The mechanism of Fc-mediated interaction of eosinophils with immobilized immune complexes. II. Identification of two membrane proteins, modified by the interaction

1982 ◽  
Vol 56 (1) ◽  
pp. 357-369
Author(s):  
K.J. Thorne ◽  
J. Free ◽  
D. Franks ◽  
R.C. Oliver
Keyword(s):  
Cell Surface ◽  
Heavy Chain ◽  
Plasma Membranes ◽  
Human Peripheral Blood ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Surface Proteins ◽  
Agar Layer ◽  
Coated Surfaces

Human peripheral blood eosinophils attach to and flatten down onto antibody-coated surfaces and subsequently degranulate. An antibody-coated surface was prepared by treating a layer of agar, containing tetanus toxoid antigen and eosinophil chemotactic factor (ECF), with human anti-tetanus immunoglobin. Changes in eosinophil surface proteins during attachment to the antibody-coated agar layer were detected by lactoperoxidase catalysed iodination. Purified eosinophils were pre-treated with unlabelled iodide, lactoperoxidase and hydrogen peroxide to block pre-existing accessible tyrosine residues on the cell surface. They were then allowed to interact with the agar layer, and subsequently treated with lactoperoxidase and 125I-labelled iodide to label newly accessible surface proteins. Separation of the radioactive proteins by sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed that, while incubation of the cells in suspension restored the major proteins to the cell surface, interaction with the antibody-coated agar layer caused the appearance of additional proteins of apparent molecular weight 55K, 30K, 28K and 18K. The 55K, 28K and 18K proteins were greatly reduced when antibody was absent, but the 55K protein was distinguishable from immunoglobulin G (IgG) heavy chain, since it could be detected in low amounts even in the absence of antibody. It was found in purified plasma membranes and it could be separated from IgG heavy chain by iso-electric focusing. The possibility is discussed that this protein is either linked to the receptor for the Fc portion of IgG, or that it is itself the receptor. The 18K protein required both antibody and ECF for maximum expression, but was seen in limited amounts with ECF alone. Possibly it is concerned with an ECF-mediated recognition of IgG. Unlike the 55K protein, it binds concanavalin A. Plasma membranes were prepared from eosinophils by lysis in borate, followed by purification on a glass-bead column. Both the 55K and the 18K proteins were found to be major components of the eosinophil membrane.

The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3494-3504 ◽  
Author(s):  
M Hanspal ◽  
JS Hanspal
Keyword(s):  
Plasma Membranes ◽  
Cell Attachment ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Heparin Binding ◽  
Erythroid Cells ◽  
Heparin Binding Protein

Abstract Although the association of erythroblasts with macrophages has been well documented in the human bone marrow, the function and identification of the intimate contacts occurring between the membranes of these two cell types in the physiology of erythropoiesis is not known. Using in vitro cultures of human peripheral blood derived erythroid progenitors, we have shown the presence of erythroblastic islands consisting of a central macrophage surrounded by a ring of erythroblasts that undergo terminal maturation leading to enucleation. However, when cultures were carried in the absence of intact macrophages, erythroid cells matured to the late erythroblast stage but failed to enucleate. Furthermore, the number of erythroid cells was markedly reduced in macrophage-depleted cultures, suggesting that the erythroblast-macrophage contact promotes proliferation and terminal maturation of erythroid cells leading to their enucleation. To examine the molecule(s) involved in the interaction between erythroblasts and macrophages, we have used a cell attachment assay involving incubation of solubilized surface-labeled erythroblasts with macrophage membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Erythroblast surface proteins specifically attached to a 30-kD protein from macrophage membranes, whereas no adhesion was seen to the protein standards. An apparently similar protein of 30 kD was also detected on erythroblasts and was shown to mediate erythroblast-erythroblast contact in addition to the erythroblast-macrophage contact. The extraction of plasma membranes with Triton X-100 showed that the 30-kD protein is linked to the membrane skeleton via an integral membrane protein both in erythroblasts and macrophages. Furthermore, our results show that the cell:cell interactions mediated by the 30-kD protein are calcium-independent and could be specifically inhibited by heparin. We conclude that the association of erythroblasts with macrophages promotes erythroid proliferation and maturation leading to erythroblast enucleation and that a 30-kD heparin-binding protein present on the surface of macrophages and erythroblasts is involved in this contact. This protein is capable of binding homotypic and heterotypic cells.

