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‣ Construção de sistema que permite a ancoragem de proteína recombinante à superfície celular de levedura.; Construction of a system that allows anchoring of recombinant protein to the cell surface of yeast.

Navarro, Jessica Paola Fuentes Rivera
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 03/07/2008 Português
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Sistemas do tipo cell surface display vêm sendo desenvolvidos para expressão de proteínas heterólogas ancoradas à superfície celular de microrganismos. Várias aplicações foram reportadas destes sistemas, incluindo o emprego como biocatalizador celular, desenvolvimento de vacinas e biosorventes celulares. Neste trabalho foi desenvolvido um sistema que permite ancoragem da proteína glicoamilase de Aspergillus awamori à superfície da parede celular da levedura Saccharomyces cerevisiae. O gene codificador da glicoamilase com sua seqüência sinal foi fusionado ao fragmento do gene codificador da região C-terminal da proteína Flo1p (Flo428), que foi utilizada como âncora (fragmento CG*FC). As células de levedura foram transformadas com o fragmento híbrido CG*FC e os transformantes foram capazes de degradar amido e liberar glicose. A atividade da glicoamilase não foi detectada no meio de cultura, porém está presente no sedimento celular. Estes resultados demonstram que a glicoamilase foi ancorada à parede celular da nova linhagem recombinante de levedura.; Cell surface display systems have being developed for expression of heterologous proteins anchored to the cell surface of microorganisms. Several applications of these systems have been reported...

‣ Identification and Characterization of a Novel 38.5-Kilodalton Cell Surface Protein of Staphylococcus aureus with Extended-Spectrum Binding Activity for Extracellular Matrix and Plasma Proteins

Hussain, Muzaffar; Becker, Karsten; von Eiff, Christof; Schrenzel, Jacques; Peters, Georg; Herrmann, Mathias
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2001 Português
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The ability to attach to host ligands is a well-established pathogenic factor in invasive Staphylococcus aureus disease. In addition to the family of adhesive proteins bound to the cell wall via the sortase A (srtA) mechanism, secreted proteins such as the fibrinogen-binding protein Efb, the extracellular adhesion protein Eap, or coagulase have been found to interact with various extracellular host molecules. Here we describe a novel protein, the extracellular matrix protein-binding protein (Emp) initially identified in Western ligand blots as a 40-kDa protein due to its broad-spectrum recognition of fibronectin, fibrinogen, collagen, and vitronectin. Emp is expressed in the stationary growth phase and is closely associated with the cell surface and yet is extractable by sodium dodecyl sulfate. The conferring gene emp (1,023 nucleotides) encodes a signal peptide of 26 amino acids and a mature protein of a calculated molecular mass of 35.5 kDa. Using PCR, emp was demonstrated in all 240 S. aureus isolates of a defined clinical strain collection as well as in 6 S. aureus laboratory strains, whereas it is lacking in all 10 S. epidermidis strains tested. Construction of an allelic replacement mutant (mEmp50) revealed the absence of Emp in mEmp50...

‣ Identification and Characterization of a Novel Heme-Associated Cell Surface Protein Made by Streptococcus pyogenes

Lei, Benfang; Smoot, Laura M.; Menning, Heather M.; Voyich, Jovanka M.; Kala, Subbarao V.; Deleo, Frank R.; Reid, Sean D.; Musser, James M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2002 Português
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Analysis of the genome sequence of a serotype M1 group A Streptococcus (GAS) strain identified a gene encoding a previously undescribed putative cell surface protein. The gene was cloned from a serotype M1 strain, and the recombinant protein was overexpressed in Escherichia coli and purified to homogeneity. The purified protein was associated with heme in a 1:1 stoichiometry. This streptococcal heme-associated protein, designated Shp, was produced in vitro by GAS, located on the bacterial cell surface, and accessible to specific antibody raised against the purified recombinant protein. Mice inoculated subcutaneously with GAS and humans with invasive infections and pharyngitis caused by GAS seroconverted to Shp, indicating that Shp was produced in vivo. The blood of mice actively immunized with Shp had significantly higher bactericidal activity than the blood of unimmunized mice. The shp gene was cotranscribed with eight contiguous genes, including homologues of an ABC transporter involved in iron uptake in gram-negative bacteria. Our results indicate that Shp is a novel cell surface heme-associated protein.

