immunosorbent spot (ELISPOT) assay in a cohort of P. falciparum-exposed nonpregnant Ghanaian women. The antigens used were a VAR2CSA-type PfEMP1 (IT4VAR04) with expression restricted to parasites infecting the placenta, as well as two commonly recognized PfEMP1 proteins (HB3VAR06 and IT4VAR60) implicated in rosetting and not pregnancy restricted. This enabled, for the first time, a direct comparison in the same individuals of immune responses specific for a clinically important parasite antigen expressed only during well-defined periods (pregnancy) to responses specific for comparable antigens expressed independent of pregnancy. Our data indicate that PfEMP1-specific B-cell memory is adequately acquired even when antigen exposure is infrequent (e.g., VAR2CSA-type PfEMP1). Furthermore, immunological memory specific for VAR2CSA-type PfEMP1 can be maintained for many years without antigen reexposure and after circulating antigen-specific IgG has disappeared. The study provides evidence that natural exposure to P. falciparum leads to formation of durable B-cell immunity to clinically important PfEMP1 antigens. This has encouraging implications for current efforts to develop PfEMP1-based vaccines. PMID:24566620

  8. Evidence that membrane transduction of oligoarginine does not require vesicle formation

    SciTech Connect

    Zaro, Jennica L.; Shen Weichiang . E-mail: weishen@usc.edu

    2005-07-01

    The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 deg. C as compared to the 37 deg. C control, and was only partially inhibited at 4 deg. C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine.

  9. Required water quality for the use of high-performance membranes.

    PubMed

    Aoike, Ikuo

    2011-01-01

    The clinical benefits of high-performance membrane (HPM) dialyzers have often been reported since the advent of the synthetic polyacrylonitrile dialysis membrane. HPMs, which have high water permeability, eliminate a wide spectrum of uremic toxins, and offer excellent biocompatibility, are now essential for hemodialysis, hemofiltration, and hemodiafiltration. For HPMs whose mean pore size is enlarged to secure better dialysis membrane performance, however, the dialyzing fluid must be highly purified to prevent contamination. Blood purification therapies using highly purified dialyzing fluid and HPM are expected to improve the prognosis of future patients undergoing dialysis. PMID:21865776

  10. Requirement for membrane potential in active transport of glutamine by Escherichia coli.

    PubMed Central

    Plate, C A

    1979-01-01

    The effect of reducing the membrane potential on glutamine transport in cells of Escherichia coli has been investigated. Addition of valinomycin to tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-treated E. coli cells in the presence of 20 mM exogenous potassium reduced the membrane potential, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium, and caused a complete inhibition of glutamine transport. Valinomycin plus potassium also caused a rapid decrease in the intracellular levels of ATP of normal E. coli cells, but had little if any effect on the ATP levels of two mutants of E. coli carrying lesions in the energy-transducing ATP complex (unc mutants). Yet both the membrane potential and the capacity to transport glutamine were depressed in the unc mutants by valinomycin and potassium. These findings are consistent with the hypothesis that both ATP and a membrane potential are essential to the active transport of glutamine by E. coli cells. PMID:153897

  11. Screening for lipid requirements of membrane proteins by combining cell-free expression with nanodiscs.

    PubMed

    Henrich, Erik; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free (CF) protein expression has emerged as one of the most efficient production platforms for membrane proteins. Central bottlenecks prevalent in conventional cell-based expression systems such as mistargeting, inclusion body formation, degradation as well as product toxicity can be addressed by taking advantage of the reduced complexity of CF expression systems. However, the open accessibility of CF reactions offers the possibility to design customized artificial expression environments by supplying synthetic hydrophobic compounds such as micelles or membranes of defined composition. The open nature of CF systems therefore generally allows systematic screening approaches for the identification of efficient cotranslational solubilization environments of membrane proteins. Synergies exist in particular with the recently developed nanodisc (ND) technology enabling the synthesis of stable and highly soluble particles containing membrane discs of defined composition. Specific types of lipids frequently modulate folding, stability, and activity of integrated membrane proteins. One recently reported example are phospho-MurNAc-pentapeptide (MraY) translocases that catalyze a crucial step in bacterial peptidoglycan biosynthesis making them interesting as future drug targets. Production of functionally active MraY homologues from most human pathogens in conventional cellular production systems was so far not successful due to their obviously strict lipid dependency for functionally folding. We demonstrate that the combination of CF expression with ND technologies is an efficient strategy for the production of folded MraY translocases, and we present a general protocol for the rapid screening of lipid specificities of membrane proteins. PMID:25857790

  12. Dissecting the membrane cholesterol requirement for mycobacterial entry into host cells.

    PubMed

    Viswanathan, Gopinath; Jafurulla, Md; Kumar, G Aditya; Raghunand, Tirumalai R; Chattopadhyay, Amitabha

    2015-07-01

    Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, and are a major cause of mortality and morbidity worldwide. The molecular mechanism involved in the internalization of mycobacteria is poorly understood. In this work, we have explored the role of host membrane cholesterol in the entry of the avirulent surrogate mycobacterial strain Mycobacterium smegmatis into THP-1 macrophages. Our results show that depletion of host membrane cholesterol using methyl-β-cyclodextrin results in a significant reduction in the entry of M. smegmatis into host cells. More importantly, we show that the inhibition in the ability of M. smegmatis to enter host macrophages could be reversed upon replenishment of membrane cholesterol. To the best of our knowledge, these results constitute the first report showing that membrane cholesterol replenishment can reverse the inhibition in the entry of mycobacteria into host cells. In addition, we demonstrate that cholesterol complexation using amphotericin B (without physical depletion) is sufficient to inhibit mycobacterial entry. Importantly, we observed a significant reduction in mycobacterial entry upon enrichment of host membrane cholesterol. Taken together, our results demonstrate, for the first time, that an optimum host plasma membrane cholesterol is necessary for the entry of mycobacteria. These results assume relevance in the context of developing novel therapeutic strategies targeting cholesterol-mediated mycobacterial host cell entry. PMID:26021693

  13. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  14. European Sero-Epidemiology Network 2: standardisation of immunoassay results for pertussis requires homogeneity in the antigenic preparations.

    PubMed

    Giammanco, Anna; Nardone, Antony; Pebody, Richard; Kafatos, George; Andrews, Nick; Chiarini, Alfredo; Taormina, Susanna; de Ory, Fernando; Prosenc, Katarina; Krize, Bohumir; Hallander, Hans; Ljungman, Margaretha; Marva, Esther; Tsakris, Athanassios; O'Flanagan, Darina; Schneider, François; Griskevicius, Algirdas; Vranckx, Robert; Karacs, Ildiko

    2008-08-18

    A standardisation process, already developed during the earlier European Sero-Epidemiology Network (ESEN) project, was employed with a more robust algorithm to harmonise results of pertussis serological assays performed in 12 European and non-European countries. Initially, results from each country's own assay were compared with those obtained at the reference laboratory by means of an in-house pertussis toxin (PT)-based ELISA: seven countries used in-house or commercial PT-ELISAs; the other countries used assays based on Bordetella pertussis whole cell extracts (WCE) (three countries) or on combined PT-FHA (filamentous haemagglutinin) antigenic preparations (two countries). The WCE assays, although admitted for diagnostic purposes, confirmed their low correlation with the PT-ELISAs and their results could not be used for standardisation; the PT-FHA ELISAs gave results that were suitable for standardisation in one country but unsatisfactory in the other; the use of purified PT in serological assays confirmed its better reliability than other preparations and all PT-ELISAs results could be calibrated against those of the reference centre. In the standardisation process two high-titre cut-offs indicative of likelihood of recent infection (from within 4 weeks of disease onset up to 1 year after) were included for evaluations as they are suggested to be more useful, for the sero-epidemiological assays of immunity to pertussis, than the cut-off of protection, commonly employed, but still not defined for pertussis. Providing PT-ELISAs are used, standardisation of pertussis assay results is always possible and, when standardisation is performed, evaluation and comparison of the impact of different interventions can be also allowed, by measuring at the distribution of high antibody titres in the populations. PMID:18602434

  15. tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation*

    PubMed Central

    Shamas-Din, Aisha; Bindner, Scott; Zhu, Weijia; Zaltsman, Yehudit; Campbell, Clinton; Gross, Atan; Leber, Brian; Andrews, David W.; Fradin, Cécile

    2013-01-01

    Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death. PMID:23744079

  16. A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.

    PubMed Central

    Porrás, A; Bennett, J; Howe, A; Tokos, K; Bouck, N; Henglein, B; Sathyamangalam, S; Thimmapaya, B; Rundell, K

    1996-01-01

    At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays. PMID:8794333

  17. 89Zr-DFO-J591 for immunoPET imaging of prostate-specific membrane antigen (PSMA) expression in vivo

    PubMed Central

    Holland, Jason P.; Divilov, Vadim; Bander, Neil H.; Smith-Jones, Peter M.; Larson, Steven M.; Lewis, Jason S.

    2010-01-01

    Zirconium-89 (t1/2 = 3.27 days) is a positron emitting radionuclide which displays excellent potential for use in the design and synthesis of radioimmunoconjugates for immunoPET. In these studies we report the preparation of 89Zr-DFO-J591, a novel 89Zr -labeled monoclonal antibody (mAb) construct for targeted immunoPET imaging and quantification of prostate-specific membrane antigen (PSMA) expression in vivo. Methods The in vivo behavior of [89Zr]Zr-chloride, [89Zr]Zr-oxalate and [89Zr]Zr-DFO was investigated by using PET imaging. High level computational studies using density functional theory (DFT) calculations have been used to investigate the electronic structure of [89Zr]Zr-DFO and probe the nature of the complex in aqueous conditions. J591 was functionalized with the hexadentate, tris-hydroxamate ligand desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature. ImmunoPET imaging experiments in male, athymic nu/nu mice bearing sub-cutaneous LNCaP (PSMA positive) or PC-3 (PSMA negative) tumors were conducted. The change in 89Zr-DFO-J591 tissue uptake in response to high- and low-specific-activity formulations in the two tumor models was measured by using acute biodistribution studies and immunoPET. Results Basic characterization of three important reagents, [89Zr]Zr-chloride and [89Zr]Zr-oxalate, as well as the complex, [89Zr]Zr-DFO, demonstrated that the nature of the 89Zr species has a dramatic effect on the biodistribution and pharmacokinetics. DFT calculations provide a rationale for the observed high in vivo stability of 89Zr-DFO-labeled mAbs and suggest that in aqueous conditions, [89Zr]Zr-DFO forms a thermodynamically stable, 8-coordinate complex by coordination of two water molecules. 89Zr-DFO-J591 was produced in high radiochemical yield (>77%) with radiochemical purity >99% and a specific-activity of 181.7±1.1 MBq/mg (4.91±0.03 mCi/mg). In vitro assays demonstrated that 89Zr-DFO-J591 had an initial immunoreactive fraction of

  18. The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin.

    PubMed

    Inoue, Mayumi; Chang, Louise; Hwang, Joseph; Chiang, Shian-Huey; Saltiel, Alan R

    2003-04-10

    Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion. PMID:12687004

  19. Mitochondrial Outer Membrane Proteome of Trypanosoma brucei Reveals Novel Factors Required to Maintain Mitochondrial Morphology*

    PubMed Central

    Niemann, Moritz; Wiese, Sebastian; Mani, Jan; Chanfon, Astrid; Jackson, Christopher; Meisinger, Chris; Warscheid, Bettina; Schneider, André

    2013-01-01

    Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei. PMID:23221899

  20. Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum.

    PubMed

    Gehde, Nina; Hinrichs, Corinna; Montilla, Irine; Charpian, Stefan; Lingelbach, Klaus; Przyborski, Jude M

    2009-02-01

    Plasmodium falciparum traffics a large number of proteins to its host cell, the mature human erythrocyte. How exactly these proteins gain access to the red blood cell is poorly understood. Here we have investigated the effect of protein folding on the transport of model substrate proteins to the host cell. We find that proteins must pass into the erythrocyte cytoplasm in an unfolded state. Our data strongly support the presence of a protein-conducting channel in the parasitophorous vacuolar membrane, and additionally imply an important role for molecular chaperones in keeping parasite proteins in a 'translocation competent' state prior to membrane passage. PMID:19040635

  1. Identification of an 11-residue portion of CTP-phosphocholine cytidylyltransferase that is required for enzyme-membrane interactions.

    PubMed Central

    Yang, J; Wang, J; Tseu, I; Kuliszewski, M; Lee, W; Post, M

    1997-01-01

    CTP-phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in the biosynthesis of phosphatidylcholine (PC) in many cells. Enzyme-membrane interactions appear to play an important role in CT activation. A putative membrane-binding domain appears to be located between residues 236 and 293 from the N-terminus. To map the membrane-binding domain more precisely, glutathione S-transferase fusion proteins were prepared that contained deletions of various domains in this putative lipid-binding region. The fusion proteins were assessed for their binding of [3H]PC/oleic acid vesicles. Fusion proteins encompassing residues 267-277 bound to PC/oleic acid vesicles, whereas fragments lacking this region exhibited no specific binding to the lipid vesicles. The membrane-binding characteristics of the CT fusion proteins were also examined using intact lung microsomes. Only fragments encompassing residues 267-277 competed with full-length 125I-labelled CT, expressed in recombinant Sf9 insect cells, for microsomal membrane binding. To investigate the role of this region in PC biosynthesis, A549 and L2 cells were transfected with cDNA for CT mutants under the control of a glucocorticoid-inducible long terminal repeat (LTR) promoter. Induction of CT mutants containing residues 267-277 in transfectants resulted in reduced PC synthesis. The decrease in PC synthesis was accompanied by a shift in endogenous CT activity from the particulate to the soluble fraction. Expression of CT mutants lacking this region in A549 and L2 cells did not affect PC formation and subcellular distribution of CT activity. These results suggest that the CT region located between residues 267 and 277 from the N-terminus is required for the interaction of CT with membranes. PMID:9224626

  2. Chronic mould exposure as a risk factor for severe community acquired pneumonia in a patient requiring extra corporeal membrane oxygenation.

    PubMed

    Thomas, Stephanie; Hassan, Ibrahim; Barker, Julian; Ashworth, Alan; Barnes, Anita; Fedor, Igor; Feddy, Lee; Hayes, Tim; Malagon, Ignacio; Stirling, Sarah; Szentgyorgyi, Lajos; Mutton, Ken; Richardson, Malcolm

    2015-01-01

    A previously fit and well man developed acute respiratory failure due to environmental mould exposure from living in damp rental accommodation. Despite aggressive intensive care management he rapidly deteriorated and required respiratory and cardiac Extracorporeal Membrane Oxygenation. We hypothesize that poor domiciliary conditions may make an underestimated contribution to community respiratory disease. These conditions may present as acute and severe illness with non-typical pathogens identified. PMID:26236598

  3. Chronic mould exposure as a risk factor for severe community acquired pneumonia in a patient requiring extra corporeal membrane oxygenation

    PubMed Central

    Thomas, Stephanie; Hassan, Ibrahim; Barker, Julian; Ashworth, Alan; Barnes, Anita; Fedor, Igor; Feddy, Lee; Hayes, Tim; Malagon, Ignacio; Stirling, Sarah; Szentgyorgyi, Lajos; Mutton, Ken; Richardson, Malcolm

    2015-01-01

    A previously fit and well man developed acute respiratory failure due to environmental mould exposure from living in damp rental accommodation. Despite aggressive intensive care management he rapidly deteriorated and required respiratory and cardiac Extracorporeal Membrane Oxygenation. We hypothesize that poor domiciliary conditions may make an underestimated contribution to community respiratory disease. These conditions may present as acute and severe illness with non-typical pathogens identified. PMID:26236598

  4. GNA33 of Neisseria meningitidis Is a Lipoprotein Required for Cell Separation, Membrane Architecture, and Virulence

    PubMed Central

    Adu-Bobie, Jeannette; Lupetti, Pietro; Brunelli, Brunella; Granoff, Dan; Norais, Nathalie; Ferrari, Germano; Grandi, Guido; Rappuoli, Rino; Pizza, Mariagrazia

    2004-01-01

    GNA33 is a membrane-bound lipoprotein with murein hydrolase activity that is present in all Neisseria species and well conserved in different meningococcal isolates. The protein shows 33% identity to a lytic transglycolase (MltA) from Escherichia coli and has been shown to be involved in the degradation of both insoluble murein sacculi and unsubstituted glycan strands. To study the function of the gene and its role in pathogenesis and virulence, a knockout mutant of a Neisseria meningitidis serogroup B strain was generated. The mutant exhibited retarded growth in vitro. Transmission electron microscopy revealed that the mutant grows in clusters which are connected by a continuous outer membrane, suggesting a failure in the separation of daughter cells. Moreover, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatant revealed that the mutant releases several proteins in the medium. The five most abundant proteins, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, belong to the outer membrane protein family. Finally, the mutant showed an attenuated phenotype, since it was not able to cause bacteremia in the infant rat model. We conclude that GNA33 is a highly conserved lipoprotein which plays an important role in peptidoglycan metabolism, cell separation, membrane architecture, and virulence. PMID:15039310

  5. Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids

    PubMed Central

    Shvets, Elena; Bitsikas, Vassilis; Howard, Gillian; Hansen, Carsten Gram; Nichols, Benjamin J.

    2015-01-01

    Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid. PMID:25897946

  6. Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly.