scholarly journals The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3494-3504 ◽  
Author(s):  
M Hanspal ◽  
JS Hanspal
Keyword(s):  
Plasma Membranes ◽  
Cell Attachment ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Heparin Binding ◽  
Erythroid Cells ◽  
Heparin Binding Protein

Although the association of erythroblasts with macrophages has been well documented in the human bone marrow, the function and identification of the intimate contacts occurring between the membranes of these two cell types in the physiology of erythropoiesis is not known. Using in vitro cultures of human peripheral blood derived erythroid progenitors, we have shown the presence of erythroblastic islands consisting of a central macrophage surrounded by a ring of erythroblasts that undergo terminal maturation leading to enucleation. However, when cultures were carried in the absence of intact macrophages, erythroid cells matured to the late erythroblast stage but failed to enucleate. Furthermore, the number of erythroid cells was markedly reduced in macrophage-depleted cultures, suggesting that the erythroblast-macrophage contact promotes proliferation and terminal maturation of erythroid cells leading to their enucleation. To examine the molecule(s) involved in the interaction between erythroblasts and macrophages, we have used a cell attachment assay involving incubation of solubilized surface-labeled erythroblasts with macrophage membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Erythroblast surface proteins specifically attached to a 30-kD protein from macrophage membranes, whereas no adhesion was seen to the protein standards. An apparently similar protein of 30 kD was also detected on erythroblasts and was shown to mediate erythroblast-erythroblast contact in addition to the erythroblast-macrophage contact. The extraction of plasma membranes with Triton X-100 showed that the 30-kD protein is linked to the membrane skeleton via an integral membrane protein both in erythroblasts and macrophages. Furthermore, our results show that the cell:cell interactions mediated by the 30-kD protein are calcium-independent and could be specifically inhibited by heparin. We conclude that the association of erythroblasts with macrophages promotes erythroid proliferation and maturation leading to erythroblast enucleation and that a 30-kD heparin-binding protein present on the surface of macrophages and erythroblasts is involved in this contact. This protein is capable of binding homotypic and heterotypic cells.

scholarly journals Cell-surface radioiodination with the sparingly soluble catalyst Iodogen. Difference between the dividing and non-dividing populations of rodent thymocytes

1981 ◽  
Vol 194 (1) ◽  
pp. 351-355 ◽  
Author(s):  
J G Salisbury ◽  
J M Graham
Keyword(s):  
Cell Surface ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Surface Proteins ◽  
New Method

The surface proteins of dividing and non-dividing subpopulations of rat and mouse thymocytes have been labelled by using a new method of radioiodination. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography of the labelled proteins shows distinct differences in labelling between the mouse and rat cells and also, in the case of the rat, between the dividing and non-dividing populations.

Electrophoretic Analysis of Polypeptides of Plasma Membranes from Bovine Pituitary Gland

1974 ◽  
Vol 52 (7) ◽  
pp. 620-630
Author(s):  
André Lemay ◽  
Fernand Labrie
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Electrophoretic Pattern ◽  
Apparent Molecular Weight ◽  
Electrophoretic Analysis ◽  
Polyacrylamide Gel ◽  
Protein Components ◽  
Membrane Components