‣ Cell Surface Protein of Pseudomonas (Hydrogenomonas) facilis

Rittenhouse, Harry G.; McFadden, Bruce A.; Shumway, Lewis K.; Heptinstali, John
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1973 Português
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Intact cells of Pseudomonas facilis contain one major molecular weight class of protein that is exposed at the cell surface as revealed by lactoperoxidase-catalyzed iodination with 125I. All molecular weight classes of protein in derived cell envelope preparations are apparently saturated by iodination by lactoperoxidase after prolonged sonic treatment. The molecular weight of the predominantly exposed protein in intact cells is approximately 16,000, which is the minimal molecular weight of a cell envelope protein that precipitates as a complex with phospholipid from extracts of P. facilis. The isolation of labeled phospholipoprotein (PLP) after labeling intact cells with 125I corroborates previous experiments which suggested a surface location for the protein portion of the phospholipoprotein (PPLP). Solvent extraction of cells and immunological evidence, including studies with ferritin-coupled antibodies, indicate that PPLP is located at the cell surface and may also be within the cell envelope. These experiments suggest that PPLP is the major cell surface protein in P. facilis.

‣ Herpes simplex virus binding and entry modulate cell surface protein mobility.

Rosenthal, K S; Leuther, M D; Barisas, B G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1984 Português
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Fluorescence photobleaching recovery measurements showed that herpes simplex virus type 1 attachment to target cells rapidly induced an anchorage modulation of cell surface protein mobility, an activity mediated by the cytoskeleton and associated with the multivalent attachment of other ligands (e.g., cells, lectins, or anti-immunoglobulin) to cell surfaces. The restriction in cell surface protein mobility was released concurrently with virus penetration. The effects of attachment and penetration on cell surface protein mobility and cytoskeletal function are some of the earliest cellular changes induced by herpes simplex virus infection.

‣ A monoclonal anti-double-stranded DNA autoantibody binds to a 94-kDa cell-surface protein on various cell types via nucleosomes or a DNA-histone complex.

Jacob, L; Viard, J P; Allenet, B; Anin, M F; Slama, F B; Vandekerckhove, J; Primo, J; Markovits, J; Jacob, F; Bach, J F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1989 Português
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A crude supernatant of hybridoma secreting a monoclonal anti-double-stranded (ds)DNA antibody (PME77 mAb), used to stain fibroblasts (CVI cells) in immunofluorescence, gives a punctuated staining of variable intensity. We had suggested that anti-DNA antibodies bind to cell-surface protein(s) of several cells. When the mAb of this crude supernatant was purified on a dsDNA-cellulose column and a histone-Trisacryl column, the mAb no longer bound to the cell surface. Only when dsDNA plus purified histones was added to the purified antibody did the immune complex strongly and uniformly stain again the cell surface of CVI cells. No significant staining was observed if either DNA or histones were omitted. A signal 94-kDa protein from membrane fractions of CVI, Raji, and RINm cell lines was visualized in immunoblots when mAb-DNA-histone complexes were applied to the nitrocellulose strips. No polypeptide was seen if one component was omitted. This 94-kDa protein behaved like a plasma membrane protein since it required the use of detergent to be solubilized and was quantitatively recovered in the Triton X-114 detergent-rich phase. Moreover, a brief treatment of living cells with trypsin cleared off this protein. Purified nucleosomes could be substituted to DNA-histone complexes...

‣ Expression of the human B-cell surface protein CD20: alteration by phorbol 12-myristate 13-acetate.

Valentine, M A; Cotner, T; Gaur, L; Torres, R; Clark, E A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1987 Português
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The monoclonal antibody 1F5 recognizes human B-cell surface protein CD20 and can activate resting B cells; with this antibody we found CD20 to be a 35/37-kDa non-disulfide-linked protein. The protein has a pI of 7.5-8.0 and is phosphorylated in B-cell lines, tonsillar B cells, and peripheral blood B cells. Both CD20 surface expression and phosphorylation are increased on buoyant tonsillar B cells activated in vivo. Because phorbol 12-myristate 13-acetate (PMA) supports the activation signal initiated by monoclonal antibody 1F5, we studied the effect of PMA on CD20 expression. After brief incubation with mitogenic levels of PMA, the number of dense tonsillar B cells positive for CD20 protein transiently decreased. Paradoxically, the cells remaining positive had more surface CD20 than did control cells, and these remaining surface CD20 molecules were hyperphosphorylated. Furthermore, PMA not only induced phosphorylation of CD20 protein on Raji cells but also increased the internalization of CD20 molecules; both phosphorylation and internalization of CD20 molecules were decreased with the protein kinase C inhibitor palmitoyl carnitine. Conditions that increase CD20 phosphorylation are shown also to increase surface mobility of the molecule...

‣ The mouse neuronal cell surface protein F3: a phosphatidylinositol- anchored member of the immunoglobulin superfamily related to chicken contactin

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/08/1989 Português
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Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated...