    PubMed

    Villacorte, Mylah; Delmarcelle, Anne-Sophie; Lernoux, Manon; Bouquet, Mahé; Lemoine, Pascale; Bolsée, Jennifer; Umans, Lieve; de Sousa Lopes, Susana Chuva; Van Der Smissen, Patrick; Sasaki, Takako; Bommer, Guido; Henriet, Patrick; Refetoff, Samuel; Lemaigre, Frédéric P; Zwijsen, An; Courtoy, Pierre J; Pierreux, Christophe E

    2016-06-01

    Thyroid follicles, the functional units of the thyroid gland, are delineated by a monolayer of thyrocytes resting on a continuous basement membrane. The developmental mechanisms of folliculogenesis, whereby follicles are formed by the reorganization of a non-structured mass of non-polarized epithelial cells, are largely unknown. Here we show that assembly of the epithelial basement membrane is crucial for folliculogenesis and is controlled by endothelial cell invasion and by BMP-Smad signaling in thyrocytes. Thyroid-specific Smad1 and Smad5 double-knockout (Smad1/5(dKO)) mice displayed growth retardation, hypothyroidism and defective follicular architecture. In Smad1/5(dKO) embryonic thyroids, epithelial cells remained associated in large clusters and formed small follicles. Although similar follicular defects are found in Vegfa knockout (Vegfa(KO)) thyroids, Smad1/5(dKO) thyroids had normal endothelial cell density yet impaired endothelial differentiation. Interestingly, both Vegfa(KO) and Smad1/5(dKO) thyroids displayed impaired basement membrane assembly. Furthermore, conditioned medium (CM) from embryonic endothelial progenitor cells (eEPCs) rescued the folliculogenesis defects of both Smad1/5(dKO) and Vegfa(KO) thyroids. Laminin α1, β1 and γ1, abundantly released by eEPCs into CM, were crucial for folliculogenesis. Thus, epithelial Smad signaling and endothelial cell invasion promote folliculogenesis via assembly of the basement membrane. PMID:27068110

  7. The newly identified yeast GRD genes are required for retention of late-Golgi membrane proteins.

    PubMed Central

    Nothwehr, S F; Bryant, N J; Stevens, T H

    1996-01-01

    Processing of A-ALP, a late-Golgi membrane protein constructed by fusing the cytosolic domain of dipeptidyl aminopeptidase A to the transmembrane and lumenal domains of alkaline phosphatase (ALP), serves as a convenient assay for loss of retention of late-Golgi membrane proteins in Saccharomyces cerevisiae. In this study, a large group of novel grd (for Golgi retention defective) yeast mutants, representing 18 complementation groups, were identified on the basis of their mislocalization of A-ALP to the vacuole, where it was proteolytically processed and thus became enzymatically activated. All of the grd mutants exhibited significant mislocalization of A-ALP, as measured by determining the kinetics of A-ALP processing and by analyzing its PMID:8649377

  8. Severe H1N1 virus in pregnancy requiring extracorporeal membrane oxygenation and lobectomy

    PubMed Central

    McNamee, K; Dawood, F

    2010-01-01

    Prompt diagnosis and treatment of H1N1 is crucial during pregnancy to prevent major morbidity and mortality as the virus poses an increased risk of severe illness in pregnant women. Currently, there is limited obstetric literature concerning pregnancy and the pandemic swine flu outbreak in the UK. Although there was a concerted effort to stockpile the HIN1 virus vaccinations, critical care adult extracorporeal membrane oxygenation is only available in one centre in the UK.

  9. Virus-mimetic nanovesicles as a versatile antigen-delivery system

    PubMed Central

    Zhang, Pengfei; Chen, Yixin; Zeng, Yun; Shen, Chenguang; Li, Rui; Guo, Zhide; Li, Shaowei; Zheng, Qingbing; Chu, Chengchao; Wang, Zhantong; Zheng, Zizheng; Tian, Rui; Ge, Shengxiang; Zhang, Xianzhong; Xia, Ning-Shao; Liu, Gang; Chen, Xiaoyuan

    2015-01-01

    It is a critically important challenge to rapidly design effective vaccines to reduce the morbidity and mortality of unexpected pandemics. Inspired from the way that most enveloped viruses hijack a host cell membrane and subsequently release by a budding process that requires cell membrane scission, we genetically engineered viral antigen to harbor into cell membrane, then form uniform spherical virus-mimetic nanovesicles (VMVs) that resemble natural virus in size, shape, and specific immunogenicity with the help of surfactants. Incubation of major cell membrane vesicles with surfactants generates a large amount of nano-sized uniform VMVs displaying the native conformational epitopes. With the diverse display of epitopes and viral envelope glycoproteins that can be functionally anchored onto VMVs, we demonstrate VMVs to be straightforward, robust and tunable nanobiotechnology platforms for fabricating antigen delivery systems against a wide range of enveloped viruses. PMID:26504197

  10. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ.

    PubMed

    Pagliuso, Alessandro; Valente, Carmen; Giordano, Lucia Laura; Filograna, Angela; Li, Guiling; Circolo, Diego; Turacchio, Gabriele; Marzullo, Vincenzo Manuel; Mandrich, Luigi; Zhukovsky, Mikhail A; Formiggini, Fabio; Polishchuk, Roman S; Corda, Daniela; Luini, Alberto

    2016-01-01

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself). PMID:27401954

  11. Golgi membrane fission requires the CtBP1-S/BARS-induced activation of lysophosphatidic acid acyltransferase δ

    PubMed Central

    Pagliuso, Alessandro; Valente, Carmen; Giordano, Lucia Laura; Filograna, Angela; Li, Guiling; Circolo, Diego; Turacchio, Gabriele; Marzullo, Vincenzo Manuel; Mandrich, Luigi; Zhukovsky, Mikhail A.; Formiggini, Fabio; Polishchuk, Roman S.; Corda, Daniela; Luini, Alberto

    2016-01-01

    Membrane fission is an essential cellular process by which continuous membranes split into separate parts. We have previously identified CtBP1-S/BARS (BARS) as a key component of a protein complex that is required for fission of several endomembranes, including basolateral post-Golgi transport carriers. Assembly of this complex occurs at the Golgi apparatus, where BARS binds to the phosphoinositide kinase PI4KIIIβ through a 14-3-3γ dimer, as well as to ARF and the PKD and PAK kinases. We now report that, when incorporated into this complex, BARS binds to and activates a trans-Golgi lysophosphatidic acid (LPA) acyltransferase type δ (LPAATδ) that converts LPA into phosphatidic acid (PA); and that this reaction is essential for fission of the carriers. LPA and PA have unique biophysical properties, and their interconversion might facilitate the fission process either directly or indirectly (via recruitment of proteins that bind to PA, including BARS itself). PMID:27401954

  12. Membrane association of the CD3ε signaling domain is required for optimal T cell development and function1

    PubMed Central

    Bettini, Matthew L.; Guy, Clifford; Dash, Pradyot; Vignali, Kate M.; Hamm, David E.; Dobbins, Jessica; Gagnon, Etienne; Thomas, Paul G.; Wucherpfennig, Kai W.; Vignali, Dario A.A.

    2014-01-01

    The T cell receptor (TCR):CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). Here we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4−CD8− double negative-3 (DN3) to DN4 thymocyte transition, due to enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function. PMID:24899501

  13. Proliferating Cell Nuclear Antigen (PCNA) Is Required for Cell Cycle-regulated Silent Chromatin on Replicated and Nonreplicated Genes*

    PubMed Central

    Miller, Andrew; Chen, Jiji; Takasuka, Taichi E.; Jacobi, Jennifer L.; Kaufman, Paul D.; Irudayaraj, Joseph M. K.; Kirchmaier, Ann L.

    2010-01-01

    In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793–809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications. PMID:20813847

  14. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG.

    PubMed

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG's inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  15. Sialic Acid Is Required for Neuronal Inhibition by Soluble MAG but not for Membrane Bound MAG

    PubMed Central

    Al-Bashir, Najat; Mellado, Wilfredo; Filbin, Marie T.

    2016-01-01

    Myelin-Associated Glycoprotein (MAG), a major inhibitor of axonal growth, is a member of the immunoglobulin (Ig) super-family. Importantly, MAG (also known as Siglec-4) is a member of the Siglec family of proteins (sialic acid-binding, immunoglobulin-like lectins), MAG binds to complex gangliosides, specifically GD1a and/or GT1b. Therefore, it has been proposed as neuronal receptors for MAG inhibitory effect of axonal growth. Previously, we showed that MAG binds sialic acid through domain 1 at Arg118 and is able to inhibit axonal growth through domain 5. We developed a neurite outgrowth (NOG) assay, in which both wild type MAG and mutated MAG (MAG Arg118) are expressed on cells. In addition we also developed a soluble form NOG in which we utilized soluble MAG-Fc and mutated MAG (Arg118-Fc). Only MAG-Fc is able to inhibit NOG, but not mutated MAG (Arg118)-Fc that has been mutated at its sialic acid binding site. However, both forms of membrane bound MAG- and MAG (Arg118)- expressing cells still inhibit NOG. Here, we review various results from different groups regarding MAG’s inhibition of axonal growth. Also, we propose a model in which the sialic acid binding is not necessary for the inhibition induced by the membrane form of MAG, but it is necessary for the soluble form of MAG. This finding highlights the importance of understanding the different mechanisms by which MAG inhibits NOG in both the soluble fragmented form and the membrane-bound form in myelin debris following CNS damage. PMID:27065798

  16. Cytomegalovirus Myocarditis Required Extracorporeal Membrane Oxygenation Support Followed by Ganciclovir Treatment in Infant

    PubMed Central

    Kim, Bong Jun; Jung, Jo Won; Shin, Yu Rim; Park, Han Ki; Park, Young Hwan; Shin, Hong Ju

    2016-01-01

    A 7-month-old girl with no medical history was treated with mechanical circulatory support due to myocarditis. Her cardiac contractility did not improve despite more than one week of extracorporeal membrane oxygenation treatment. Thus, we planned a heart transplant. However, a high level of cytomegalovirus was found in blood laboratory results by quantitative polymerase chain reaction. The patient’s heart contractility recovered to normal range four days after ganciclovir treatment. She was discharged with slightly decreased cardiac contractility with a left ventricular ejection fraction of 45%. PMID:27298799

  17. The Role of Positron Emission Tomography With (68)Gallium (Ga)-Labeled Prostate-specific Membrane Antigen (PSMA) in the Management of Patients With Organ-confined and Locally Advanced Prostate Cancer Prior to Radical Treatment and After Radical Prostatectomy.

    PubMed

    Rai, Bhavan Prasad; Baum, Richard Paul; Patel, Amit; Hughes, Robert; Alonzi, Roberto; Lane, Tim; Adshead, Jim; Vasdev, Nikhil

    2016-09-01

    The role of positron emission tomography (PET) with (68)Gallium (Ga)-labeled prostate-specific membrane antigen (PSMA) imaging for prostate cancer is gaining prominence. Current imaging strategies, despite having progressed significantly, have limitations, in particular their ability to diagnose metastatic lymph node involvement. Preliminary results of PET with (68)Ga-labeled PSMA have shown encouraging results, particularly in the recurrent prostate cancer setting. Furthermore, the ability of PET with (68)Ga-labeled PSMA of playing a dual diagnostic and therapeutic setting (theranostics) is currently being investigated as well. PET with (68)Ga-labeled PSMA certainly has a role to play in bridging some of the voids in contemporary prostate cancer imaging tools. PMID:26790588

  18. PERP, a Host Tetraspanning Membrane Protein, is Required for Salmonella-Induced Inflammation

    PubMed Central

    Hallstrom, Kelly N.; Srikanth, C.V.; Agbor, Terence A.; Dumont, Christopher M.; Peters, Kristen; Paraoan, Luminita; Casanova, James E.; Boll, Erik J.; McCormick, Beth A.

    2016-01-01

    Summary Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of PMN transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast-two-hybrid screen, we identified the tetraspanning membrane protein, PERP (p53-effector related to PMP-22), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface. PMID:25486861

  19. The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

    PubMed

    Schneider, Anja; Steinberger, Iris; Herdean, Andrei; Gandini, Chiara; Eisenhut, Marion; Kurz, Samantha; Morper, Anna; Hoecker, Natalie; Rühle, Thilo; Labs, Mathias; Flügge, Ulf-Ingo; Geimer, Stefan; Schmidt, Sidsel Birkelund; Husted, Søren; Weber, Andreas P M; Spetea, Cornelia; Leister, Dario

    2016-04-01

    In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance. PMID:27020959

  20. Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function

    PubMed Central

    Kim, Gukhan; Luján, Rafael; Schwenk, Jochen; Kelley, Melissa H; Aguado, Carolina; Watanabe, Masahiko; Fakler, Bernd; Maylie, James; Adelman, John P

    2016-01-01

    Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca2+ influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels. DOI: http://dx.doi.org/10.7554/eLife.12637.001 PMID:26880549

  1. Severe Tumor Lysis Syndrome and Acute Pulmonary Edema Requiring Extracorporeal Membrane Oxygenation Following Initiation of Chemotherapy for Metastatic Alveolar Rhabdomyosarcoma.

    PubMed

    Sanford, Ethan; Wolbrink, Traci; Mack, Jennifer; Grant Rowe, R

    2016-05-01

    We present an 8-year-old male with metastatic alveolar rhabdomyosarcoma (ARMS) who developed precipitous cardiopulmonary collapse with severe tumor lysis syndrome (TLS) 48 hr after initiation of chemotherapy. Despite no detectable pulmonary metastases, acute hypoxemic respiratory failure developed, requiring extracorporeal membrane oxygenation (ECMO). Although TLS has been reported in disseminated ARMS, this singular case of life-threatening respiratory deterioration developing after initiation of chemotherapy presented unique therapeutic dilemmas. We review the clinical aspects of this case, including possible mechanisms of respiratory failure, and discuss the role of ECMO utilization in pediatric oncology. PMID:26713672

  2. Biologic activity of antigen receptors artificially incorporated onto B lymphocytes.

    PubMed

    Peacock, J S; Londo, T R; Roess, D A; Barisas, B G

    1986-09-15

    .2 monoclonal antibody and complement causes only a slight reduction in the number of receptor-decorated B cells that respond to the relatively thymus-independent antigen DNP-POL. This type of phenomenon is also seen with natural antigen-specific B cells. Thus, polyclonal activation of receptor-decorated B cells exhibits the same gross helper cell requirements as antigenic activation of natural antigen-specific B cells. The results of this study are discussed in the context of the role of membrane-bound surface Ig in antigen-dependent B cell activation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2427584

  3. An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors

    SciTech Connect

    Levar, Caleb E.; Chan, Chi Ho; Mehta-Kolte, Misha G.; Bond, Daniel R.

    2014-10-28

    Dissimilatory metal-reducing bacteria, such as Geobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption of imcH, encoding an inner membrane c-type cytochrome, eliminated the ability of G. sulfurreducens to reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but the imcH mutant retained the ability to reduce Fe(III) oxides with potentials of ≤–0.1 V versus SHE. The imcH mutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to –0.1 V versus SHE triggered exponential growth. At potentials of ≤–0.1 V versus SHE, both the wild type and the imcH mutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not trigger imcH mutant growth. These results demonstrate that G. sulfurreducens possesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. Redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments where Geobacter is found.

  4. An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors

    DOE PAGESBeta

    Levar, Caleb E.; Chan, Chi Ho; Mehta-Kolte, Misha G.; Bond, Daniel R.

    2014-10-28

    Dissimilatory metal-reducing bacteria, such as Geobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption of imcH, encoding an inner membrane c-type cytochrome, eliminated the ability of G. sulfurreducens to reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentialsmore » greater than 0.1 V versus the standard hydrogen electrode (SHE), but the imcH mutant retained the ability to reduce Fe(III) oxides with potentials of ≤–0.1 V versus SHE. The imcH mutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to –0.1 V versus SHE triggered exponential growth. At potentials of ≤–0.1 V versus SHE, both the wild type and the imcH mutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not trigger imcH mutant growth. These results demonstrate that G. sulfurreducens possesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. Redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments where Geobacter is found.« less

  5. Modulatory Effect of Taurine on 7,12-Dimethylbenz(a)Anthracene-Induced Alterations in Detoxification Enzyme System, Membrane Bound Enzymes, Glycoprotein Profile and Proliferative Cell Nuclear Antigen in Rat Breast Tissue.

    PubMed

    Vanitha, Manickam Kalappan; Baskaran, Kuppusamy; Periyasamy, Kuppusamy; Selvaraj, Sundaramoorthy; Ilakkia, Aruldoss; Saravanan, Dhiravidamani; Venkateswari, Ramachandran; Revathi Mani, Balasundaram; Anandakumar, Pandi; Sakthisekaran, Dhanapal

    2016-08-01

    The modulatory effect of taurine on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in rats was studied. DMBA (25 mg/kg body weight) was administered to induce breast cancer in rats. Protein carbonyl levels, activities of membrane bound enzymes (Na(+) /K(+) ATPase, Ca(2+) ATPase, and Mg(2+) ATPase), phase I drug metabolizing enzymes (cytochrome P450, cytochrome b5, NADPH cytochrome c reductase), phase II drug metabolizing enzymes (glutathione-S-transferase and UDP-glucuronyl transferase), glycoprotein levels, and proliferative cell nuclear antigen (PCNA) were studied. DMBA-induced breast tumor bearing rats showed abnormal alterations in the levels of protein carbonyls, activities of membrane bound enzymes, drug metabolizing enzymes, glycoprotein levels, and PCNA protein expression levels. Taurine treatment (100 mg/kg body weight) appreciably counteracted all the above changes induced by DMBA. Histological examination of breast tissue further supported our biochemical findings. The results of the present study clearly demonstrated the chemotherapeutic effect of taurine in DMBA-induced breast cancer. PMID:27091720

  6. Inhibition of Class II Major Histocompatibility Complex Antigen Processing by Escherichia coli Heat-Labile Enterotoxin Requires an Enzymatically Active A Subunit

    PubMed Central

    Matousek, Milita P.; Nedrud, John G.; Cieplak, Witold; Harding, Clifford V.

    1998-01-01

    Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity. PMID:9632629

  7. Ankyrin-B directs membrane tethering of periaxin and is required for maintenance of lens fiber cell hexagonal shape and mechanics.

    PubMed

    Maddala, Rupalatha; Walters, Mark; Brophy, Peter J; Bennett, Vann; Rao, Ponugoti V

    2016-01-15

    Periaxin (Prx), a PDZ domain protein expressed preferentially in myelinating Schwann cells and lens fibers, plays a key role in membrane scaffolding and cytoarchitecture. Little is known, however, about how Prx is anchored to the plasma membrane. Here we report that ankyrin-B (AnkB), a well-characterized adaptor protein involved in linking the spectrin-actin cytoskeleton to integral membrane proteins, is required for membrane association of Prx in lens fibers and colocalizes with Prx in hexagonal fiber cells. Under AnkB haploinsufficiency, Prx accumulates in the soluble fraction with a concomitant loss from the membrane-enriched fraction of mouse lenses. Moreover, AnkB haploinsufficiency induced age-dependent disruptions in fiber cell hexagonal geometry and radial alignment and decreased compressive stiffness in mouse lenses parallel to the changes observed in Prx null mouse lens. Both AnkB- and Prx-deficient mice exhibit disruptions in membrane organization of the spectrin-actin network and the dystrophin-glycoprotein complex in lens fiber cells. Taken together, these observations reveal that AnkB is required for Prx membrane anchoring and for maintenance of lens fiber cell hexagonal geometry, membrane skeleton organization, and biomechanics. PMID:26538089

  8. Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae

    PubMed Central

    Fagarasanu, Monica; Fagarasanu, Andrei; Tam, Yuen Yi C.; Aitchison, John D.; Rachubinski, Richard A.