Purified plasma membranes from bovine hypophyseal tissue have been fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under various conditions of pH and acrylamide concentrations. The best separation of protein components is achieved at a concentration of 7.5% acrylamide and at pH 7.1. Under these conditions, the electrophoretic pattern consistently shows 36 protein bands ranging in molecular weights from 250 000 to 15 000. Only one broad band, having an apparent molecular weight of 150 000, stains for glycoproteins by the period acid – Schiff technique. After electrophoresis on a two-dimensional polyacrylamide gel system using disc gels containing urea and Triton X-100 in the first dimension and SDS in the second dimension, approximately 45 different protein components can be identified. Less than 12% of the membrane proteins are solubilized by washing the membranes with 1 M KCl or NH4Cl. Denaturating agents like urea and lithium 3,4-diiodosalycilate solubilize 55–60% of membrane components. Adenohypophyseal plasma membranes show an eleetrophoretic pattern completely different from that obtained with membranes isolated from the intermediate or posterior pituitary lobes.

Isolation and partial characterization of host cell surface agglutinin and its role in attachment of a biotrophic mycoparasite

1991 ◽  
Vol 37 (5) ◽  
pp. 377-383 ◽  
Author(s):  
M. S. Manocha ◽  
Y. Chen
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Carbohydrate Binding ◽  
Protein Extract ◽  
Host Cell Wall ◽  
Host Cell Surface ◽  
Faint Band

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, of the mycoparasite Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 agglutination units/mg) compared with that of the nonhost extract (21 agglutination units/mg). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the crude extract of the host revealed four bands, a, b, c, and d, with respective Mr of 117 000, 100 000, 85 000 and 64 000; these bands except for a faint band c, were absent from the nonhost surface. Deletion of proteins b or c from the crude protein extract of the host significantly reduced its agglutinating activity. Proteins b and c, purified by a series of procedures, were shown to be glycoproteins with glucose and N-acetylglucosamine as major saccharides. The agglutinating activity of a mixture of pure proteins b and c was over 500 times that of either glycoprotein alone, suggesting an involvement of both glycoproteins in the agglutination process. Further characterization showed that the two glycoproteins were heat-resistant with respect to their agglutinin function, which could be totally inhibited by three sugars: arabinose, glucose and N-acetyglucosamine. It is suggested that glycoproteins b and c are the two subunits of a carbohydrate-binding agglutinin present at the host cell surface and involved in agglutination and attachment of the mycoparasite germ tubes. Key words: agglutinin, attachment, cell surface, sugars, glycoproteins, mycoparasitism.

scholarly journals Localized Tufts of Fibrils on Staphylococcus epidermidis NCTC 11047 Are Comprised of the Accumulation-Associated Protein

2007 ◽  
Vol 189 (7) ◽  
pp. 2793-2804 ◽  
Author(s):  
Miriam A. Banner ◽  
John G. Cunniffe ◽  
Robin L. Macintosh ◽  
Timothy J. Foster ◽  
Holger Rohde ◽  
...  
Keyword(s):  
Cell Surface ◽  
Staphylococcus Epidermidis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Opportunistic Pathogen ◽  
Lateral Position ◽  
Full Length ◽  
Surface Proteins ◽  
Immunogold Staining ◽  
Reverse Transcription Pcr

ABSTRACT Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 ± 10.8 nm long) are usually in a lateral position on the cells. Fibrillar (Fib+) and nonfibrillar (Fib−) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% Fib+ cells and the less hydrophobic cells from the buffer phase entirely comprised Fib− cells. The Fib+ and Fib− subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, Fib+, and Fib− cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and Fib+ cells that were absent from the Fib− cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogold staining with anti-Aap antibodies labeled tuft fibrils of the WT and Fib+ cells but not the cell surface of Fib− cells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and Fib+ S. epidermidis NCTC 11047 populations. Reverse transcription-PCR showed that aap was transcribed in both Fib+ and Fib− cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.