‣ Protein rib: a novel group B streptococcal cell surface protein that confers protective immunity and is expressed by most strains causing invasive infections

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/06/1993 Português
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The group B Streptococcus, an important cause of invasive infections in the neonate, is classified into four major serotypes (Ia, Ib, II, and III) based on the structure of the polysaccharide capsule. Since the capsule is a known virulence factor, it has been extensively studied, in particular in type III strains, which cause the majority of invasive infections. Two cell surface proteins, alpha and beta, have also been studied in detail since they confer protective immunity, but these proteins are usually not expressed by type III strains. We describe here a cell surface protein, designated protein Rib (resistance to proteases, immunity, group B), that confers protective immunity and is expressed by most strains of type III. Protein Rib was first identified as a distinct 95-kD protein in extracts of a type III strain, and was purified to homogeneity from that strain. Rabbit antiserum to protein Rib was used to demonstrate that it is expressed on the cell surface of 31 out of 33 type III strains, but only on 1 out of 25 strains representing the other three serotypes. Mouse protection tests showed that antiserum to protein Rib protects against lethal infection with three different strains expressing this antigen, including a strain representing a recently identified high virulence type III clone. Protein Rib is immunologically unrelated to the alpha and beta proteins...

‣ Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

Maurel, Damien; Comps-Agrar, Laetitia; Brock, Carsten; Rives, Marie-Laure; Bourrier, Emmanuel; Ayoub, Mohammed Akli; Bazin, Hervé; Tinel, Norbert; Durroux, Thierry; Prézeau, Laurent; Trinquet, Eric; Pin, Jean-Philippe
Fonte: Nature Pub. Group Publicador: Nature Pub. Group
Tipo: Artigo de Revista Científica
Português
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Cell surface proteins play key roles in cell-cell communication. They assemble into hetero-complexes that include different receptors and effectors. Demonstrating and manipulating such protein complexes will certainly offer new ways for new therapeutics. Here we developed reagents to quantitatively analyze in a high throughput format protein-protein interaction at the surface of living cells. Using this approach we examined whether G-protein coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers, a matter of intense debates. We bring new evidence for the oligomeric state of both class A and class C GPCRs. We also report a different quaternary structure of the GPCRs for the two major neurotransmitters. Whereas metabotropic glutamate receptors assemble into strict dimers, the GABAB receptor spontaneously form dimers of heterodimers offering a way to modulate G-protein coupling efficacy. This approach will be useful to systematically analyze the dynamics of cell surface protein complexes in living cells.

‣ The Cell Surface Protein Gene ecm33+ Is a Target of the Two Transcription Factors Atf1 and Mbx1 and Negatively Regulates Pmk1 MAPK Cell Integrity Signaling in Fission Yeast

Takada, Hirofumi; Nishida, Aiko; Domae, Mitsuhiro; Kita, Ayako; Yamano, Yuki; Uchida, Atsushi; Ishiwata, Shunji; Fang, Yue; Zhou, Xin; Masuko, Takashi; Kinoshita, Mitsuhiro; Kakehi, Kazuaki; Sugiura, Reiko
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em 15/02/2010 Português
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We identified and characterized ecm33+, which encodes a GPI-anchored cell surface protein as a transcriptional target of Atf1 and Mbx1. Here, we show that Ecm33 is involved in the negative regulation of Pmk1 MAPK signaling and demonstrates real-time monitoring of the activation of the cell integrity MAPK signaling pathway.

‣ Hexabromocyclododecane Decreases Tumor-cell-binding Capacity and Cell-Surface Protein Expression of Human Natural Killer Cells

Hinkson, Natasha C.; Whalen, Margaret M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/2010 Português
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Hexabromocyclododecane (HBCD) is a flame retardant that decreases the lytic function of human natural killer (NK) cells. NK cells defend against tumor cells and virally infected cells. Thus, HBCD has the potential to increase cancer incidence and viral infections. NK cells must bind to their targets for lysis to occur. Thus, concentrations of HBCD that decrease lytic function were examined for their ability to alter NK binding to tumor targets. Levels of HBCD that caused a loss of binding function were examined for effects on expression of cell surface proteins needed for binding. NK cells exposed to HBCD for 24 h, 48 h, or 6 days or to HBCD for 1 h followed by 24 h, 48 h, or 6 days in HBCD-free media were examined for binding function and cell surface protein expression. The results indicated that exposure of NK cells to 10 μM HBCD for 24 h (which caused a greater than 90% loss of lytic function) caused a very significant decrease in NK cell binding function (70.9%), and in CD16 and CD56 cell-surface protein expression (57.8%, and 24.6% respectively). NK cells exposed to 10 μM HBCD for 1 h followed by 24 h in HBCD-free media (which caused a 89.3% loss of lytic function) showed decreased binding function (79.2%), and CD 16 expression (48.1%). Results indicate that HBCD exposures decreased binding function as well as cell-surface marker expression in NK cells and that these changes may explain the losses of lytic function induced by certain HBCD exposures.