    2005-01-01

    Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance. PMID:15928207

  9. SOM 1, a small new gene required for mitochondrial inner membrane peptidase function in Saccharomyces cerevisiae.

    PubMed

    Esser, K; Pratje, E; Michaelis, G

    1996-09-25

    IMP1 encodes a subunit of the mitochondrial inner membrane peptidase responsible for the proteolytic processing of cytochrome oxidase subunit 2 (Cox2) and cytochrome b2 (Cytb2). The molecular defect in an imp1 mutation and the characterisation of a high-copy-number suppressor is described. A deletion of the suppressor region causes respiration deficiency. The DNA sequence revealed three very small overlapping ORFs. Constructs which carried termination codons within the ORFs or lacked ATG initiation codons still retained complementing activity on a high-copy-number plasmid. Nevertheless, the possibility that the suppressor acts at DNA or RNA level could be excluded. Subcloning of the ORFs, complementation analysis in low-copy-number plasmids and transcript mapping identified the 222 bp ORF as the suppressor gene designated SOM1. The SOM1 gene is transcribed into a 375 bp polyadenylated RNA and the deduced amino acid sequence predicts a small protein of 8.4 kDa with no significant sequence similarity to known proteins. In the som1 deletion mutant, proteolytic processing of the Cox2 precursor is prevented and Cytb2 is strongly reduced. SOM1 represents a new small gene which encodes a novel factor that is essential for the correct function of the Imp1 peptidase and/or the protein sorting machinery. PMID:8879245

  10. Electrostatically induced recruitment of membrane peptides into clusters requires ligand binding at both interfaces.

    PubMed

    Antonenko, Yuri N; Horner, Andreas; Pohl, Peter

    2012-01-01

    Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin). Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i) by the decreased diffusional PLL mobility D(PLL) and (ii) by an increased lifetime τ(PLL) of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither D(PLL) nor τ(P) changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors. PMID:23285199

  11. Electrostatically Induced Recruitment of Membrane Peptides into Clusters Requires Ligand Binding at Both Interfaces

    PubMed Central

    Pohl, Peter

    2012-01-01

    Protein recruitment to specific membrane locations may be governed or facilitated by electrostatic attraction, which originates from a multivalent ligand. Here we explored the energetics of a model system in which this simple electrostatic recruitment mechanism failed. That is, basic poly-L-lysine binding to one leaflet of a planar lipid bilayer did not recruit the triply-charged peptide (O-Pyromellitylgramicidin). Clustering was only observed in cases where PLL was bound to both channel ends. Clustering was indicated (i) by the decreased diffusional PLL mobility DPLL and (ii) by an increased lifetime τPLL of the clustered channels. In contrast, if PLL was bound to only one leaflet, neither DPLL nor τP changed. Simple calculations suggest that electrostatic repulsion of the unbound ends prevented neighboring OPg dimers from approaching each other. We believe that a similar mechanism may also operate in cell signaling and that it may e.g. contribute to the controversial results obtained for the ligand driven dimerization of G protein-coupled receptors. PMID:23285199

  12. Membrane tension and membrane fusion.

    PubMed

    Kozlov, Michael M; Chernomordik, Leonid V

    2015-08-01

    Diverse cell biological processes that involve shaping and remodeling of cell membranes are regulated by membrane lateral tension. Here we focus on the role of tension in driving membrane fusion. We discuss the physics of membrane tension, forces that can generate the tension in plasma membrane of a cell, and the hypothesis that tension powers expansion of membrane fusion pores in late stages of cell-to-cell and exocytotic fusion. We propose that fusion pore expansion can require unusually large membrane tensions or, alternatively, low line tensions of the pore resulting from accumulation in the pore rim of membrane-bending proteins. Increase of the inter-membrane distance facilitates the reaction. PMID:26282924

  13. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    PubMed

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion. PMID:26764596

  14. Nonviable mutants of simian virus 40 with deletions near the 3' end of gene A define a function for large T antigen required after onset of viral DNA replication.

    PubMed Central

    Tornow, J; Cole, C N

    1983-01-01

    Deletion mutants of simian virus 40 (SV40) with lesions at the three DdeI sites near the 3' end of the early region were constructed. Mutants with deletions at 0.203 and 0.219 map units (mu) which did not change the large T antigen reading frame were viable. This extends slightly the upstream boundary for the location of viable mutants with deletions in the 3' end of the A gene. Mutants with frameshift deletions at 0.193 and 0.219 mu were nonviable. These are the first nonviable mutants with deletions in this portion of the A gene. None of the three nonviable mutants with deletions at 0.219 mu produced progeny viral DNA. These three mutants all used the alternate reading frame located in this portion of the SV40 early region. The mutant with a deletion at 0.193 mu, dlA2459, was positive for viral DNA replication and was defective for adenovirus helper function. All of these mutations were located in the portion of the SV40 large T antigen which has no homology to the polyoma T antigens. These results indicate that this portion of large T antigen is required for some late step in the viral growth cycle and suggest that adenovirus helper function is required for productive infection by SV40. Images PMID:6312080

  15. Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

    PubMed

    Cattelan, Natalia; Villalba, María Inés; Parisi, Gustavo; Arnal, Laura; Serra, Diego Omar; Aguilar, Mario; Yantorno, Osvaldo

    2016-02-01

    Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition. PMID:26673448

  16. Non-classical antigen processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4+ T cells

    PubMed Central

    Matsuzaki, Junko; Tsuji, Takemasa; Luescher, Immanuel; Old, Lloyd J.; Shrikant, Protul; Gnjatic, Sacha; Odunsi, Kunle

    2014-01-01

    Tumor antigen-specific CD4+ T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4+ T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157–170 peptide vaccination in ovarian cancer patients. While both subsets similarly recognized exogenous NY-ESO-1 protein pulsed on DP04+ target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4+ T cells more efficiently recognized the short 8–9-mer peptides than the non-tumor-recognizing CD4+ T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways such as the proteasomal degradation and transporter-associated with antigen-processing (TAP)-mediated peptide transport were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacological inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrated that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple non-classical antigen-processing pathways. Harnessing direct tumor-recognizing ability of CD4+ T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment. PMID:24764581

  17. Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells.

    PubMed

    Matsuzaki, Junko; Tsuji, Takemasa; Luescher, Immanuel; Old, Lloyd J; Shrikant, Protul; Gnjatic, Sacha; Odunsi, Kunle

    2014-04-01

    Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment. PMID:24764581

  18. Drainin required for membrane fusion of the contractile vacuole in Dictyostelium is the prototype of a protein family also represented in man.

    PubMed Central

    Becker, M; Matzner, M; Gerisch, G

    1999-01-01

    The contractile vacuole expels water by forming a channel with the plasma membrane and thus enables cells to survive in a hypo-osmotic environment. Here we characterize drainin, a Dictyostelium protein involved in this process, as the first member of a protein family represented in fission yeast, Caenorhabditis elegans and man. Gene replacement in Dictyostelium shows that drainin acts at a checkpoint of channel formation between the contractile vacuole and the plasma membrane. A green fluorescent protein fusion of drainin localizes specifically to the contractile vacuole and rescues its periodic discharge in drainin-null cells. Drainin is a peripheral membrane protein, requiring a short hydrophobic stretch in its C-terminal region for localization and function. We suggest that drainin acts in a signaling cascade that couples a volume-sensing device in the vacuolar membrane to the membrane fusion machinery. PMID:10369671

  19. A novel fixed fibre biofilm membrane process for on-site greywater reclamation requiring no fouling control.

    PubMed

    Jabornig, Simon; Podmirseg, Sabine Marie

    2015-03-01

    On-site greywater treatment and reuse in urban areas bears the potential to reduce huge quantities of wastewater and lower freshwater shortages. Until now dissemination of small, single household applications has been rather limited as simple and high quality water producing, but also cost-effective treatment units have not been developed so far. This paper proposes a new process, based on a concurrently working hollow-fibre membrane as fixed biofilm support and filtration device. Bioreactor characteristics, influence of different aeration rates, membrane flux development, as well as structure and composition of biofilm were monitored to evaluate the performance of the tested pilot unit. The introduced process achieved international water reuse guidelines, worked soundly and could, compared to conventional micro MBR, significantly reduce energy demand (<1.4 kWh m(-3)). Fouling control by air scouring and chemical cleaning was not required once flux had stabilized. The biofilm analysis showed a porous, spongy-like structure. Microbiological investigation revealed a community of sheathed bacteria and nematodes that could play an important role in the flux stabilisation effect. In general, the study confirmed the suitability of the presented process for greywater treatment and provides valuable design data for future optimization and systematic analysis. PMID:25220525

  20. MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.

    PubMed Central

    Iida, H; Nakamura, H; Ono, T; Okumura, M S; Anraku, Y

    1994-01-01

    By establishing a unique screening method, we have isolated yeast mutants that die only after differentiating into cells with a mating projection, and some of them are also defective in Ca2+ signaling. The mutants were classified into five complementation groups, one of which we studied extensively. This mutation defines a new gene, designated MID1, which encodes an N-glycosylated, integral plasma membrane protein with 548 amino acid residues. The mid1-1 mutant has low Ca2+ uptake activity, loses viability after receiving mating pheromones, and escapes death when incubated with high concentrations of CaCl2. The MID1 gene is nonessential for vegetative growth. The efficiency of mating between MATa mid1-1 and MAT alpha mid1-1 cells is low. These results demonstrate that MID1 is required for Ca2+ influx and mating. Images PMID:7526155

  1. Cytostructural Localization of a Tumor-Associated Antigen

    NASA Astrophysics Data System (ADS)

    Howard, Donald R.; Batsakis, John G.

    1980-10-01

    Tumor cell membrane glycoproteins may be involved in the induction of tumor immunity or in the escape of tumors from immunologic defense mechanisms. Forty-four benign and malignant breast lesions were examined for the presence of a carbohydrate precursor antigen (T antigen) of the human blood group system MN. T antigen was demonstrated by means of an immunohistochemical technique to detect tissue binding of peanut agglutinin, a plant lectin, with affinity for T antigen. Malignant breast lesions showed a pattern of T antigen expression different from that of benign breast tissues. A possible role for T antigen in the modulation of the immune response to breast carcinoma is suggested.

  2. Cross-reactivity of anti-PLA2R1 autoantibodies to rabbit and mouse PLA2R1 antigens and development of two novel ELISAs with different diagnostic performances in idiopathic membranous nephropathy.

    PubMed

    Seitz-Polski, Barbara; Dolla, Guillaume; Payré, Christine; Tomas, Nicola M; Lochouarn, Marine; Jeammet, Louise; Mariat, Christophe; Krummel, Thierry; Burtey, Stéphane; Courivaud, Cécile; Schlumberger, Wolfgang; Zorzi, Kévin; Benzaken, Sylvia; Bernard, Ghislaine; Esnault, Vincent L M; Lambeau, Gérard

    2015-11-01

    About 70% of patients with idiopathic membranous nephropathy (iMN) have autoantibodies to the phospholipase A2 receptor PLA2R1. We screened sera from iMN patients for their cross-reactivity to human (h), rabbit (rb) and mouse (m) PLA2R1 by western blot (WB) and antigen-specific ELISAs. All iMN patients recognized hPLA2R1 and rbPLA2R1 by WB, and a rbPLA2R1 ELISA was as sensitive as the standardized hPLA2R1 ELISA to monitor anti-PLA2R1 in patients with active disease or in drug-induced remission. In contrast, only 51% of patients were reactive to mPLA2R1 by WB, and a maximum of 78% were weakly to highly positive in the mPLA2R1 ELISA, suggesting that iMN patients exhibit different subsets of anti-PLA2R1 autoantibodies against epitopes that are shared or not among PLA2R1 orthologs. In a cohort of 41 patients with a mean follow-up of 42 months from anti-PLA2R1 assay, the detection of anti-mPLA2R1 autoantibodies was an independent predictor of clinical outcome in multivariate analysis (p = 0.009), and a ROC curve analysis identified a threshold of 605 RU/mL above which 100% of patients (12 patients) had a poor renal outcome (p < 0.001). A similar threshold could not be defined in hPLA2R1 and rbPLA2R1 ELISAs. We conclude that rbPLA2R1 is an alternative antigen to hPLA2R1 to measure anti-PLA2R1 in active disease while mPLA2R1 is a unique antigen that can detect a subset of anti-PLA2R1 autoantibodies present at high levels (>605 RU/mL) only in iMN patients at risk of poor prognosis, and is thus useful to predict iMN outcome. PMID:26296473

  3. Purification of integral outer-membrane protein OmpC, a surface antigen from Salmonella typhi for structure-function studies: a method applicable to enterobacterial major outer-membrane protein.

    PubMed

    Arockiasamy, A; Krishnaswamy, S

    2000-07-15

    Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure-function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins. PMID:10929809

  4. HIV-1 antibodies and vaccine antigen selectively interact with lipid domains.

    PubMed

    Hardy, Gregory J; Wong, Gene C; Nayak, Rahul; Anasti, Kara; Hirtz, Michael; Shapter, Joseph G; Alam, S Munir; Zauscher, Stefan

    2014-10-01

    The rare, broadly neutralizing antibodies, 4E10 and 2F5, that target the HIV-1 membrane proximal external region also associate with HIV-1 membrane lipids as part of a required first-step in HIV-1 neutralization. HIV-1 virions have high concentration of cholesterol and sphingomyelin, which are able to organize into liquid-ordered domains (i.e., lipid rafts), and could influence the interaction of neutralizing antibodies with epitopes proximal to the membrane. The objective of this research is to understand how these lipid domains contribute to 2F5/4E10 membrane interactions and to antigen presentation in liposomal form of HIV-1 vaccines. To this end we have engineered biomimetic supported lipid bilayers and are able to use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions. Our results demonstrate that 2F5/4E10 do not interact with highly ordered gel and liquid-ordered domains and exclusively bind to a liquid-disordered lipid phase. This suggests that vaccine liposomes that contain key viral membrane components, such as high cholesterol content, may not be advantageous for 2F5/4E10 vaccine strategies. Rather, vaccine liposomes that primarily contain a liquid-disordered phase may be more likely to elicit production of lipid reactive, 2F5- and 4E10-like antibodies. PMID:25019685

  5. Utility of biological membranes as indicators for radiation exposure: alterations in membrane structure and function over time.

    PubMed

    Dainiak, N; Tan, B J

    1995-05-01

    In addition to interacting with genomic DNA, ionizing radiation may directly and indirectly alter the structure and function of components of the plasma membrane of eukaryotic cells. Water radiolysis generates reactive species, including superoxide, hypochlorous acid and chloride radicals that may in turn react with biological membranes, as well as with cellular DNA. Reaction of plasma membrane lipids with molecular oxygen results in lipid peroxidation of both reconstituted membranes and biological membranes, an effect that increases with decreasing dose rate. Both ionizing radiation and ultraviolet light alter functions of membrane-anchored molecules, including adhesion molecules, histocompatibility complex antigens and membrane-bound growth factors. The latter growth factors represent a repertoire of growth and differentiation signals that are expressed in a nondiffusible fashion at the cell surface, and in soluble forms appearing after cleavage of their extracellular domain. The importance of cell-cell signaling via the membrane-anchored form of growth factors is becoming increasingly recognized. Expression of membrane-bound hematopoietic cytokines by eukaryotic cells is impaired after exposure to ultraviolet light, a defect in cell-cell signaling that may lead to impaired hematopoiesis. While studies suggest that permanent changes in membrane structure and function may result from radiation-induced injury to the plasma membrane and reconstituted "pure" membranes, reversibility of these defects over time requires additional study. PMID:7488940

  6. Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria.

    PubMed

    Hooda, Yogesh; Lai, Christine Chieh-Lin; Judd, Andrew; Buckwalter, Carolyn M; Shin, Hyejin Esther; Gray-Owen, Scott D; Moraes, Trevor F

    2016-01-01

    Lipoproteins decorate the surface of many Gram-negative bacterial pathogens, playing essential roles in immune evasion and nutrient acquisition. In Neisseria spp., the causative agents of gonorrhoea and meningococcal meningitis, surface lipoproteins (SLPs) are required for virulence and have been extensively studied as prime candidates for vaccine development. However, the machinery and mechanism that allow for the surface display of SLPs are not known. Here, we describe a transposon (Tn5)-based search for the proteins required to deliver SLPs to the surface of Neisseria meningitidis, revealing a family of proteins that we have named the surface lipoprotein assembly modulator (Slam). N. meningitidis contains two Slam proteins, each exhibiting distinct substrate preferences. The Slam proteins are sufficient to reconstitute SLP transport in laboratory strains of Escherichia coli, which are otherwise unable to efficiently display these lipoproteins on their cell surface. Immunoprecipitation and domain probing experiments suggest that the SLP, TbpB, interacts with Slam during the transit process; furthermore, the membrane domain of Slam is sufficient for selectivity and proper surface display of SLPs. Rather than being a Neisseria-specific factor, our bioinformatic analysis shows that Slam can be found throughout proteobacterial genomes, indicating a conserved but until now unrecognized virulence mechanism. PMID:27572441

  7. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    PubMed

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-01-01

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV. PMID:24517254

  8. A pore-forming toxin requires a specific residue for its activity in membranes with particular physicochemical properties.

    PubMed

    Morante, Koldo; Caaveiro, Jose M M; Tanaka, Koji; González-Mañas, Juan Manuel; Tsumoto, Kouhei

    2015-04-24

    The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physiochemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol-rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes composed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane. PMID:25759390

  9. A Pore-Forming Toxin Requires a Specific Residue for Its Activity in Membranes with Particular Physicochemical Properties*

    PubMed Central

    Morante, Koldo; Caaveiro, Jose M. M.; Tanaka, Koji; González-Mañas, Juan Manuel; Tsumoto, Kouhei

    2015-01-01

    The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physiochemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol-rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes composed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane. PMID:25759390

  10. Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase.

    PubMed Central

    Melloni, E; Pontremoli, S; Michetti, M; Sacco, O; Sparatore, B; Salamino, F; Horecker, B L

    1985-01-01

    In the presence of micromolar concentrations of Ca2+, both protein kinase C and a cytosolic Ca2+-requiring neutral proteinase of human neutrophils become associated with the neutrophil membrane. Binding to the membrane results in activation of the proteinase, which then catalyzes limited proteolysis of the kinase to produce a form that is fully active in the absence of Ca2+ and phospholipid. This irreversibly activated protein kinase is released from the membrane and may thus have access, in the intact cell, to intracellular protein substrates. In the absence of the proteinase, Ca2+ promotes the binding of protein kinase C, but conversion to the Ca2+/phospholipid-independent form does not occur and the kinase remains associated with the membrane fraction. PMID:2995965

  11. Membrane curvature generation by a C-terminal amphipathic helix in peripherin-2/rds, a tetraspanin required for photoreceptor sensory cilium morphogenesis

    PubMed Central

    Khattree, Nidhi; Ritter, Linda M.; Goldberg, Andrew F. X.