Cell surface protein of Salmonella typhimurium

1975 ◽  
Vol 21 (7) ◽  
pp. 1132-1136 ◽  
Author(s):  
S. Kabir
Keyword(s):  
Cell Surface ◽  
Salmonella Typhimurium ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Apparent Molecular Weight ◽  
Major Protein ◽  
Cell Surface Protein ◽  
Exterior Surface

Lactoperoxidase-catalyzed radioiodination with Na125I was performed both on intact Salmonella typhimurium 1195 and on ghost membrane isolated from the same bacterial strain. Ghost membrane was also prepared from radioiodinated whole bacteria. The labelled proteins from both these ghost membrane preparations were compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to identify the cell surface protein. From the results obtained it was concluded that one major protein with an apparent molecular weight of 12 000 – 13 000 was exposed on the exterior surface of the ghost membrane.

scholarly journals The three cortical membranes of the gregarines (parasitic protozoa). Characterization of the membrane proteins of Gregarina blaberae

1982 ◽  
Vol 201 (3) ◽  
pp. 455-464 ◽  
Author(s):  
M. Philippe ◽  
J. Schrével
Keyword(s):  
Electron Microscopy ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Parasitic Protozoa ◽  
Whole Cells ◽  
Cortical Membranes

Gregarines, which are parasitic protozoa living in invertebrates, possess a cortical structure specific to their vegetative stage: namely two additional cytomembranes are lying just under the plasma membrane. This cortical complex has been isolated by centrifugation on discontinuous sucrose gradients and characterized chemically. Its integrity was tested by electron microscopy. Ghost proteins were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. About 30 polypeptides of mol.wt. 15000–300000 were present in this fraction and four glycoproteins were detected after periodate/Schiff staining. Ten major proteins were labelled after lactoperoxidase-catalysed iodination. The GP2 glycoprotein (41000–49000 apparent mol.wt.) appears to be a major component of the cell surface. Effects of trypsin and Pronase digestion on ghosts and cells were monitored by gel electrophoresis and by electron microscopy. Ghosts treated with low trypsin or Pronase concentrations (10–25μg/ml) became drastically disorganized; many proteins were vigorously attacked in comparison with those of control ghosts. Variations in proteinase-sensitivity of proteins are pointed out. The GP3 glycoprotein (130000–160000 apparent mol.wt.) seemed to be the only glycoprotein released from the cell surface by trypsin. Whole cells treated under the same conditions or with higher proteinase concentrations (up to 1mg/ml) do not exhibit morphological modifications of the cell surface; furthermore, no discernible cleavage of membrane proteins was indicated by electrophoretograms. It is postulated that cell-surface proteins are protected by the dense carbohydrate cell coat. By using various different methods (change of ionic strength, detergent, denaturing agent, labelling experiment) it was possible to localize several major proteins within the protozoon cortical membranes.

Monoclonal antibodies against surface antigens of schistosomula of Schistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (1) ◽  
pp. 65-82 ◽  
Author(s):  
D. W. Taylor ◽  
A. F. Butterworth
Keyword(s):  
Monoclonal Antibodies ◽  
Gel Electrophoresis ◽  
Primary Infection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Surface Antigens ◽  
Soft Agar ◽  
Surface Proteins ◽  
Spleen Cells

SUMMARYMonoclonal antibodies have been produced after fusion of NS-1 murine myeloma cells with spleen cells from mice immunized either by chronic primary infection or with irradiated cercariae: in both cases, animals were challenged with live cercariae 7 days before fusion. The initial cultures were screened for anti-schistosomular antibodies both by a radioimmunoassay with whole schistosomulum extracts and by immunofluorescence. There was no correlation between the two techniques and subsequent screening was carried out by immunofluorescence. Cloning was carried out in soft agar and 7 cloned cell lines, from 5 initial cultures, were selected for detailed study. Products of 6 of these 7 lines were monoclonal, as judged by isoelectricfocusing of [35S]methionine-labelled supernatant fluids, and their binding to live schistosomula was specific. None of the antibodies showed detectable activity in mediating eosinophil- or complement-dependent damage to schistosomula in vitro. However, 2 antibodies were successfully used to isolate surface proteins with an apparent molecular weight of 24000 on SDS-polyacrylamide gel electrophoresis.

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