‣ Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry

Bi, Shuguang; Hong, Patrick W.; Lee, Benhur; Baum, Linda G.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
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Interaction of cell surface glycoproteins with endogenous lectins on the cell surface regulates formation and maintenance of plasma membrane domains, clusters signaling complexes, and controls the residency time of glycoproteins on the plasma membrane. Galectin-9 is a soluble, secreted lectin that binds to glycoprotein receptors to form galectin–glycoprotein lattices on the cell surface. Whereas galectin-9 binding to specific glycoprotein receptors induces death of CD4 Th1 cells, CD4 Th2 cells are resistant to galectin-9 death due to alternative glycosylation. On Th2 cells, galectin-9 binds cell surface protein disulfide isomerase (PDI), increasing retention of PDI on the cell surface and altering the redox status at the plasma membrane. Cell surface PDI regulates integrin function on platelets and also enhances susceptibility of T cells to infection with HIV. We find that galectin-9 binding to PDI on Th2 cells results in increased cell migration through extracellular matrix via β3 integrins, identifying a unique mechanism to regulate T-cell migration. In addition, galectin-9 binding to PDI on T cells potentiates infection with HIV. We identify a mechanism for regulating cell surface redox status via a galectin–glycoprotein lattice...

‣ New Cell Surface Protein Involved in Biofilm Formation by Streptococcus parasanguinis ▿

Liang, Xiaobo; Chen, Yi-Ywan M.; Ruiz, Teresa; Wu, Hui
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2011 Português
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Dental biofilm formation is critical for maintaining the healthy microbial ecology of the oral cavity. Streptococci are predominant bacterial species in the oral cavity and play important roles in the initiation of plaque formation. In this study, we identified a new cell surface protein, BapA1, from Streptococcus parasanguinis FW213 and determined that BapA1 is critical for biofilm formation. Sequence analysis revealed that BapA1 possesses a typical cell wall-sorting signal for cell surface-anchored proteins from Gram-positive bacteria. No functional orthologue was reported in other streptococci. BapA1 possesses nine putative pilin isopeptide linker domains which are crucial for pilus assembly in a number of Gram-positive bacteria. Deletion of the 3′ portion of bapA1 generated a mutant that lacks surface-anchored BapA1 and abolishes formation of short fibrils on the cell surface. The mutant failed to form biofilms and exhibited reduced adherence to an in vitro tooth model. The BapA1 deficiency also inhibited bacterial autoaggregation. The N-terminal muramidase-released-protein-like domain mediated BapA1-BapA1 interactions, suggesting that BapA1-mediated cell-cell interactions are important for bacterial autoaggregation and biofilm formation. Furthermore...

‣ A Mass Spectrometric-Derived Cell Surface Protein Atlas

Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P.; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L.; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anett
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 20/04/2015 Português
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Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets...

‣ In silico evaluation of two mass spectrometry-based approaches for the identification of novel human leukocyte cell-surface proteins

Nicholson, I.; Ayhan, M.; Hoogenraad, N.; Zola, H.
Fonte: Federation Amer Soc Exp Biol Publicador: Federation Amer Soc Exp Biol
Tipo: Artigo de Revista Científica
Publicado em //2005 Português
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The identification and quantitation of cell-surface proteins expressed by leukocytes currently use the wide availability of monoclonal antibodies (mAb) in immunohistochemical and flow cytometric assays. Presently, ∼400 such proteins have been characterized; however, analysis of the completed human genome sequence indicates that it may contain several thousand as-yet unidentified molecules, which may be expressed on the leukocyte cell surface. Recent advances in protein isolation and analysis using mass spectrometry illustrate that it is now feasible to identify the protein composition of a complex sample such as a plasma membrane extract. Such an approach may be useful for the identification of the cell-surface proteins that have not been identified using mAb techniques. Here, we detail the results of an in silico evaluation of the peptides isolated using two methods used to label plasma membrane proteins to determine whether these methods are suitable for the identification of known leukocyte cell-surface proteins by mass spectrometry. The labeling of cell-surface proteins before isolation and characterization is a valuable means of differentiating between plasma membrane and internal membrane proteins The results indicate that although the majority of cell-surface proteins can be identified using either of the approaches...