    2013-01-01

    Summary Vertebrate vision requires photon absorption by photoreceptor outer segments (OSs), structurally elaborate membranous organelles derived from non-motile sensory cilia. The structure and function of OSs depends on a precise stacking of hundreds of membranous disks. Each disk is fully (as in rods) or partially (as in cones) bounded by a rim, at which the membrane is distorted into an energetically unfavorable high-curvature bend; however, the mechanism(s) underlying disk rim structure is (are) not established. Here, we demonstrate that the intrinsically disordered cytoplasmic C-terminus of the photoreceptor tetraspanin peripherin-2/rds (P/rds) can directly generate membrane curvature. A P/rds C-terminal domain and a peptide mimetic of an amphipathic helix contained within it each generated curvature in liposomes with a composition similar to that of OS disks and in liposomes generated from native OS lipids. Association of the C-terminal domain with liposomes required conical phospholipids, and was promoted by membrane curvature and anionic surface charge, results suggesting that the P/rds C-terminal amphipathic helix can partition into the cytosolic membrane leaflet to generate curvature by a hydrophobic insertion (wedging) mechanism. This activity was evidenced in full-length P/rds by its induction of small-diameter tubulovesicular membrane foci in cultured cells. In sum, the findings suggest that curvature generation by the P/rds C-terminus contributes to the distinctive structure of OS disk rims, and provide insight into how inherited defects in P/rds can disrupt organelle structure to cause retinal disease. They also raise the possibility that tethered amphipathic helices can function for shaping cellular membranes more generally. PMID:23886945

  12. Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits.

    PubMed

    Rangasamy, Sneha Priya; Menon, Veena; Dhopeshwarkar, Priyanka; Pal, Ranajit; Vaniambadi, Kalyanaraman S; Mahalingam, Sundarasamy

    2016-05-01

    The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine. PMID:27032514

  13. Antigenic sites in carcinoembryonic antigen.

    PubMed

    Hammarstrom, S; Shively, J E; Paxton, R J; Beatty, B G; Larsson, A; Ghosh, R; Bormer, O; Buchegger, F; Mach, J P; Burtin, P

    1989-09-01

    The epitope reactivities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen from 11 different research groups were studied using competitive solid-phase immunoassays. About 60% of all possible combinations of Mabs as inhibitors and as the primary binding antibody were investigated. The inhibition data were analyzed by a specially developed computer program "EPITOPES" which measures concordance and discordance in inhibition patterns between Mabs. The analysis showed that 43 of the 52 Mabs (83%) could be classified into one of five essentially noninteracting epitope groups (GOLD 1-5) containing between four and 15 Mabs each. The epitopes recognized by the Mabs belonging to groups 1 to 5 were peptide in nature. With one or two possible exceptions non-classifiable Mabs were either directed against carbohydrate epitopes (4 Mabs) or were inactive in the tests used. Within epitope groups GOLD 1, 4, and 5 two partially overlapping subgroups were distinguished. Mabs with a high degree of carcinoembryonic antigen specificity generally belonged to epitope groups GOLD 1 and 3. PMID:2474375

  14. Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability[W

    PubMed Central

    Schapire, Arnaldo L.; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A.

    2008-01-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca2+-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca2+-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  15. Requirement of kinesin-mediated membrane transport of WAVE2 along microtubules for lamellipodia formation promoted by hepatocyte growth factor.

    PubMed

    Takahashi, Kazuhide; Suzuki, Katsuo

    2008-07-01

    Lamellipodia formation necessary for epithelial cell migration and invasion is accomplished by rearrangement of the actin cytoskeleton at the leading edge through membrane transport of WAVE2. However, how WAVE2 is transported to the cell periphery where lamellipodia are formed remains to be established. We report here that hepatocyte growth factor (HGF) promoted lamellipodia formation and intracellular transport of WAVE2 to the cell periphery, depending on Rac1 activity, in MDA-MB-231 human breast cancer cells. Immunoblot analyses indicating the coimmunoprecipitation of WAVE2 with kinesin heavy chain KIF5B, one of the motor proteins, and IQGAP1 suggest that KIF5B and IQGAP1 formed a complex with WAVE2 in serum-starved cells and increased in their amount after HGF stimulation. Both downregulation of KIF5B by the small interfering RNA and depolymerization of microtubules with nocodazole abrogated the HGF-induced lamellipodia formation and WAVE2 transport. Therefore, we propose here that the promotion of lamellipodia formation by HGF in MDA-MB-231 cells is Rac1-dependent and requires KIF5B-mediated transport of WAVE2 and IQGAP1 to the cell periphery along microtubules. PMID:18514191

  16. Myosin Ia is required for CFTR brush border membrane trafficking and ion transport in the mouse small intestine.

    PubMed

    Kravtsov, Dmitri V; Caputo, Christina; Collaco, Anne; Hoekstra, Nadia; Egan, Marie E; Mooseker, Mark S; Ameen, Nadia A

    2012-08-01

    In enterocytes of the small intestine, endocytic trafficking of CFTR channels from the brush border membrane (BBM) to the subapical endosomes requires the minus-end motor, myosin VI (Myo6). The subapical localization of Myo6 is dependent on myosin Ia (Myo1a) the major plus-end motor associated with the BBM, suggestive of functional synergy between these two motors. In villus enterocytes of the Myo1a KO mouse small intestine, CFTR accumulated in syntaxin-3 positive subapical endosomes, redistributed to the basolateral domain and was absent from the BBM. In colon, where villi are absent and Myo1a expression is low, CFTR exhibited normal localization to the BBM in the Myo1a KO similar to WT. cAMP-stimulated CFTR anion transport in the small intestine was reduced by 58% in the KO, while anion transport in the colon was comparable to WT. Co-immunoprecipitation confirmed the association of CFTR with Myo1a. These data indicate that Myo1a is an important regulator of CFTR traffic and anion transport in the BBM of villus enterocytes and suggest that Myo1a may power apical CFTR movement into the BBM from subapical endosomes. Alternatively, it may anchor CFTR channels in the BBM of villus enterocytes as was proposed for Myo1a's role in BBM localization of sucrase-isomaltase. PMID:22510086

  17. Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions*

    PubMed Central

    Tittel, Janine; Welz, Tobias; Czogalla, Aleksander; Dietrich, Susanne; Samol-Wolf, Annette; Schulte, Markos; Schwille, Petra; Weidemann, Thomas; Kerkhoff, Eugen

    2015-01-01

    Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes. PMID:25564607

  18. Evidence for multiple mechanisms for membrane binding and integration via carboxyl-terminal insertion sequences.

    PubMed

    Kim, P K; Janiak-Spens, F; Trimble, W S; Leber, B; Andrews, D W

    1997-07-22

    Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated into the correct membrane. The mechanism of membrane integration of cytochrome b5 has been shown to be promiscuous, spontaneous, nonsaturable, and independent of membrane proteins. Thus endoplasmic reticulum localization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane. Here direct comparison of this mechanism with that of three other proteins integrated into membranes via carboxyl-terminal insertion sequences [vesicle-associated membrane protein 1(Vamp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b5, membrane selectivity for these molecules is conferred at least in part by the mechanisms of membrane integration. Bcl-2 membrane integration was similar to that of cytochrome b5 except that insertion into lipid vesicles was inefficient. Unlike cytochrome b5 and Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms for correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also demonstrate that, contrary to expectation, hydrophobicity is not sufficient for insertion sequence-mediated membrane integration. We suggest that the structure of the insertion sequence determines whether or not specific membrane-bound receptor proteins are required for membrane integration. PMID:9220974

  19. Phosphatidylglycerol biosynthesis is required for the development of embryos and normal membrane structures of chloroplasts and mitochondria in Arabidopsis.

    PubMed

    Tanoue, Ryo; Kobayashi, Megumi; Katayama, Kenta; Nagata, Noriko; Wada, Hajime

    2014-05-01

    Phosphatidylglycerophosphate (PGP) synthase, encoded by PGP1 and PGP2 in Arabidopsis, catalyzes a committed step in the biosynthesis of phosphatidylglycerol (PG). In this study, we isolated a pgp1pgp2 double mutant of Arabidopsis to study the function of PG. In this mutant, embryo development was delayed and the majority of seeds did not germinate. Thylakoid membranes did not develop in plastids, mitochondrial membrane structures were abnormal in the mutant embryos, and radiolabeling of phospholipids showed that radioactivity was not significantly incorporated into PG. These results demonstrated that PG biosynthesis is essential for the development of embryos and normal membrane structures of chloroplasts and mitochondria. PMID:24632290

  20. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

    PubMed

    McWilliam, Hamish E G; Eckle, Sidonia B G; Theodossis, Alex; Liu, Ligong; Chen, Zhenjun; Wubben, Jacinta M; Fairlie, David P; Strugnell, Richard A; Mintern, Justine D; McCluskey, James; Rossjohn, Jamie; Villadangos, Jose A

    2016-05-01

    The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability. PMID:27043408

  1. Asparagine endopeptidase is not essential for class II MHC antigen presentation but is required for processing of cathepsin L in mice.

    PubMed

    Maehr, René; Hang, Howard C; Mintern, Justine D; Kim, You-Me; Cuvillier, Armelle; Nishimura, Mikio; Yamada, Kenji; Shirahama-Noda, Kanae; Hara-Nishimura, Ikuko; Ploegh, Hidde L

    2005-06-01

    Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation. PMID:15905550

  2. Linkage between anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prevention of bacterial infections via immunization presents particular challenges. While outer membrane extracts are often protective; they are difficult and expensive to isolate and standardize, and thus often impractical for development and implementation in vaccination programs. In contrast, ind...

  3. Phosphofructokinase muscle-specific isoform requires caveolin-3 expression for plasma membrane recruitment and caveolar targeting: implications for the pathogenesis of caveolin-related muscle diseases.

    PubMed

    Sotgia, Federica; Bonuccelli, Gloria; Minetti, Carlo; Woodman, Scott E; Capozza, Franco; Kemp, Robert G; Scherer, Philipp E; Lisanti, Michael P

    2003-12-01

    Previous co-immunoprecipitation studies have shown that endogenous PFK-M (phosphofructokinase, muscle-specific isoform) associates with caveolin (Cav)-3 under certain metabolic conditions. However, it remains unknown whether Cav-3 expression is required for the plasma membrane recruitment and caveolar targeting of PFK-M. Here, we demonstrate that recombinant expression of Cav-3 dramatically affects the subcellular localization of PFK-M, by targeting PFK-M to the plasma membrane, and by trans-locating PFK-M to caveolae-enriched membrane domains. In addition, we show that the membrane recruitment and caveolar targeting of PFK-M appears to be strictly dependent on the concentration of extracellular glucose. Interestingly, recombinant expression of PFK-M with three Cav-3 mutants [DeltaTFT (63 to 65), P104L, and R26Q], which harbor the same mutations as seen in the human patients with Cav-3-related muscle diseases, causes a substantial reduction in PFK-M expression levels, and impedes the membrane recruitment of PFK-M. Analysis of skeletal muscle tissue samples from Cav-3(-/-) mice directly demonstrates that Cav-3 expression regulates the phenotypic behavior of PFK-M. More specifically, in Cav-3-null mice, PFK-M is no longer targeted to the plasma membrane, and is excluded from caveolar membrane domains. As such, our current results may be important in understanding the pathogenesis of Cav-3-related muscle diseases, such as limb-girdle muscular dystrophy-1C, distal myopathy, and rippling muscle disease, that are caused by mutations within the human Cav-3 gene. PMID:14633633

  4. Aptamer-targeted Antigen Delivery

    PubMed Central

    Wengerter, Brian C; Katakowski, Joseph A; Rosenberg, Jacob M; Park, Chae Gyu; Almo, Steven C; Palliser, Deborah; Levy, Matthew

    2014-01-01

    Effective therapeutic vaccines often require activation of T cell-mediated immunity. Robust T cell activation, including CD8 T cell responses, can be achieved using antibodies or antibody fragments to direct antigens of interest to professional antigen presenting cells. This approach represents an important advance in enhancing vaccine efficacy. Nucleic acid aptamers present a promising alternative to protein-based targeting approaches. We have selected aptamers that specifically bind the murine receptor, DEC205, a C-type lectin expressed predominantly on the surface of CD8α+ dendritic cells (DCs) that has been shown to be efficient at facilitating antigen crosspresentation and subsequent CD8+ T cell activation. Using a minimized aptamer conjugated to the model antigen ovalbumin (OVA), DEC205-targeted antigen crosspresentation was verified in vitro and in vivo by proliferation and cytokine production by primary murine CD8+ T cells expressing a T cell receptor specific for the major histocompatibility complex (MHC) I-restricted OVA257–264 peptide SIINFEKL. Compared with a nonspecific ribonucleic acid (RNA) of similar length, DEC205 aptamer-OVA-mediated antigen delivery stimulated strong proliferation and production of interferon (IFN)-γ and interleukin (IL)-2. The immune responses elicited by aptamer-OVA conjugates were sufficient to inhibit the growth of established OVA-expressing B16 tumor cells. Our results demonstrate a new application of aptamer technology for the development of effective T cell-mediated vaccines. PMID:24682172

  5. Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis

    PubMed Central

    Jouannet, Virginie; Moreno, Ana Beatriz; Elmayan, Taline; Vaucheret, Hervé; Crespi, Martin D; Maizel, Alexis

    2012-01-01

    Formation of trans-acting small interfering RNAs (ta-siRNAs) from the TAS3 precursor is triggered by the AGO7/miR390 complex, which primes TAS3 for conversion into double-stranded RNA by the RNA-dependent RNA polymerase RDR6 and SGS3. These ta-siRNAs control several aspects of plant development. The mechanism routing AGO7-cleaved TAS3 precursor to RDR6/SGS3 and its subcellular organization are unknown. We show that AGO7 accumulates together with SGS3 and RDR6 in cytoplasmic siRNA bodies that are distinct from P-bodies. siRNA bodies colocalize with a membrane-associated viral protein and become positive for stress-granule markers upon stress-induced translational repression, this suggests that siRNA bodies are membrane-associated sites of accumulation of mRNA stalled during translation. AGO7 congregates with miR390 and SGS3 in membranes and its targeting to the nucleus prevents its accumulation in siRNA bodies and ta-siRNA formation. AGO7 is therefore required in the cytoplasm and membranous siRNA bodies for TAS3 processing, revealing a hitherto unknown role for membrane-associated ribonucleoparticles in ta-siRNA biogenesis and AGO action in plants. PMID:22327216

  6. Limited Density of an Antigen Presented by RMA-S Cells Requires B7-1/CD28 Signaling to Enhance T-Cell Immunity at the Effector Phase

    PubMed Central

    Li, Xiao-Lin; Sluijter, Marjolein; Doorduijn, Elien M.; Kale, Shubha P.; McFerrin, Harris; Liu, Yong-Yu; Li, Yan; Mottamal, Madhusoodanan; Yao, Xin; Du, Fengkun; Gu, Baihan; Hoang, Kim; Nguyen, Yen H.; Taylor, Nichelle; Stephens, Chelsea R.; van Hall, Thorbald; Zhang, Qian-Jin

    2014-01-01

    The association of B7-1/CD28 between antigen presenting cells (APCs) and T-cells provides a second signal to proliferate and activate T-cell immunity at the induction phase. Many reports indicate that tumor cells transfected with B7-1 induced augmented antitumor immunity at the induction phase by mimicking APC function; however, the function of B7-1 on antitumor immunity at the effector phase is unknown. Here, we report direct evidence of enhanced T-cell antitumor immunity at the effector phase by the B7-1 molecule. Our experiments in vivo and in vitro indicated that reactivity of antigen-specific monoclonal and polyclonal T-cell effectors against a Lass5 epitope presented by RMA-S cells is increased when the cells expressed B7-1. Use of either anti-B7-1 or anti-CD28 antibodies to block the B7-1/CD28 association reduced reactivity of the T effectors against B7-1 positive RMA-S cells. Transfection of Lass5 cDNA into or pulse of Lass5 peptide onto B7-1 positive RMA-S cells overcomes the requirement of the B7-1/CD28 signal for T effector response. To our knowledge, the data offers, for the first time, strong evidence that supports the requirement of B7-1/CD28 secondary signal at the effector phase of antitumor T-cell immunity being dependent on the density of an antigenic peptide. PMID:25383875

  7. The bacteriocin AS-48 requires dimer dissociation followed by hydrophobic interactions with the membrane for antibacterial activity.

    PubMed

    Cebrián, Rubén; Martínez-Bueno, Manuel; Valdivia, Eva; Albert, Armando; Maqueda, Mercedes; Sánchez-Barrena, María José

    2015-05-01

    The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer. PMID:25816760

  8. PilF Is an Outer Membrane Lipoprotein Required for Multimerization and Localization of the Pseudomonas aeruginosa Type IV Pilus Secretin

    SciTech Connect

    Koo, J.; Tammam, S; Ku, S; Samplaeanu, L; Burrows, L; Howell, P

    2008-01-01

    Type IV pili (T4P) are retractile appendages that contribute to the virulence of bacterial pathogens. PilF is a Pseudomonas aeruginosa lipoprotein that is essential for T4P biogenesis. Phenotypic characterization of a pilF mutant confirmed that T4P-mediated functions are abrogated: T4P were no longer present on the cell surface, twitching motility was abolished, and the mutant was resistant to infection by T4P retraction-dependent bacteriophage. The results of cellular fractionation studies indicated that PilF is the outer membrane pilotin required for the localization and multimerization of the secretin, PilQ. Mutation of the putative PilF lipidation site untethered the protein from the outer membrane, causing secretin assembly in both inner and outer membranes. T4P-mediated twitching motility and bacteriophage susceptibility were moderately decreased in the lipidation site mutant, while cell surface piliation was substantially reduced. The tethering of PilF to the outer membrane promotes the correct localization of PilQ and appears to be required for the formation of stable T4P. Our 2.0-A structure of PilF revealed a superhelical arrangement of six tetratricopeptide protein-protein interaction motifs that may mediate the contacts with PilQ during secretin assembly. An alignment of pseudomonad PilF sequences revealed three highly conserved surfaces that may be involved in PilF function.

  9. A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

    PubMed Central

    Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

    2011-01-01

    To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634

  10. Recombinant hepatitis B triple antigen vaccine: Hepacare.

    PubMed

    Zuckerman, Jane N; Zuckerman, Arie J

    2002-08-01

    Infection with hepatitis B virus is a public health problem throughout the world. Hepatitis B vaccines are now included in national immunization programmes of infants and/or adolescents in 129 countries. Current single antigen vaccines, that are plasma-derived or produced by recombinant DNA technology are highly effective, but between 5-10% or more of healthy immunocompetent subjects do not mount an antihepatitis B surface antibody protective response and others respond poorly (hyporesponders). The inclusion of pre-S1 and -S2 components of hepatitis B surface antigen in addition to the single antigen (triple antigen) in a novel vaccine, Hepacare, Medeva Pharma Plc, Speke, UK, overcomes nonresponsiveness and hyporesponsiveness in a significant number of individuals. The triple antigen is indicated for vaccination of nonresponders (and hyporesponders) to the current single antigen vaccines and for persons who require rapid protection against hepatitis B infection. PMID:12901552

  11. Antigen Recognition By Variable Lymphocyte Receptors

    SciTech Connect

    Han, B.W.; Herrin, B.R.; Cooper, M.D.; Wilson, I.A.

    2009-05-18

    Variable lymphocyte receptors (VLRs) rather than antibodies play the primary role in recognition of antigens in the adaptive immune system of jawless vertebrates. Combinatorial assembly of leucine-rich repeat (LRR) gene segments achieves the required repertoire for antigen recognition. We have determined a crystal structure for a VLR-antigen complex, VLR RBC36 in complex with the H-antigen trisaccharide from human blood type O erythrocytes, at 1.67 angstrom resolution. RBC36 binds the H-trisaccharide on the concave surface of the LRR modules of the solenoid structure where three key hydrophilic residues, multiple van der Waals interactions, and the highly variable insert of the carboxyl-terminal LRR module determine antigen recognition and specificity. The concave surface assembled from the most highly variable regions of the LRRs, along with diversity in the sequence and length of the highly variable insert, can account for the recognition of diverse antigens by VLRs.

  12. [Polyagglutinability due to Hempas antigen].

    PubMed

    Rochant, H; Gerbal, A

    1976-03-01

    A new antigen has been recently discoverd in patients with congenital dyserythropoietic anemia type II. The acronyme Hempas was proposed for this disease as a remind of the main morphological feature of erythroblasts (hereditary erythroblastic multinuclearity) and the characteristic serological findings (positive acidified serum test). The patients red cells are agglutinated and lysed by an IgM cold reacting antibody present in the serum of most normal subjects and not previously recognized. This behaviour is thus reminding of cells carrying antigens such as T, Tn, Cad or acquired B. As for T and Tn cells, sialic acid and electrophoretic mobility are reduced, but in contrast, agglutinability of Hempas cells is enhanced by enzyme treatment. Agglutination by anti H and anti Pr specific reagents is reduced. I and mainly i activity are strongly increased. The relationship between the membrane abnormalities of Hempas red cells and the failure of normoblasts to divide their cytoplasm i still largely unknown. PMID:788106

  13. Membrane microdomain sphingolipids are required for anti-CD20-induced death of chronic lymphocytic leukemia B cells

    PubMed Central

    Hammadi, Mariam; Youinou, Pierre; Tempescul, Adrian; Tobón, Gabriel; Berthou, Christian; Bordron, Anne; Pers, Jacques-Olivier

    2012-01-01

    Background Chronic lymphocytic leukemia remains incurable, despite the addition of rituximab to chemotherapy as an available means of treatment. The resistance of certain patients to this monoclonal antibody prompted us to set up in vitro studies of another CD20-specific monoclonal antibody, B1 (later termed tositumomab). We hypothesized that the membrane lipid organization of leukemic B cells might be instrumental in the cells’ sensitivity to the B1 monoclonal antibody. Design and Methods B lymphocytes from 36 patients with chronic lymphocytic leukemia and 13 patients with non-Hodgkin’s lymphoma were investigated for B1-triggered cell death. Membrane components, such as sphingomyelin and ganglioside M1, were investigated by flow cytometry, immunofluorescence and co-immunoprecipitation, together with the Csk-binding protein. Results Chronic lymphocytic leukemia patients segregated into two groups: B cells from one group were sensitive to B1, whereas those from the second group were not. Further results ascribed the resistance of these latter cases to a defective recruitment of Csk-binding protein, resulting in a lack of sphingomyelin and ganglioside M1 at the outer leaflet of the plasma membrane of their malignant B cells. Sphingolipids were indeed retained in the cytoplasm, because of lowered activity of P-glycoprotein. Supporting this mechanism, rifampicin, an inducer of P-glycoprotein, improved the activity of this transmembrane efflux pump, normalized the quantity of sphingomyelin within the membrane, and thereby restored the efficacy of the B1 monoclonal antibody in the formerly B1-resistant cases of chronic lymphocytic leukemia. Conclusions The lipid organization of membranes of B cells from patients with chronic lymphocytic leukemia differs from one patient to another. In practice, given the relevance of the membrane lipid distribution to the efficacy of biotherapies, this observation is of potential importance. PMID:22058197

  14. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    PubMed Central

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  15. Interleukin-1-induced gene expression requires the membrane-raft-dependent internalization of the interleukin-1 receptor.

    PubMed

    Windheim, Mark

    2016-10-01

    Interleukin-1 (IL-1) binding to its receptor triggers signaling events at the plasma membrane that are essential but not sufficient for the induction of the IL-1-dependent gene expression. In addition, the ligand-induced endocytosis of the IL-1 receptor and signaling events that are initiated after the internalization of the IL-1 receptor presumably involving signaling endosomes are critical for the IL-1-induced gene expression. In this study, we investigate the role of membrane domains, commonly denoted as lipid rafts, in the IL-1-induced signal transduction. We demonstrate that the internalization of the IL-1 receptor depends on the integrity of lipid rafts and that the disruption of lipid rafts strongly reduces the IL-1-induced gene expression. Interestingly, the IL-1-dependent signaling events activated at the plasma membrane are not influenced by the disruption of lipid rafts suggesting that IL-1 signaling is initiated in a non-raft domain of the plasma membrane. Subsequently, the IL-1 receptor is translocated to lipid rafts where receptor endocytosis occurs to enable the internalization-dependent IL-1 signaling to activate the IL-1-induced gene expression. PMID:27327966

  16. Epstein-Barr Virus-Encoded Latent Membrane Protein 2A Promotes the Epithelial-Mesenchymal Transition in Nasopharyngeal Carcinoma via Metastatic Tumor Antigen 1 and Mechanistic Target of Rapamycin Signaling Induction

    PubMed Central

    Lin, Zhe; Wan, Xin; Jiang, Runqiu; Deng, Lei; Gao, Yun; Tang, Junwei; Yang, Yu; Zhao, Wei; Yan, Xin; Yao, Kun

    2014-01-01

    ABSTRACT Epstein-Barr virus-encoded latent membrane protein 2A (LMP2A) promotes the epithelial-mesenchymal transition (EMT) of nasopharyngeal carcinoma (NPC), thereby increasing tumor invasion. Recently, the dysregulation of metastatic tumor antigen 1 (MTA1) was found to enhance tumor metastasis in a variety of cancers. A molecular connection between these two proteins has been proposed but not firmly established. In this study, we reported the overexpression of MTA1 in 29/60 (48.3%) NPC patients, and the overexpression of MTA1 significantly correlated with tumor metastasis. The overexpression of MTA1 promoted EMT via the Wnt1 pathway and β-catenin activation. We demonstrated that LMP2A reinforces the expression of MTA1 via the mechanistic target of rapamycin (mTOR) pathway to promote EMT in NPC. Furthermore, by knocking down 4EBP1 in combination with the new mTOR inhibitor INK-128 treatment, we discovered that LMP2A expression activates the 4EBP1-eIF4E axis and increases the expression of MTA1 at the translational level partially independent of c-myc. These findings provided novel insights into the correlation between the LMP2A and MTA1 proteins and reveal a novel function of the 4EBP1-eIF4E axis in EMT of nasopharyngeal carcinoma. IMPORTANCE Prevention of the recurrence and metastasis of NPC is critical to achieving a successful NPC treatment. As we all know, EMT has a vital role in metastasis of malignancies. LMP2A, an oncoprotein of Epstein-Barr virus, a well-known NPC activator, induces EMT and has been proved to exert a promoting effect in tumor metastasis. Our study demonstrated that LMP2A could induce EMT by activating MTA1 at the translational level via activating mTOR signaling and the 4EBP1-eIF4E axis. Taken together, our findings bridge the gap between the NPC-specific cell surface molecule and the final phenotype of the NPC cells. Additionally, our findings indicate that LMP2A and mTOR will serve as targets for NPC therapy in the future. PMID:25100829

  17. Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

    PubMed Central

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A. Pedro; Starr, Trevor L.

    2015-01-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

  18. Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.

    PubMed

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A Pedro; Starr, Trevor L; Glass, N Louise

    2015-03-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

  19. Diffusion of nitric oxide across cell membranes of the vascular wall requires specific connexin-based channels.

    PubMed

    Figueroa, Xavier F; Lillo, Mauricio A; Gaete, Pablo S; Riquelme, Manuel A; Sáez, Juan C

    2013-12-01

    NO is generated within cells and frequently must be transferred to responsive neighboring cells, as occurs in the endothelium-dependent relaxation of smooth muscle cells observed in blood vessels. It is thought that NO diffuses freely across cell membranes, but it may also permeate through low resistant membrane pathways. Here, we describe the participation of connexin (Cx)-formed channels in the NO transport across cell membranes and between endothelial and smooth muscle cells. We used a water-soluble NO donor of high molecular weight (S-nitrosylated albumin, BSA-NO) that does not permeate through cell membranes or Cx-based channels and the NO-sensitive dye 4,5-diaminofluorescein diacetate to detect changes of intracellular NO concentration. We found that NO generated in the extracellular space was not detected intracellularly in Cx-deficient HeLa cells, suggesting that cell membrane represents a significant diffusion barrier for NO transfer. However, Cx-based channels provide efficient pathways for NO signaling because NO opened and permeated hemichannels expressed in HeLa cells transfected with Cx43, Cx40, or Cx37. In contrast, NO closed hemichannels of HeLa-Cx32 cells, which otherwise are permeable to NO if are opened by a divalent cation-free extracellular solution. Consistent with this, blockade of Cx-based channels abolished the myoendothelial NO transfer and associated NO-dependent vasodilation induced by acethylcholine. These results indicate that Cx-based channels play a key role in the NO-dependent tonic control of vascular function and may direct the NO signal to specific targets, which provides a novel mechanistic basis for the critical role of Cxs in cell-cell communication in the vessel wall. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'. PMID:23499665

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

    SciTech Connect

    Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J. . E-mail: matthias.schnell@jefferson.edu

    2006-09-30

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen 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. Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements.

    PubMed Central

    Taylor, S J; Exton, J H

    1987-01-01

    The effect of the GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with adenosine 5'-[gamma-thio]triphosphate, and was inhibited by guanosine 5'-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is

  2. Rotavirus antigen test

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003349.htm Rotavirus antigen test To use the sharing features on this page, please enable JavaScript. The rotavirus antigen test detects rotavirus in the feces. This ...

  3. Evaluation of hepatocellular carcinoma aggressiveness by a panel of extracellular matrix antigens.

    PubMed Central

    Grigioni, W. F.; Garbisa, S.; D'Errico, A.; Baccarini, P.; Stetler-Stevenson, W. G.; Liotta, L. A.; Mancini, A. M.

    1991-01-01

    Invasion and metastasis requires a series of interactions between malignant cells and the extracellular matrix (ECM). Antigen markers that relate to these interactions were evaluated for prognostic correlation in human hepatocellular carcinoma. Basement membrane type IV collagen (cIV), type IV collagenase (cIVase), laminin, and laminin receptors (LRs)--all ECM antigens previously proposed to be modulated in association with tumor aggressiveness--were immunohistochemically investigated in 30 cases of hepatocellular carcinomas (HCCs). The pattern of antigen expression was correlated with 1) 36 months' clinical follow-up and 2) the pathologic grade. As a means of estimating the proliferation fraction, an additional antigen, Ki67, was also studied in this series. There were major differences in the distribution of cIV and laminin, and in the quantity of cIVase-, LR-, and Ki67-positive cells associated with grade and prognosis. A smaller quantity of cIV and laminin and a higher number of cIVase-, LR-, and Ki67-positive cells were detected in the poorly differentiated compared with the well-differentiated HCCs. The tumors with lower immunoreactivity for cIV and laminin components accompanied by a higher number of cIVase-, LR-, and Ki67-positive cells fall into a group with the poorest overall survival (P less than 0.006). The panel of antigens is proposed as a useful prognostic tool for evaluating HCC tumor aggressiveness. Images Figure 2 Figure 3 Figure 4 PMID:1848041

  4. The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis

    PubMed Central

    Guerrier, Sabrice; Coutinho-Budd, Jaeda; Sassa, Takayuki; Gresset, Aurélie; Jordan, Nicole Vincent; Cheng, Ken; Jin, Wei-Lin; Frost, Adam; Polleux, Franck

    2009-01-01

    SUMMARY During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in non-neuronal cells, filopodia dynamics decreases the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results (1) suggest that F-BAR domains are functionally diverse and (2) highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis. PMID:19737524

  5. In vitro activation of a transcription factor by gamma interferon requires a membrane-associated tyrosine kinase and is mimicked by vanadate.

    PubMed Central

    Igarashi, K; David, M; Larner, A C; Finbloom, D S

    1993-01-01

    Gamma interferon (IFN-gamma) activates the formation of a DNA-binding protein complex (FcRF gamma) that recognizes the gamma response region (GRR) of the promoter for the human high-affinity Fc gamma receptor. In a membrane-enriched fraction prepared from human peripheral blood monocytes, IFN-gamma activation of FcRF gamma occurred within 1 min and was ATP dependent. Activation of FcRF gamma required a tyrosine kinase activity, and recognition of the GRR sequence by FcRF gamma could be abrogated by treatment with a tyrosine-specific protein phosphatase. Treatment of cells with vanadate alone resulted in the formation of FcRF gamma without the need for IFN-gamma. UV cross-linking and antibody competition experiments demonstrated that the FcRF gamma complex was composed of at least two components: the 91-kDa protein of the IFN-alpha-induced transcription complex ISGF3 and a 43-kDa component that bound directly to the GRR. Therefore, specificity for IFN-induced transcriptional activation of early response genes requires at least two events: (i) ligand-induced activation of membrane-associated protein by tyrosine phosphorylation and (ii) formation of a complex composed of an activated membrane protein(s) and a sequence-specific DNA-binding component. Images PMID:8321205

  6. Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment

    PubMed Central

    Potluri, Lakshmiprasad; Karczmarek, Aneta; Verheul, Jolanda; Piette, Andre; Wilkin, Jean-Marc; Werth, Nadine; Banzhaf, Manuel; Vollmer, Waldemar; Young, Kevin D; Nguyen-Distèche, Martine; den Blaauwen, Tanneke

    2010-01-01

    The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment. PMID:20545860

  7. Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of a Genotype-Specific Immunodominant CD8+ T-Cell Epitope

    PubMed Central

    Kersting, Nadine; Fitzmaurice, Karen; Oniangue-Ndza, Cesar; Kemper, Michael N.; Humphreys, Isla; McKiernan, Susan; Kelleher, Dermot; Lohmann, Volker; Bowness, Paul; Huzly, Daniela; Rosen, Hugo R.; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Barnes, Eleanor; Roggendorf, Michael; Blum, Hubert E.; Thimme, Robert

    2015-01-01

    Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. PMID:20034048

  8. HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death

    PubMed Central

    Jacobsen, A V; Lowes, K N; Tanzer, M C; Lucet, I S; Hildebrand, J M; Petrie, E J; van Delft, M F; Liu, Z; Conos, S A; Zhang, J-G; Huang, D C S; Silke, J; Lessene, G; Murphy, J M

    2016-01-01

    Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3. PMID:26775703

  9. HSP90 activity is required for MLKL oligomerisation and membrane translocation and the induction of necroptotic cell death.

    PubMed

    Jacobsen, A V; Lowes, K N; Tanzer, M C; Lucet, I S; Hildebrand, J M; Petrie, E J; van Delft, M F; Liu, Z; Conos, S A; Zhang, J-G; Huang, D C S; Silke, J; Lessene, G; Murphy, J M

    2016-01-01

    Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90's previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3. PMID:26775703

  10. Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes.

    PubMed

    Aseeva, Elena; Ossenbühl, Friederich; Sippel, Claudia; Cho, Won K; Stein, Bernhard; Eichacker, Lutz A; Meurer, Jörg; Wanner, Gerhard; Westhoff, Peter; Soll, Jürgen; Vothknecht, Ute C

    2007-02-01

    Vipp1 (vesicle inducing protein in plastids 1) is found in cyanobacteria and chloroplasts where it is essential for thylakoid formation. Arabidopsis thaliana mutant plants with a reduction of Vipp1 to about 20% of wild type content become albinotic at an early stage. We propose that this drastic phenotype results from an inability of the remaining Vipp1 protein to assemble into a homo-oligomeric complex, indicating that oligomerization is a prerequisite for Vipp1 function. A Vipp1-ProteinA fusion protein, expressed in the Deltavipp1 mutant background, is able to reinstate oligomerization and restore photoautotrophic growth. Plants containing Vipp1-ProteinA in amounts comparable to Vipp1 in the wild type exhibit a wild type phenotype. However, plants with a reduced amount of Vipp1-ProteinA protein are growth-retarded and significantly paler than the wild type. This phenotype is caused by a decrease in thylakoid membrane content and a concomitant reduction in photosynthetic activity. To the extent that thylakoid membranes are made in these plants they are properly assembled with protein-pigment complexes and are photosynthetically active. This strongly supports a function of Vipp1 in basic thylakoid membrane formation and not in the functional assembly of thylakoid protein complexes. Intriguingly, electron microscopic analysis shows that chloroplasts in the mutant plants are not equally affected by the Vipp1 shortage. Indeed, a wide range of different stages of thylakoid development ranging from wild-type-like chloroplasts to plastids nearly devoid of thylakoids can be observed in organelles of one and the same cell. PMID:17346982

  11. Identification of Neospora antigens recognized by CD4+ T cells and immune sera from experimentally infected cattle.

    PubMed

    Marks, J; Lundén, A; Harkins, D; Innes, E

    1998-07-01

    Neospora caninum is recognized as a major cause of infectious abortion in cattle. Very little is known about immunity to Neospora. Cell mediated responses have previously been shown to be important in the development of protective immunity to the closely related parasite Toxoplasma gondii, and may therefore be an important component in the immune response to Neospora. In this paper we report that a group of low molecular weight NCI strain tachyzoite antigens (< or = 30 kDa) separated by SDS PAGE and bound to nitrocellulose membrane stimulated proliferation in vitro of CD4+ T cells from calves experimentally infected with N. caninum. Proliferation was accompanied by production of high concentrations of IFN-gamma. Several of these antigens were also recognized by antibody produced in these animals. As the most effective vaccines require the stimulation of both humoral and cell mediated immune responses, these antigens may be important in the development of a vaccine against neosporosis. PMID:9717191

  12. Pentobarbital-Induced Myocardial Stunning in Status Epilepticus Requiring Extracorporeal Membrane Oxygenation: A Case Report and Literature Review

    PubMed Central

    Molaie, Donna; Nurok, Michael; Rosengart, Axel; Lahiri, Shouri

    2016-01-01

    Introduction. Mild hypotension is a well-recognized complication of intravenous pentobarbital; however fulminant cardiopulmonary failure has not been previously reported. Case Report. A 28-year-old woman developed pentobarbital-induced cardiopulmonary failure that was successfully treated with maximal medical management including arteriovenous extracorporeal membrane oxygenation. She made an excellent cardiopulmonary and neurological recovery. Discussion and Conclusion. Pentobarbital is underrecognized as a potential cause of myocardial stunning. The mechanism involves direct myocardial depression and inhibition of autonomic neuroanatomical structures including the medulla and hypothalamus. Early recognition and implementation of aggressive cardiopulmonary support are essential to optimize the likelihood of a favorable outcome. PMID:27529037

  13. Pentobarbital-Induced Myocardial Stunning in Status Epilepticus Requiring Extracorporeal Membrane Oxygenation: A Case Report and Literature Review.

    PubMed

    Kavi, Tapan; Molaie, Donna; Nurok, Michael; Rosengart, Axel; Lahiri, Shouri

    2016-01-01

    Introduction. Mild hypotension is a well-recognized complication of intravenous pentobarbital; however fulminant cardiopulmonary failure has not been previously reported. Case Report. A 28-year-old woman developed pentobarbital-induced cardiopulmonary failure that was successfully treated with maximal medical management including arteriovenous extracorporeal membrane oxygenation. She made an excellent cardiopulmonary and neurological recovery. Discussion and Conclusion. Pentobarbital is underrecognized as a potential cause of myocardial stunning. The mechanism involves direct myocardial depression and inhibition of autonomic neuroanatomical structures including the medulla and hypothalamus. Early recognition and implementation of aggressive cardiopulmonary support are essential to optimize the likelihood of a favorable outcome. PMID:27529037

  14. Vacuole Membrane Protein 1 Is an Endoplasmic Reticulum Protein Required for Organelle Biogenesis, Protein Secretion, and Development

    PubMed Central

    Calvo-Garrido, Javier; Carilla-Latorre, Sergio; Lázaro-Diéguez, Francisco; Egea, Gustavo

    2008-01-01

    Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1− Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1− cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised. PMID:18550798

  15. Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.

    PubMed

    Schmidt, Helgo; Hansen, Guido; Singh, Sonia; Hanuszkiewicz, Anna; Lindner, Buko; Fukase, Koichi; Woodard, Ronald W; Holst, Otto; Hilgenfeld, Rolf; Mamat, Uwe; Mesters, Jeroen R

    2012-04-17

    WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA. PMID:22474366

  16. Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

    PubMed Central

    Ortiz, D F; Kreppel, L; Speiser, D M; Scheel, G; McDonald, G; Ow, D W

    1992-01-01

    In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants. Images PMID:1396551

  17. Minimum structural requirements for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s.

    PubMed

    Fujii, Kana; Morita, Daichi; Onoda, Kenji; Kuroda, Teruo; Miyachi, Hiroyuki

    2016-05-01

    Macrocyclic bis(bibenzyl)-type phenolic natural products, found exclusively in bryophytes, exhibit potent antibacterial activity towards methicillin-resistant Staphylococcus aureus (anti-MRSA activity). Here, in order to identify the minimum essential structure for cell membrane leakage-mediated anti-MRSA activity of these compounds, we synthesized acyclic fragment structures and evaluated their anti-MRSA activity. The activities of all of the acyclic fragments tested exhibited similar characteristics to those of the macrocycles, i.e., anti-MRSA bactericidal activity, an enhancing effect on influx and efflux of ethidium bromide (EtBr: fluorescent DNA-binder) in Staphylococcus aureus cells, and bactericidal activity towards a Staphylococcus aureus strain resistant to 2-phenoxyphenol (4). The latter result suggests that they have a different mechanism of action from 4, which is a FabI inhibitor previously proposed to be the minimum active fragment of riccardin-type macrocycles. Thus, cyclic structure is not a necessary condition for cell membrane leakage-mediated anti-MRSA activity of macrocyclic bis(bibenzyl)s. PMID:26995530

  18. Localization of Pom121 to the inner nuclear membrane is required for an early step of interphase nuclear pore complex assembly

    PubMed Central

    Funakoshi, Tomoko; Clever, Michaela; Watanabe, Ai; Imamoto, Naoko

    2011-01-01

    The nuclear pore complex (NPC) is a large protein assembly that mediates molecular trafficking between the cytoplasm and the nucleus. NPCs assemble twice during the cell cycle in metazoans: postmitosis and during interphase. In this study, using small interfering RNA (siRNA) in conjunction with a cell fusion–based NPC assembly assay, we demonstrated that pore membrane protein (Pom)121, a vertebrate-specific integral membrane nucleoporin, is indispensable for an early step in interphase NPC assembly. Functional domain analysis of Pom121 showed that its nuclear localization signals, which bind to importin β via importin α and likely function with RanGTP, play an essential role in targeting Pom121 to the interphase NPC. Furthermore, a region of Pom121 that interacts with the inner nuclear membrane (INM) and lamin B receptor was found to be crucial for its NPC targeting. Based on these findings and on evidence that Pom121 localizes at the INM in the absence of a complete NPC structure, we propose that the nuclear migration of Pom121 and its subsequent interaction with INM proteins are required to initiate interphase NPC assembly. Our data also suggest, for the first time, the importance of the INM as a seeding site for “prepores” during interphase NPC assembly. PMID:21289085

  19. VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals.

    PubMed

    Taubenblatt, P; Dedieu, J C; Gulik-Krzywicki, T; Morel, N

    1999-10-01

    Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin. After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoichiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process

  20. Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

    PubMed Central

    Claudi, Beatrice; Mazé, Alain; Schemmer, Anne K.; Kirchhoff, Dennis; Schmidt, Alexander; Burton, Neil; Bumann, Dirk

    2012-01-01

    Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens. PMID:23093937

  1. Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins.

    PubMed

    Eisenbach, L; Ramanathan, R; Nelson, D L

    1983-11-01

    The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross-reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i-antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential. PMID:6415066

  2. Atomic structure of anthrax protective antigen pore elucidates toxin translocation.

    PubMed

    Jiang, Jiansen; Pentelute, Bradley L; Collier, R John; Zhou, Z Hong

    2015-05-28

    Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Protective antigen forms oligomeric prepores that undergo conversion to membrane-spanning pores by endosomal acidification, and these pores translocate the enzymes lethal factor and oedema factor into the cytosol of target cells. Protective antigen is not only a vaccine component and therapeutic target for anthrax infections but also an excellent model system for understanding the mechanism of protein translocation. On the basis of biochemical and electrophysiological results, researchers have proposed that a phi (Φ)-clamp composed of phenylalanine (Phe)427 residues of protective antigen catalyses protein translocation via a charge-state-dependent Brownian ratchet. Although atomic structures of protective antigen prepores are available, how protective antigen senses low pH, converts to active pore, and translocates lethal factor and oedema factor are not well defined without an atomic model of its pore. Here, by cryo-electron microscopy with direct electron counting, we determine the protective antigen pore structure at 2.9-Å resolution. The structure reveals the long-sought-after catalytic Φ-clamp and the membrane-spanning translocation channel, and supports the Brownian ratchet model for protein translocation. Comparisons of four structures reveal conformational changes in prepore to pore conversion that support a multi-step mechanism by which low pH is sensed and the membrane-spanning channel is formed. PMID:25778700

  3. T cell-dependent IgM memory B cells generated during bacterial infection are required for IgG responses to antigen challenge.

    PubMed

    Yates, Jennifer L; Racine, Rachael; McBride, Kevin M; Winslow, Gary M

    2013-08-01

    Immunological memory has long considered to be harbored in B cells that express high-affinity class-switched IgG. IgM-positive memory B cells can also be generated following immunization, although their physiological role has been unclear. In this study, we show that bacterial infection elicited a relatively large population of IgM memory B cells that were uniquely identified by their surface expression of CD11c, CD73, and programmed death-ligand 2. The cells lacked expression of cell surface markers typically expressed by germinal center B cells, were CD138 negative, and did not secrete Ab ex vivo. The population was also largely quiescent and accumulated somatic mutations. The IgM memory B cells were located in the region of the splenic marginal zone and were not detected in blood or other secondary lymphoid organs. Generation of the memory cells was CD4 T cell dependent and required IL-21R signaling. In vivo depletion of the IgM memory B cells abrogated the IgG recall responses to specific Ag challenge, demonstrating that the cell population was required for humoral memory, and underwent class-switch recombination following Ag encounter. Our findings demonstrate that T cell-dependent IgM memory B cells can be elicited at high frequency and can play an important role in maintaining long-term immunity during bacterial infection. PMID:23804710

  4. Plasma Membrane –Cell Wall Adhesion Is Required for Expression of Plant Defense Responses during Fungal Penetration

    PubMed Central

    Mellersh, Denny G.; Heath, Michèle C.

    2001-01-01

    Fungal pathogens almost invariably trigger cell wall–associated defense responses, such as extracellular hydrogen peroxide generation and callose deposition, when they attempt to penetrate either resistant or susceptible plant cells. In the current study, we provide evidence that the expression of these defenses is dependent on adhesion between the plant cell wall and the plasma membrane. Peptides containing an Arg-Gly-Asp (RGD) motif, which interfered with plasma membrane–cell wall adhesion as shown by the loss of the thin plasma membrane–cell wall connections known as Hechtian strands, reduced the expression of cell wall–associated defense responses during the penetration of nonhost plants by biotrophic fungal pathogens. This reduction was associated with increased fungal penetration efficiency. Neither of these effects was seen after treatment with similar peptides lacking the RGD motif. Disruption of plant microfilaments had no effect on Hechtian strands but mimicked the effect of RGD peptides on wall defenses, suggesting that the expression of cell wall–associated defenses involves communication between the plant cell wall and the cytosol across the plasma membrane. To visualize the state of the plasma membrane–cell wall interaction during fungal penetration, we observed living cells during sucrose-induced plasmolysis. In interactions that were characterized by the early expression of cell wall–associated defenses, there was no change, or an increase, in plasma membrane–cell wall adhesion under the penetration point as the fungus grew through the plant cell wall. In contrast, for rust fungus interactions with host plants, there was a strong correlation between a lack of cell wall–associated defenses and a localized decrease in plasma membrane–cell wall adhesion under the penetration point. Abolition of this localized decreased adhesion by previous inoculation with a fungus that increased plasma membrane–cell wall adhesion resulted in

  5. Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

    PubMed

    Matsuda, Shuichi; Takano, Sho; Sato, Moeko; Furukawa, Kaoru; Nagasawa, Hidetaka; Yoshikawa, Shoko; Kasuga, Jun; Tokuji, Yoshihiko; Yazaki, Kazufumi; Nakazono, Mikio; Takamure, Itsuro; Kato, Kiyoaki

    2016-03-01

    Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure. PMID:26708605

  6. Direct Contacts Between Extracellular Membrane-Proximal Domains are Required for VEGF Receptor Activation and Cell Signaling

    SciTech Connect

    Yang, Y.; Xie, P; Opatowsky, Y; Schlessinger, J

    2010-01-01

    Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 {angstrom}. The structure shows that homotypic D7 contacts are mediated by salt bridges and van der Waals contacts formed between Arg726 of one protomer and Asp731 of the other protomer. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.

  7. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    PubMed

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

  8. Suppression of Apoptosis by Basement Membrane Requires three-dimensional Tissue Organization and Withdrawal from the Cell Cycle

    SciTech Connect

    Boudreau, N.; Werb, Z.; Bissell, M.J.

    1995-12-28

    The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce {beta}-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the G1 cyclin kinase inhibitor p21/WAF-I and positive proliferative signals including c-myc and cyclin Dl were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor a and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-{beta}1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

  9. Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

    SciTech Connect

    Kao, K.J.

    1988-01-01

    To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I-125-labeled platelets showed that only beta 2-M but not other I-125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I-125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins.

  10. The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress

    PubMed Central

    Johnson, Kristen A.; Taghon, Geoffrey J. F.; Scott, Jordan L.; Stahelin, Robert V.

    2016-01-01

    VP40 is one of eight proteins encoded by the Ebola Virus (EBOV) and serves as the primary matrix protein, forming virus like particles (VLPs) from mammalian cells without the need for other EBOV proteins. While VP40 is required for viral assembly and budding from host cells during infection, the mechanisms that target VP40 to the plasma membrane are not well understood. Phosphatidylserine is required for VP40 plasma membrane binding, VP40 hexamer formation, and VLP egress, However, PS also becomes exposed on the outer membrane leaflet at sites of VP40 budding, raising the question of how VP40 maintains an interaction with the plasma membrane inner leaflet when PS is flipped to the opposite side. To address this question, cellular and in vitro assays were employed to determine if phosphoinositides are important for efficient VP40 localization to the plasma membrane. Cellular studies demonstrated that PI(4,5)P2 was an important component of VP40 assembly at the plasma membrane and subsequent virus like particle formation. Additionally, PI(4,5)P2 was required for formation of extensive oligomers of VP40, suggesting PS and PI(4,5)P2 have different roles in VP40 assembly where PS regulates formation of hexamers from VP40 dimers and PI(4,5)P2 stabilizes and/or induces extensive VP40 oligomerization at the plasma membrane. PMID:26753796

  11. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  12. Meningococcal vaccine antigen diversity in global databases

    PubMed Central

    Brehony, C; Hill, DM; Lucidarme, J; Borrow, R; Maiden, MC

    2016-01-01

    The lack of an anti-capsular vaccine against serogroup B meningococcal disease has necessitated the exploration of alternative vaccine candidates, mostly proteins exhibiting varying degrees of antigenic variation. Analysis of variants of antigen-encoding genes is facilitated by publicly accessible online sequence repositories, such as the Neisseria PubMLST database and the associated Meningitis Research Foundation Meningococcus Genome Library (MRF-MGL). We investigated six proposed meningococcal vaccine formulations by deducing the prevalence of their components in the isolates represented in these repositories. Despite high diversity, a limited number of antigenic variants of each of the vaccine antigens were prevalent, with strong associations of particular variant combinations with given serogroups and genotypes. In the MRF-MGL and globally, the highest levels of identical sequences were observed with multicomponent/multivariant vaccines. Our analyses further demonstrated that certain combinations of antigen variants were prevalent over periods of decades in widely differing locations, indicating that vaccine formulations containing a judicious choice of antigen variants have potential for long-term protection across geographic regions. The data further indicated that formulations with multiple variants would be especially relevant at times of low disease incidence, as relative diversity was higher. Continued surveillance is required to monitor the changing prevalence of these vaccine antigens. PMID:26676305

  13. The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

    PubMed Central

    LIU, Chaohong; FALLEN, Margaret K.; MILLER, Heather; UPADHYAYA, Arpita; SONG, Wenxia

    2014-01-01

    The B cell antigen receptor (BCR) is the sensor on the B cell surface that surveys foreign molecules (antigen) in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR-mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling. PMID:24999354

  14. THE N-TERMINAL AMPHIPATHIC REGION OF THE ESCHERICHIA COLI TYPE III SECRETION SYSTEM PROTEIN EspD IS REQUIRED FOR MEMBRANE INSERTION AND FUNCTION

    PubMed Central

    Dasanayake, Dayal; Richaud, Manon; Cyr, Normand; Caballero-Franco, Celia; Pitroff, Sabrina; Finn, Ron M.; Ausió, Juan; Luo, Wensheng; Donnenberg, Michael S.; Jardim, Armando

    2011-01-01

    Enterohemorrhagic E. coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1–171, contains two amphipathic domains spanning residues 24–41 and 66–83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His6-EspD1-171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His6-EspD1-171 undergoes a pH depended conformational change that increases the α-helix content of this protein ~7-fold. This change coincides with insertion of the region circumscribing Trp47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His6-EspD1-171 forms a homodimer that is postulated to promote EspD-EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66-83 gene showed that this protein was secreted but non-functional. PMID:21651628

  15. A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

    PubMed Central

    Ward IV, Robert E.; Lamb, Rebecca S.; Fehon, Richard G.

    1998-01-01

    The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members. PMID:9508778

  16. Unusual N-glycan Structures Required for Trafficking Toxoplasma gondii GAP50 to the Inner Membrane Complex Regulate Host Cell Entry Through Parasite Motility*

    PubMed Central

    Fauquenoy, Sylvain; Hovasse, Agnès; Sloves, Pierre-Julien; Morelle, Willy; Alayi, Tchilabalo Dilezitoko; Slomianny, Christian; Werkmeister, Elisabeth; Schaeffer, Christine; Van Dorsselaer, Alain; Tomavo, Stanislas

    2011-01-01

    Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry. PMID:21610105

  17. Insights into the Function of YciM, a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

    PubMed Central

    Nicolaes, Valérie; El Hajjaji, Hayat; Davis, Rebecca M.; Van der Henst, Charles; Depuydt, Matthieu; Leverrier, Pauline; Aertsen, Abram; Haufroid, Vincent; Ollagnier de Choudens, Sandrine; De Bolle, Xavier; Ruiz, Natividad

    2013-01-01

    The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function. PMID:24187084

  18. Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

    DOE PAGESBeta

    Stromberg, Loreen R.; Hengartner, Nicolas W.; Swingle, Kirstie L.; Moxley, Rodney A.; Graves, Steven W.; Montano, Gabriel A.; Mukundan, Harshini; Gasset, Maria

    2016-05-26

    Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assaysmore » to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection

  19. Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the Dynamics of Amphiphile-in-Lipid Assays

    PubMed Central

    Hengartner, Nicolas W.; Swingle, Kirstie L.; Moxley, Rodney A.; Graves, Steven W.

    2016-01-01

    Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. We also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures. PMID

  20. Relationship between Fc receptors, antigen-binding sites on T and B cells, and H-2 complex-associated determinants.

    PubMed

    Basten, A; Miller, J F; Abraham, R

    1975-03-01

    The relationship between H-2 complex-associated determinants, Fc receptors, and specific antigen-recognition sites on T and B cells was examined by binding and functional assays. The Fc receptor was detected by radiolabeled immune complexes or aggregated human IgG. Both these reagents selectively bound to B cells, not to T cells. When spleen cells, from mice primed to several antigens, were exposed to highly substituted radioactive aggregates, their capacity to transfer both a direct and indirect plaque-forming cell response to these antigens was abrogated. Addition of B cells, but not of T cells, restored responsiveness. Complexed Ig binding to Fc receptors was prevented by pretreatment of mixed lymphoid cell populations with antisera directed against membrane components on the same cell (e.g., H-2) and on other cells (e.g., theta). The lack of specificity of inhibition was thought to be due to the formation on cell surfaces of antigen-antibody complexes which would then attach to the Fc receptor during the incubation precedure. Specific blockade of the Fc receptor during the incubation procedure. Specific blockade of the Fc receptor however occurred when B cells were pretreated with the Fab fragments of anti-H-2 antibody. This was demonstrated autoradiographically and by inhibition of aggregate-induced suicide. The blocking activity of ante-H-2 Fab was removed by absorption with spleen cells from thymectomized irradiated mice but not with thymus cells of appropriate specificity. This suggested that the antibodies involved had specificity for determinants on the B-cell membrane distinct from those coded by the K or D end of the H-2 complex, and either absent from, or poorly represented on, thymus cells. Specific antigen-induced suicide of B cells was achieved simply by incubating the cells with radioactive antigen in the cold. T-cell suicide on the other hand required that the 125I-labeled antigen be presented to the T cells at 37 degrees-C on the surface of

  1. A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor

    PubMed Central

    Fujimura-Kamada, Konomi; Nouvet, Franklin J.; Michaelis, Susan

    1997-01-01

    Many secreted bioactive signaling molecules, including the yeast mating pheromones a-factor and α-factor, are initially synthesized as precursors requiring multiple intracellular processing enzymes to generate their mature forms. To identify new gene products involved in the biogenesis of a-factor in Saccharomyces cerevisiae, we carried out a screen for MATa-specific, mating-defective mutants. We have identified a new mutant, ste24, in addition to previously known sterile mutants. During its biogenesis in a wild-type strain, the a-factor precursor undergoes a series of COOH-terminal CAAX modifications, two sequential NH2-terminal cleavage events, and export from the cell. Identification of the a-factor biosynthetic intermediate that accumulates in the ste24 mutant revealed that STE24 is required for the first NH2-terminal proteolytic processing event within the a-factor precursor, which takes place after COOH-terminal CAAX modification is complete. The STE24 gene product contains multiple predicted membrane spans, a zinc metalloprotease motif (HEXXH), and a COOH-terminal ER retrieval signal (KKXX). The HEXXH protease motif is critical for STE24 activity, since STE24 fails to function when conserved residues within this motif are mutated. The identification of Ste24p homologues in a diverse group of organisms, including Escherichia coli, Schizosaccharomyces pombe, Haemophilus influenzae, and Homo sapiens, indicates that Ste24p has been highly conserved throughout evolution. Ste24p and the proteins related to it define a new subfamily of proteins that are likely to function as intracellular, membrane-associated zinc metalloproteases. PMID:9015299

  2. The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in Aspergillus nidulans

    PubMed Central

    Osmani, Aysha H.; Osmani, Stephen A.

    2015-01-01

    How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in Aspergillus nidulans and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term “reboot regulation” to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated. PMID:26147902

  3. The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in Aspergillus nidulans.

    PubMed

    Liu, Hui-Lin; Osmani, Aysha H; Osmani, Stephen A

    2015-01-01

    How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in Aspergillus nidulans and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term "reboot regulation" to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated. PMID:26147902

  4. Bioengineering of photosynthetic membranes. Requirement of magnesium for the conversion of chlorophyllide a to chlorophyll a during the greening of etiochloroplasts in vitro

    SciTech Connect

    Daniell, H.; Rebeiz, C.A.

    1984-01-01

    The massive conversion of delta-aminolevulinic acid (ALA) to protochlorophyllide (Pchlide) and the massive conversion of chlorophyllide a (Chlide a) to chlorophyll a (Chl a) are two essential conditions for the ALA-dependent assembly of photosynthetic membranes in vitro. In this work, the authors describe the development of a cell-free system capable of the forementioned biosynthetic activities at rates higher than in vivo, for the first 2 h of dark-incubation. The cell-free system consisted of 1) etiochloroplasts prepared from kinetin and gibberellic-acid-pretreated cucumber cotyledons, and 2) cofactors and additives described elsewhere and which are needed for the massive conversion of ALA to Pchlide, 3) high concentrations of ATP, MgCl/sub 2/, and an isoprenol alcohol such as phytol, were required for the massive conversion of Chlide a to Chl a. An absolute and novel requirement of Mg/sup 2 +/ for the conversion of Chlide a to Chl a was also demonstrated. In addition to the role of phytol as a substrate for the conversion of Chlide a to Chl a, the data suggested that this alcohol may also be involved in the regulation of the reactions between ALA and Pchlide. It is proposed that during greening, the conversion of Chlide a to Chl a may follow different biosynthetic rates, having different substrate and cofactor requirements, depending on the stage of plastid development.

  5. Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin.

    PubMed

    Garcia-Castillo, Maria Daniela; Tran, Thi; Bobard, Alexandre; Renard, Henri-François; Rathjen, Stefan J; Dransart, Estelle; Stechmann, Bahne; Lamaze, Christophe; Lord, Mike; Cintrat, Jean-Christophe; Enninga, Jost; Tartour, Eric; Johannes, Ludger

    2015-07-01

    Antigen-presenting cells have the remarkable capacity to transfer exogenous antigens to the cytosol for processing by proteasomes and subsequent presentation on major histocompatibility complex class-I (MHC-I) molecules, a process termed cross-presentation. This is the target of biomedical approaches that aim to trigger a therapeutic immune response. The receptor-binding B-subunit of Shiga toxin (STxB) has been developed as an antigen delivery tool for such immunotherapy applications. In this study, we have analyzed pathways and trafficking factors that are involved in this process. A covalent conjugate between STxB and saporin was generated to quantitatively sample the membrane translocation step to the cytosol in differentiated monocyte-derived THP-1 cells. We have found that retrograde trafficking to the Golgi complex was not required for STxB-saporin translocation to the cytosol or for STxB-dependent antigen cross-presentation. Depletion of endosomal Rab7 inhibited, and lowering membrane cholesterol levels favored STxB-saporin translocation. Interestingly, experiments with reducible and non-reducible linker-arm-STxB conjugates led to the conclusion that after translocation, STxB remains associated with the cytosolic membrane leaflet. In summary, we report new facets of the endosomal escape process bearing relevance to antigen cross-presentation. PMID:25977475

  6. A comparative study of natural immune responses against Plasmodium vivax C-terminal merozoite surface protein-1 (PvMSP-1) and apical membrane antigen-1 (PvAMA-1) in two endemic settings

    PubMed Central

    Xia, Hui; Fang, Qiang; Jangpatarapongsa, Kulachart; Zhiyong, Tao; Cui, Liwang; Li, Baiqing; Udomsangpetch, Rachanee

    2015-01-01

    The mechanisms of cellular and humoral immune responses against P. vivax parasite remain poorly understood. Several malaria immunological studies have been conducted in endemic regions where both P. falciparum and P. vivax parasites co-exist. In this study, a comparative analysis of immunity to Plasmodium vivax antigens in different geography and incidence of Plasmodium spp. infection was performed. We characterised antibodies against two P. vivax antigens, PvMSP-1 and PvAMA-1, and the cross-reactivity between these antigens using plasma from acute malaria infected patients living in the central region of China and in the western border of Thailand. P. vivax endemicity is found in central China whereas both P. vivax and P. falciparum are endemic in Thailand. There was an increased level of anti-PvMSP-1/anti-PvAMA-1 in both populations. An elevated level of antibodies to total P. vivax proteins and low level of antibodies to total P. falciparum proteins was found in acute P. vivax infected Chinese, suggesting antibody cross-reactivity between the two species. P. vivax infected Thai patients had both anti-P. vivax and anti-P. falciparum antibodies as expected since both species are present in Thailand. More information on humoral and cell mediated immunity during acute P. vivax-infection in the area where only single P. vivax species existed is of great interest in the relation of building up anti-disease severity caused by P. falciparum. This knowledge will support vaccine development in the future. PMID:26713085

  7. Surface co-expression of two different PfEMP1 antigens on single plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1.

    PubMed

    Joergensen, Louise; Bengtsson, Dominique C; Bengtsson, Anja; Ronander, Elena; Berger, Sanne S; Turner, Louise; Dalgaard, Michael B; Cham, Gerald K K; Victor, Michala E; Lavstsen, Thomas; Theander, Thor G; Arnot, David E; Jensen, Anja T R

    2010-01-01

    The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required. PMID:20824088

  8. Nanoengineered membrane electrode assembly interface

    DOEpatents

    Song, Yujiang; Shelnutt, John A

    2013-08-06

    A membrane electrode structure suitable for use in a membrane electrode assembly (MEA) that comprises membrane-affixed metal nanoparticles whose formation is controlled by a photochemical process that controls deposition of the metal nanoparticles using a photocatalyst integrated with a polymer electrolyte membrane, such as an ionomer membrane. Impregnation of the polymer membrane with the photocatalyst prior to metal deposition greatly reduces the required amount of metal precursor in the deposition reaction solution by restricting metal reduction substantially to the formation of metal nanoparticles affixed on or near the surface of the polymer membrane with minimal formation of metallic particles not directly associated with the membrane.

  9. Human immune response to Mycobacterium tuberculosis antigens.

    PubMed Central

    Havlir, D V; Wallis, R S; Boom, W H; Daniel, T M; Chervenak, K; Ellner, J J

    1991-01-01

    and recombinant antigens in healthy tuberculin reactors and tuberculous patients. Antigens which stimulated prominent lymphocyte blastogenic responses were identified in seven fractions on T-cell Western blot analysis. Two of these may represent previously characterized proteins; the others may contain immunodominant proteins that will require further characterization. Images PMID:1898911

  10. Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM.

    PubMed Central

    Wienands, J; Hombach, J; Radbruch, A; Riesterer, C; Reth, M

    1990-01-01

    Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain. Images Fig. 3. Fig. 5. Fig. 6. Fig. 7. PMID:2303036

  11. Lipid membranes for membrane proteins.

    PubMed

    Kukol, Andreas

    2015-01-01

    The molecular dynamics (MD) simulation of membrane proteins requires the setup of an accurate representation of lipid bilayers. This chapter describes the setup of a lipid bilayer system from scratch using generally available tools, starting with a definition of the lipid molecule POPE, generation of a lipid bilayer, energy minimization, MD simulation, and data analysis. The data analysis includes the calculation of area and volume per lipid, deuterium order parameters, self-diffusion constant, and the electron density profile. PMID:25330959

  12. Medically Relevant Acinetobacter Species Require a Type II Secretion System and Specific Membrane-Associated Chaperones for the Export of Multiple Substrates and Full Virulence

    PubMed Central

    Harding, Christian M.; Kinsella, Rachel L.; Palmer, Lauren D.; Skaar, Eric P.; Feldman, Mario F.

    2016-01-01

    Acinetobacter baumannii, A. nosocomialis, and A. pittii have recently emerged as opportunistic human pathogens capable of causing severe human disease; however, the molecular mechanisms employed by Acinetobacter to cause disease remain poorly understood. Many pathogenic members of the genus Acinetobacter contain genes predicted to encode proteins required for the biogenesis of a type II secretion system (T2SS), which have been shown to mediate virulence in many Gram-negative organisms. Here we demonstrate that Acinetobacter nosocomialis strain M2 produces a functional T2SS, which is required for full virulence in both the Galleria mellonella and murine pulmonary infection models. Importantly, this is the first bona fide secretion system shown to be required for virulence in Acinetobacter. Using bioinformatics, proteomics, and mutational analyses, we show that Acinetobacter employs its T2SS to export multiple substrates, including the lipases LipA and LipH as well as the protease CpaA. Furthermore, the Acinetobacter T2SS, which is found scattered amongst five distinct loci, does not contain a dedicated pseudopilin peptidase, but instead relies on the type IV prepilin peptidase, reinforcing the common ancestry of these two systems. Lastly, two of the three secreted proteins characterized in this study require specific chaperones for secretion. These chaperones contain an N-terminal transmembrane domain, are encoded adjacently to their cognate effector, and their disruption abolishes type II secretion of their cognate effector. Bioinformatic analysis identified putative chaperones located adjacent to multiple previously known type II effectors from several Gram-negative bacteria, which suggests that T2SS chaperones constitute a separate class of membrane-associated chaperones mediating type II secretion. PMID:26764912

  13. SERS immunoassay based on the capture and concentration of antigen-assembled gold nanoparticles.

    PubMed

    Lopez, Arielle; Lovato, Francis; Oh, Soon Hwan; Lai, Yen H; Filbrun, Seth; Driskell, Elizabeth A; Driskell, Jeremy D

    2016-01-01

    A simple, rapid, and sensitive immunoassay has been developed based on antigen-mediated aggregation of gold nanoparticles (AuNP) and surface-enhanced Raman spectroscopy (SERS). Central to this platform is the extrinsic Raman label (ERL), which consists of a gold nanoparticle modified with a mixed monolayer of a Raman active molecule and an antibody. ERLs are mixed with sample, and antigen induces the aggregation of the ERLs. A membrane filter is then used to isolate and concentrate the ERL aggregates for SERS analysis. Preliminary work to establish proof-of-principle of the platform technology utilized mouse IgG as a model antigen. The effects of membrane pore diameter and AuNP size on the analytical performance of the assay were systematically investigated, and it was determined that a pore diameter of 200 nm and AuNP diameter of 80 nm provide maximum sensitivity while minimizing signal from blank samples. Optimization of the assay provided a detection limit of 1.9 ng/mL, 20-fold better than the detection limit achieved by an ELISA employing the same antibody-antigen system. Furthermore, this assay required only 60 min compared to 24h for the ELISA. To validate this assay, mouse serum was directly analyzed to accurately quantify IgG. Collectively, these results demonstrate the potential advantages of this technology over current diagnostic tests for protein biomarkers with respect to time, simplicity, and detection limits. Thus, this approach provides a framework for prospective development of new and more powerful tools that can be designed for point-of-care diagnostic or point-of-need detection. PMID:26695280

  14. Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors.

    PubMed

    Cornelissen, C N; Biswas, G D; Tsai, J; Paruchuri, D K; Thompson, S A; Sparling, P F

    1992-09-01

    The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor. PMID:1325963

  15. Safety of targeting tumor endothelial cell antigens.

    PubMed

    Wagner, Samuel C; Riordan, Neil H; Ichim, Thomas E; Szymanski, Julia; Ma, Hong; Perez, Jesus A; Lopez, Javier; Plata-Munoz, Juan J; Silva, Francisco; Patel, Amit N; Kesari, Santosh

    2016-01-01

    The mechanisms underlying discrimination between "self" and "non-self", a central immunological principle, require careful consideration in immune oncology therapeutics where eliciting anti-cancer immunity must be weighed against the risk of autoimmunity due to the self origin of tumors. Whole cell vaccines are one promising immunotherapeutic avenue whereby a myriad of tumor antigens are introduced in an immunogenic context with the aim of eliciting tumor rejection. Despite the possibility collateral damage to healthy tissues, cancer immunotherapy can be designed such that off target autoimmunity remains limited in scope and severity or completely non-existent. Here we provide an immunological basis for reconciling the safety of cancer vaccines, focusing on tumor endothelial cell vaccines, by discussing the following topics: (a) Antigenic differences between neoplastic and healthy tissues that can be leveraged in cancer vaccine design; (b) The layers of tolerance that control T cell responses directed against antigens expressed in healthy tissues and tumors; and, (c) The hierarchy of antigenic epitope selection and display in response to whole cell vaccines, and how antigen processing and presentation can afford a degree of selectivity against tumors. We conclude with an example of early clinical data utilizing ValloVax™, an immunogenic placental endothelial cell vaccine that is being advanced to target the tumor endothelium of diverse cancers, and we report on the safety and efficacy of ValloVax™ for inducing immunity against tumor endothelial antigens. PMID:27071457

  16. How to Make a Non-Antigenic Protein (Auto) Antigenic: Molecular Complementarity Alters Antigen Processing and Activates Adaptive-Innate Immunity Synergy.

    PubMed

    Root-Bernstein, Robert

    2015-01-01

    Evidence is reviewed that complementary proteins and peptides form complexes with increased antigenicity and/or autoimmunogenicity. Five case studies are highlighted: 1) diphtheria toxin-antitoxin (antibody), which induces immunity to the normally non-antigenic toxin, and autoimmune neuritis; 2) tryptophan peptide of myelin basic protein and muramyl dipeptide ("adjuvant peptide"), which form a complex that induces experimental allergic encephalomyelitis; 3) an insulin and glucagon complex that is far more antigenic than either component individually; 4) various causes of experimental autoimmune myocarditis such as C protein in combination with its antibody, or coxsackie B virus in combination with the coxsackie and adenovirus receptor; 5) influenza A virus haemagglutinin with the outer membrane protein of the Haemophilus influenzae, which increases antigenicity. Several mechanisms cooperate to alter immunogenicity. Complexation alters antigen processing, protecting the components against proteolysis, altering fragmentation and presenting novel antigens to the immune system. Complementary antigens induce complementary adaptive immune responses (complementary antibodies and/or T cell receptors) that produce circulating immune complexes (CIC). CIC stimulate innate immunity. Concurrently, complementary antigens stimulate multiple Toll-like receptors that synergize to over-produce cytokines, which further stimulate adaptive immunity. Thus innate and adaptive immunity form a positive feedback loop. If components of the complex mimic a host protein, then autoimmunity may result. Enhanced antigenicity for production of improved vaccines and/or therapeutic autoimmunity (e.g., against cancer cells) might be achieved by using information from antibody or TCR recognition sites to complement an antigen; by panning for complements in randomized peptide libraries; or using antisense peptide strategies to design complements. PMID:26179268

  17. Comparing multi-module connections in membrane chromatography scale-up.

    PubMed

    Yu, Zhou; Karkaria, Tishtar; Espina, Marianela; Hunjun, Manjeet; Surendran, Abera; Luu, Tina; Telychko, Julia; Yang, Yan-Ping

    2015-07-20

    Membrane chromatography is increasingly used for protein purification in the biopharmaceutical industry. Membrane adsorbers are often pre-assembled by manufacturers as ready-to-use modules. In large-scale protein manufacturing settings, the use of multiple membrane modules for a single batch is often required due to the large quantity of feed material. The question as to how multiple modules can be connected to achieve optimum separation and productivity has been previously approached using model proteins and mass transport theories. In this study, we compare the performance of multiple membrane modules in series and in parallel in the production of a protein antigen. Series connection was shown to provide superior separation compared to parallel connection in the context of competitive adsorption. PMID:25907833

  18. Presentation of hepatocellular antigens

    PubMed Central

    Grakoui, Arash; Crispe, Ian Nicholas

    2016-01-01

    The liver is an organ in which antigen-specific T-cell responses manifest a bias toward immune tolerance. This is clearly seen in the rejection of allogeneic liver transplants, and multiple other phenomena suggest that this effect is more general. These include tolerance toward antigens introduced via the portal vein, immune failure to several hepatotropic viruses, the lack of natural liver-stage immunity to malaria parasites, and the frequent metastasis of cancers to the liver. Here we review the mechanisms by which T cells engage with hepatocellular antigens, the context in which such encounters occur, and the mechanisms that act to suppress a full T-cell response. While many mechanisms play a role, we will argue that two important processes are the constraints on the cross-presentation of hepatocellular antigens, and the induction of negative feedback inhibition driven by interferons. The constant exposure of the liver to microbial products from the intestine may drive innate immunity, rendering the local environment unfavorable for specific T-cell responses through this mechanism. Nevertheless, tolerance toward hepatocellular antigens is not monolithic and under specific circumstances allows both effective immunity and immunopathology. PMID:26924525

  19. Identification of Protective Antigens for Vaccination against Systemic Salmonellosis

    PubMed Central

    Bumann, Dirk

    2014-01-01

    There is an urgent medical need for improved vaccines with broad serovar coverage and high efficacy against systemic salmonellosis. Subunit vaccines offer excellent safety profiles but require identification of protective antigens, which remains a challenging task. Here, I review crucial properties of Salmonella antigens that might help to narrow down the number of potential candidates from more than 4000 proteins encoded in Salmonella genomes, to a more manageable number of 50–200 most promising antigens. I also discuss complementary approaches for antigen identification and potential limitations of current pre-clinical vaccine testing. PMID:25157252

  20. Overcoming Antigen Escape with CAR T-cell Therapy.

    PubMed

    Jackson, Hollie J; Brentjens, Renier J

    2015-12-01

    Sotillo and colleagues describe the molecular events associated with apparent loss of target antigen expression following CAR T-cell therapy. We propose that broader immune activation is required to prevent outgrowth of tumor antigen escape variants following targeted therapies. PMID:26637657

  1. Membrane stabilizer

    DOEpatents

    Mingenbach, William A.

    1988-01-01

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material.

  2. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  3. A Novel Ankyrin-Repeat Membrane Protein, IGN1, Is Required for Persistence of Nitrogen-Fixing Symbiosis in Root Nodules of Lotus japonicus1[OA

    PubMed Central

    Kumagai, Hirotaka; Hakoyama, Tsuneo; Umehara, Yosuke; Sato, Shusei; Kaneko, Takakazu; Tabata, Satoshi; Kouchi, Hiroshi

    2007-01-01

    Nitrogen-fixing symbiosis of legume plants with Rhizobium bacteria is established through complex interactions between two symbiotic partners. Similar to the mutual recognition and interactions at the initial stages of symbiosis, nitrogen fixation activity of rhizobia inside root nodules of the host legume is also controlled by specific interactions during later stages of nodule development. We isolated a novel Fix− mutant, ineffective greenish nodules 1 (ign1), of Lotus japonicus, which forms apparently normal nodules containing endosymbiotic bacteria, but does not develop nitrogen fixation activity. Map-based cloning of the mutated gene allowed us to identify the IGN1 gene, which encodes a novel ankyrin-repeat protein with transmembrane regions. IGN1 expression was detected in all organs of L. japonicus and not enhanced in the nodulation process. Immunoanalysis, together with expression analysis of a green fluorescent protein-IGN1 fusion construct, demonstrated localization of the IGN1 protein in the plasma membrane. The ign1 nodules showed extremely rapid premature senescence. Irregularly enlarged symbiosomes with multiple bacteroids were observed at early stages (8–9 d post inoculation) of nodule formation, followed by disruption of the symbiosomes and disintegration of nodule infected cell cytoplasm with aggregation of the bacteroids. Although the exact biochemical functions of the IGN1 gene are still to be elucidated, these results indicate that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes, thus being essential for functional symbiosis. PMID:17277093

  4. Internal cleavages of the autoinhibitory prodomain are required for membrane type 1 matrix metalloproteinase activation, although furin cleavage alone generates inactive proteinase.

    PubMed

    Golubkov, Vladislav S; Cieplak, Piotr; Chekanov, Alexei V; Ratnikov, Boris I; Aleshin, Alexander E; Golubkova, Natalya V; Postnova, Tatiana I; Radichev, Ilian A; Rozanov, Dmitri V; Zhu, Wenhong; Motamedchaboki, Khatereh; Strongin, Alex Y

    2010-09-01

    The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP. PMID:20605791

  5. Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation, although Furin Cleavage Alone Generates Inactive Proteinase*

    PubMed Central

    Golubkov, Vladislav S.; Cieplak, Piotr; Chekanov, Alexei V.; Ratnikov, Boris I.; Aleshin, Alexander E.; Golubkova, Natalya V.; Postnova, Tatiana I.; Radichev, Ilian A.; Rozanov, Dmitri V.; Zhu, Wenhong; Motamedchaboki, Khatereh; Strongin, Alex Y.

    2010-01-01

    The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD↓L50 site initiates the MT1-MMP activation, whereas the 108RRKR111↓Y112 cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP. PMID:20605791

  6. Vacuolar Protein Sorting Protein 13A, TtVPS13A, Localizes to the Tetrahymena thermophila Phagosome Membrane and Is Required for Efficient Phagocytosis▿†

    PubMed Central

    Samaranayake, Haresha S.; Cowan, Ann E.; Klobutcher, Lawrence A.

    2011-01-01

    Vacuolar protein sorting 13 (VPS13) proteins have been studied in a number of organisms, and mutations in VPS13 genes have been implicated in two human genetic disorders, but the function of these proteins is poorly understood. The TtVPS13A protein was previously identified in a mass spectrometry analysis of the Tetrahymena thermophila phagosome proteome (M. E. Jacobs et al., Eukaryot. Cell 5:1990–2000, 2006), suggesting that it is involved in phagocytosis. In this study, we analyzed the structure of the macronuclear TtVPS13A gene, which was found to be composed of 17 exons spanning 12.5 kb and was predicted to encode a protein of 3,475 amino acids (aa). A strain expressing a TtVPS13A-green fluorescent protein (GFP) fusion protein was constructed, and the protein was found to associate with the phagosome membrane during the entire cycle of phagocytosis. In addition, Tetrahymena cells with a TtVPS13A knockout mutation displayed impaired phagocytosis. Specifically, they grew slowly under conditions where phagocytosis is essential, they formed few phagosomes, and the digestion of phagosomal contents was delayed compared to wild-type cells. Overall, these results provide evidence that the TtVPS13A protein is required for efficient phagocytosis. PMID:21764909

  7. YND1, a homologue of GDA1, encodes membrane-bound apyrase required for Golgi N- and O-glycosylation in Saccharomyces cerevisiae.

    PubMed

    Gao, X D; Kaigorodov, V; Jigami, Y

    1999-07-23

    The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membrane-bound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ATP, GDP/GTP, and UDP/UTP and approximately 50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The Deltaynd1 mutant cells were defective in O- and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in Deltagda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the Deltaynd1Deltagda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells. PMID:10409709

  8. A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

    PubMed

    Karlsson, J; Clarke, A K; Chen, Z Y; Hugghins, S Y; Park, Y I; Husic, H D; Moroney, J V; Samuelsson, G

    1998-08-10

    A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry. PMID:9482718

  9. Escherichia coli mrsC Is an Allele of hflB, Encoding a Membrane-Associated ATPase and Protease That Is Required for mRNA Decay

    PubMed Central

    Wang, Rong-fu; O’Hara, Eileen B.; Aldea, Marti; Bargmann, Cornelia I.; Gromley, Heather; Kushner, Sidney R.

    1998-01-01

    The mrsC gene of Escherichia coli is required for mRNA turnover and cell growth, and strains containing the temperature-sensitive mrsC505 allele have longer half-lives than wild-type controls for total pulse-labeled and individual mRNAs (L. L. Granger et al., J. Bacteriol. 180:1920–1928, 1998). The cloned mrsC gene contains a long open reading frame beginning at an initiator UUG codon, confirmed by N-terminal amino acid sequencing, encoding a 70,996-Da protein with a consensus ATP-binding domain. mrsC is identical to the independently identified ftsH gene except for three additional amino acids at the N terminus (T. Tomoyasu et al., J. Bacteriol. 175:1344–1351, 1993). The purified protein had a Km of 28 μM for ATP and a Vmax of 21.2 nmol/μg/min. An amino-terminal glutathione S-transferase–MrsC fusion protein retained ATPase activity but was not biologically active. A glutamic acid replacement of the highly conserved lysine within the ATP-binding motif (mrsC201) abolished the complementation of the mrsC505 mutation, confirming that the ATPase activity is required for MrsC function in vivo. In addition, the mrsC505 allele conferred a temperature-sensitive HflB phenotype, while the hflB29 mutation promoted mRNA stability at both 30 and 44°C, suggesting that the inviability associated with the mrsC505 allele is not related to the defect in mRNA decay. The data presented provide the first direct evidence for the involvement of a membrane-bound protein in mRNA decay in E. coli. PMID:9537394

  10. Lipid antigens in immunity

    PubMed Central

    Dowds, C. Marie; Kornell, Sabin-Christin

    2014-01-01

    Lipids are not only a central part of human metabolism but also play diverse and critical roles in the immune system. As such, they can act as ligands of lipid-activated nuclear receptors, control inflammatory signaling through bioactive lipids such as prostaglandins, leukotrienes, lipoxins, resolvins, and protectins, and modulate immunity as intracellular phospholipid- or sphingolipid-derived signaling mediators. In addition, lipids can serve as antigens and regulate immunity through the activation of lipid-reactive T cells, which is the topic of this review. We will provide an overview of the mechanisms of lipid antigen presentation, the biology of lipid-reactive T cells, and their contribution to immunity. PMID:23999493

  11. Multimeric C9 within C5b-9 is required for inner membrane damage to Escherichia coli J5 during complement killing.

    PubMed

    Bloch, E F; Schmetz, M A; Foulds, J; Hammer, C H; Frank, M M; Joiner, K A

    1987-02-01

    We have shown recently that an average of three or more C9 molecules must bind to C5b-8 on Escherichia coli strain J5 to cause direct complement killing in the absence of serum lysozyme. We initially confirmed and extended this observation by showing that deposition of a large number of C5b-9 complexes bearing 1C9 per C5b-8 was not bactericidal for J5. To identify the target site for bactericidal C5b-9 deposition, we measured release of periplasmic and cytoplasmic markers of different size from J5 as the C9:C5b-8 ratio was changed, because the diameter of the C5b-9 channel is known to increase as the C9:C5b-8 ratio increases. To facilitate measurement of release of the periplasmic marker beta-lactamase (BLA), J5 was transformed for high level constitutive TEM-1 BLA production (J5-Amp). Multimeric C9 within C5b-9 (C9:C5b-8 greater than 3) was required to release BLA (m.w. 28,900) from J5-Amp regardless of whether cells bore 310, 560, or 890 C5b-9/organism. Curves of both BLA release and killing vs C9:C5b-8 ratio were sigmoidal and nearly superimposable. Release of the small cytoplasmic marker 86Rb, a potassium analog, also required a minimum C9:C5b-8 ratio of 3:1; specific 86Rb release did not occur in the absence of killing. Release of the large cytoplasmic marker beta-galactosidase (m.w. 505,000) did not occur even at the highest achievable C9:C5b-8 ratio of 11:1, despite greater than 99.9% killing, indicating that there was no dissolution of the peptidoglycan layer due to incomplete removal of serum lysozyme. Complement-mediated killing of J5 requires sufficient damage to the outer membrane or formation of a sufficiently large C5b-9 channel to release the large periplasmic marker BLA. The requirement of multimeric C9 for 86Rb release suggests that at low C9:C5b-8 ratios, either C5b-9 does not have access to the cytoplasmic space or that the J5 K+ transport systems are able to compensate for putative C5b-9 channels. PMID:3100618

  12. Antigen-triggered membrane potential changes in IgE-sensitized rat basophilic leukemia cells: evidence for a repolarizing response that is important in the stimulation of cellular degranulation.

    PubMed

    Labrecque, G F; Holowka, D; Baird, B

    1989-01-01

    We have studied Ag-induced membrane potential changes of rat basophilic leukemia cells by using the potential-sensitive dye, bis-(1,3-diethylthiobarbiturate)trimethineoxonol. A rapid membrane depolarization is triggered by a multivalent Ag, and it has a bell-shaped dose dependence that parallels the degranulation response but not the extent of cross-linking of the IgE-receptor complexes. As the temperature is reduced from 37 degrees C, this depolarization response slows and decreases in magnitude until complete inhibition is observed at 15 degrees C, similar to the temperature dependence previously observed for the Ag-stimulated rise in cytoplasmic Ca2+ and for degranulation. The results imply that a highly temperature-dependent step subsequent to Ag binding and cross-linking is necessary for the depolarization response. A partial return to the resting potential is seen to follow the depolarization response to Ag. This repolarization process is inhibited by quinidine.HCl and Ba2+ in parallel with an inhibition of the degranulation response. Repolarization is not affected by 4-aminopyridine or by the absence of K+ in the external buffer. These data suggest that the repolarization is caused by a previously uncharacterized K+ channel. PMID:2909616

  13. Complex N-glycans or core 1-derived O-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or LeY in the preimplantation mouse embryo

    PubMed Central

    Williams, Suzannah A; Stanley, Pamela

    2010-01-01

    The glycan epitopes termed stage-specific embryonic antigens (SSEA) occur on glycoproteins and glycolipids in mammals. However, it is not known whether these epitopes are attached to N- or O-glycans on glycoproteins and/or on glycolipids in the developing mouse embryo. In this paper the expression of the antigens SSEA-1, SSEA-3, SSEA-4 and LeY was examined on ovulated eggs, early embryos and blastocysts lacking either complex and hybrid N-glycans or core-1 derived O-glycans. In all cases, antigen expression determined by fluorescence microscopy of bound monoclonal antibodies to embryos at the stage of development of maximal expression, was similar in mutant and control embryos. Thus, none of these developmental antigens are expressed solely on either complex N- or core 1-derived O-glycans attached to glycoproteins in the preimplantation mouse embryo. Furthermore, neither of these classes of glycan is essential for the expression of SSEA-1, SSEA-3, SSEA-4 or LeY on mouse embryos. PMID:18773292

  14. Epigenetic Control of Salmonella enterica O-Antigen Chain Length: A Tradeoff between Virulence and Bacteriophage Resistance

    PubMed Central

    Cota, Ignacio; Sánchez-Romero, María Antonia; Hernández, Sara B.; Pucciarelli, M. Graciela; García-del Portillo, Francisco; Casadesús, Josep

    2015-01-01

    The Salmonella enterica opvAB operon is a horizontally-acquired locus that undergoes phase variation under Dam methylation control. The OpvA and OpvB proteins form intertwining ribbons in the inner membrane. Synthesis of OpvA and OpvB alters lipopolysaccharide O-antigen chain length and confers resistance to bacteriophages 9NA (Siphoviridae), Det7 (Myoviridae), and P22 (Podoviridae). These phages use the O-antigen as receptor. Because opvAB undergoes phase variation, S. enterica cultures contain subpopulations of opvAB OFF and opvAB ON cells. In the presence of a bacteriophage that uses the O-antigen as receptor, the opvAB OFF subpopulation is killed and the opvAB ON subpopulation is selected. Acquisition of phage resistance by phase variation of O-antigen chain length requires a payoff: opvAB expression reduces Salmonella virulence. However, phase variation permits resuscitation of the opvAB OFF subpopulation as soon as phage challenge ceases. Phenotypic heterogeneity generated by opvAB phase variation thus preadapts Salmonella to survive phage challenge with a fitness cost that is transient only. PMID:26583926

  15. A Cronobacter turicensis O1 Antigen-Specific Monoclonal Antibody Inhibits Bacterial Motility and Entry into Epithelial Cells

    PubMed Central

    Lehner, Angelika; Dietrich, Richard; Kleinsteuber, Ina; Canals, Rocío; Zurfluh, Katrin; Weiner, Kerstin; Märtlbauer, Erwin

    2014-01-01

    Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited. PMID:25534937

  16. Detection of peste des petits ruminants virus antigen using immunofiltration and antigen-competition ELISA methods.

    PubMed

    Raj, G Dhinakar; Rajanathan, T M C; Kumar, C Senthil; Ramathilagam, G; Hiremath, Geetha; Shaila, M S

    2008-06-22

    Peste des petits ruminants (PPR) is one of the most economically important diseases affecting sheep and goats in India. An immunofiltration-based test has been developed using either mono-specific serum/monoclonal antibodies (mAb) prepared against a recombinant truncated nucleocapsid protein of rinderpest virus (RPV) cross-reactive with PPR virus. This method consists of coating ocular swab eluate from suspected animals onto a nitrocellulose membrane housed in a plastic module, which is allowed to react with suitable dilutions of a mAb or a mono-specific polyclonal antibody. The antigen-antibody complex formed on the membrane is then detected by protein A-colloidal gold conjugate, which forms a pink colour. In the immunofiltration test, concordant results were obtained using either PPRV mAb or mono-specific serum. Another test, an antigen-competition ELISA which relies on the competition between plate-coated recombinant truncated 'N' protein of RPV and the PPRV 'N' protein present in ocular swab eluates (sample) for binding to the mono-specific antibody against N protein of RPV (in liquid phase) was developed. The cut-off value for this test was established using reverse transcription polymerase chain reaction (RT-PCR) positive and negative oculo-nasal swab samples. Linear correlation between percent inhibition (PI) values in antigen-competition ELISA and virus infectivity titres was 0.992. Comparison of the immunofiltration test with the antigen-competition ELISA yielded a sensitivity of 80% and specificity of 100%. These two tests can serve as a screening (immunofiltration) and confirmatory (antigen-competition ELISA) test, respectively, in the diagnosis of PPR in sheep or goats. PMID:18182256

  17. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissue using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular methodology is chosen ...

  18. Antigen smuggling in tuberculosis.

    PubMed

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. PMID:24922567

  19. Antigen detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissues or other specimens, using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular m...

  20. Detection and Characterization of Autoantibodies to Neuronal Cell-Surface Antigens in the