‣ Charakterisierung der Oberflächensialylierung von dendritischen Zellen und T-Zellen in unterschiedlichen Funktionszuständen sowie Untersuchung der Expression und der biochemischen Eigenschaften von CD83-Liganden; Characterization of cell surface sialylation of dendritic cells and T cells in different functional states and expression and biochemical characterization of CD83 ligand

Jenner, Jutta
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Dendritische Zellen (DC) sind potente antigenpräsentierende Zellen und können gegen das jeweilige Antigen entweder eine Immunantwort oder aber für dieses immunologische Toleranz induzieren. Bisher sind hauptsächlich Protein-Protein-Wechselwirkungen untersucht worden, ohne dass schlüssig der Mechanismus der Toleranzinduktion bei unreifen DC und regulatorischen T-Zellen aufgeklärt werden konnte. In der hier vorliegenden Arbeit wurde die Sialylierung von Oberflächenproteinen mit den Lektinen SNL und MAL II, sowie die Spezifität von CD83 als Lektin untersucht. Von Monozyten abgeleitete DC reduzieren im Laufe ihrer Reifung die alpha2,6-Sialinsäuredichte auf der Zelloberfläche. Dies geht einher mit der Umstellung von Toleranzinduktion auf Immunstimulation dieser antigenpräsentierenden Zellen. Darüber hinaus wurden andere wichtige Suppressorzellen in unserem Immunsystem, die regulatorischen T-Zellen (Treg), analysiert. Auch hier wurde eine Zunahme der alpha2,6-Sialinsäuredichte auf der Zelloberfläche durch polyklonale Stimulierung gefunden. Da CD8+ T-Zellen inhibitorische Sialinsäure-bindende Rezeptoren exprimieren, kann diesem Phänotyp funktionelle Bedeutung zukommen. Im Gegensatz zu den regulatorischen Zellen zeigten immunomagnetisch aufgereinigte CD8+ zytotoxische T-Zellen bei Stimulierung eine Verringerung der alpha2...

‣ Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase

Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Luz, Simão; Amaral, Margarida D.; Farinha, Carlos M.; Jordan, Peter
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.; Fundação para a Ciência e Tecnologia

‣ Podoendin. A new cell surface protein of the podocyte and endothelium

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/07/1985 Português
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A new cell surface protein, podoendin, has been identified in Sprague- Dawley rats, and isolated using monoclonal antibody (mAb) G4. The distribution of podoendin is restricted to the surface of glomerular podocytes, urinary surface of the parietal epithelium of Bowman's capsule, and the luminal surface of endothelial cells. The antibody does not crossreact with podocytes or endothelia of human or mice. In newborn rats, the appearance of podoendin on glomerular epithelium is attendant on podocyte differentiation during glomerulogenesis of metanephrogenic vesicles. It disappears when podocytes retract and efface foot processes in tissue culture. Thus, podoendin appears to be a cell differentiation-dependent surface protein of podocytes. Podoendin is a protein of 62 kD mobility on 5% polyacrylamide gel electrophoresis. It stains intensely with Coomassie blue, but gives negative reactions to carbohydrate (periodic acid/Schiff reaction) and polyanions (alcian blue, colloidal iron, and carbocyanine). It is distinct from the major sialoglycoprotein of podocyte fuzzy coat, podocalyxin (11). Podoendin isolated and purified from endothelium of lungs appears to be identical with that from podocytes and endothelium of kidneys. Injection of mAb G4 into left ventricle of rats resulted in intense decoration of the endothelium and podocyte surface within 30 min. The decoration persisted throughout the 3-d period of observation. This was not accompanied by complement (C3) fixation. Preliminary results showed that the rats developed moderate proteinuria (100 mg/ml protein in urine)...

‣ Cell-Surface GRP78 and its Antibodies: Pathologic and Therapeutic Roles in Cancer

de Ridder, Gustaaf Gregoire
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2010 Português
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The chaperone protein GRP78 is primarily expressed in the endoplasmic reticulum, but it is also aberrantly expressed on the surface of cells under pathological conditions. One the cell membrane, GRP78 acts as a signaling molecule with unique properties. The amino-terminal domain acts as a growth factor receptor-like protein, while the carboxyl-terminal domain functions as a death-signaling receptor-like protein to extrinsically induce apoptosis. Autoantibodies that react with cell-surface GRP78 on many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer and melanoma, and when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these IgGs are merely a biomarker, or if they actually promote tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We employed the antisera from these mice for in vitro cell signaling and proliferation assays. The immunodominant epitope in human cancer patients was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth...