Phagocytosis of concanavalin A in normal and enucleated cultures of mammalian cells

1976 ◽  
Vol 22 (3) ◽  
pp. 623-632
Author(s):  
G.E. Wise
Keyword(s):  
Cell Membrane ◽  
Concanavalin A ◽  
Mammalian Cells ◽  
Ultrastructural Level ◽  
Con A ◽  
Normal Cells ◽  
Chase Period ◽  
Entire Cell ◽  
Uniform Manner ◽  
Large Clusters

The fate of concanavalin A (Con A) bound to normal and enucleated L cells was followed at the ultrastructural level over a 20-h period. In both enucleates and normal cells the Con A is seen to be distributed in a uniform manner over the entire cell surface following a 30-min pulse with a low concentration of Con A. In the subsequent chase period the cells then aggregate the Con A and Con A sites into large clusters on the cell membrane. The cells then phagocytoze the Con A and large phagocytic vacuoles containing it are observed. Thus, enucleated cells are capable of phagocytozing Con A and its sites in the same manner as normal cells.

scholarly journals Concanavalin A binding to human erythrocytes leads to alterations in properties of the membrane skeleton

1987 ◽  
Vol 241 (2) ◽  
pp. 521-525 ◽  
Author(s):  
S M Gokhale ◽  
N G Mehta
Keyword(s):  
Concanavalin A ◽  
Membrane Skeleton ◽  
Cross Linking ◽  
Dependent Manner ◽  
Con A ◽  
Normal Cells ◽  
Skeletal Protein ◽  
Low Ionic Strength ◽  
Dose Dependent ◽  
Resistance To Heat

Three properties related to the erythrocyte membrane skeleton are found to be altered after the binding of concanavalin A (Con A) to erythrocytes or their isolated membranes. Con A binding to normal erythrocytes imparts resistance to heat (49 degrees C)-induced fragmentation of the cells. The fragmentation, due to denaturation of spectrin at 49 degrees C, is prevented by Con A in a dose-dependent manner, but levels off at concentrations of Con A in excess of 100 micrograms/ml. The binding of Con A to ghosts isolated from normal, trypsin- or Pronase-treated cells prevents (completely or substantially) the elution of the skeletal protein complex when the membranes are extracted under low-ionic-strength conditions in the cold. The Con A-agglutinated membranes of trypsin- and Pronase-treated, but not normal, cells show cross-linking of skeletal proteins and band 3 with dimethyl adipimidate, a 0.86 nm (8.6 A)-span bifunctional reagent. The extent of cross-linking is greater in the Pronase-treated membrane than in the less-agglutinable trypsin-treated membranes. The results show that, after Con A has bound, rearrangements occur in the membrane that alter properties of the skeletal proteins. Additionally, redistribution of the skeletal proteins and the Con A receptor occurs in the lectin-agglutinated membranes.

Localization of lectins in legume cotyledons

1975 ◽  
Vol 19 (1) ◽  
pp. 157-167
Author(s):  
A.E. Clarke ◽  
R.B. Knox ◽  
M.A. Jermyn
Keyword(s):  
Cell Wall ◽  
High Resolution ◽  
Cell Membrane ◽  
Concanavalin A ◽  
Kidney Bean ◽  
Intercellular Spaces ◽  
Con A ◽  
Fluorescent Method ◽  
Red Kidney Bean ◽  
Specific Sugar

High-resolution techniques for the localization of lectins are described. Concanavalin A (Con A) and phytohaemagglutinin (PHA) are localized using a fluorescent method with (FITC)-labelled immunoglobulins which bind to the lectins in sections of jack and red kidney bean cotyledons. Specificity is defined by the use of specific sugar inhibitors. Both Con A and PHA are found in cytoplasmic sites. Lectins with beta-glycoside specificity are detected with red-coloured artificial carbohydrate antigens. The beta-galactosyl and beta-glucosyl antigens bind specifically to clusters of spherical bodies in the intercellular spaces, to cell wall sites, and to the periphery of the cytoplasm associated with the cell membrane.

scholarly journals Cytochemical study of concanavalin A binding sites and their mobility in normal, cystic fibrosis, and SV40 transformed human fibroblasts in vitro.

1980 ◽  
Vol 28 (6) ◽  
pp. 543-551 ◽  
Author(s):  
M Yokoyama ◽  
J P Chang ◽  
P C Moller
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Human Fibroblasts ◽  
Transformed Cells ◽  
Reaction Products ◽  
Con A ◽  
Normal Cells ◽  
Cytochemical Study

Concanavalin A (Con A) binding sites and their mobility were studied by peroxidase (Po) and ferritin labeling techniques in normal and SV40 transformed human fibroblasts. Binding sites were visualized either as osmium black of 3'3-diaminobenzidine (DAB) reactions or as ferritin particles. DAB reaction products were localized at the external surface of the plasma membrane and in some multivesicular bodies of fixed cells. The labeling was continuous in normal and SV40 transformed human fibroblasts. When living cells were treated with Con A-Po at 4 degrees C and incubated at 37 degrees C, both normal and transformed cells showed remarkable changes. The foci of membrane indentations (caps or patches) are formed on the cell surface. Many labeled internalized vacuoles and vesicles appeared within the cytoplasm and in close proximity to the Golgi region of all cell types. The cellular changes occurred more quickly in transformed cells than in normal cells. It is concluded that normal cells do cap under certain conditions and that the plasma membranes of transformed cells are more fluid than those of normal cells.

Concanavalin A-induced Aggregation of Human Blood Platelets

1975 ◽  
Vol 33 (02) ◽  
pp. 354-360 ◽  
Author(s):  
Heinrich Patscheke ◽  
Reinhard Brossmer
Keyword(s):  
Concanavalin A ◽  
Binding Capacity ◽  
Specific Binding ◽  
Blood Platelets ◽  
Residual Effect ◽  
Independent Effect ◽  
Con A ◽  
Experimental Conditions ◽  
Main Effect ◽  
Induced Aggregation

SummaryConcanavalin A (CON A) causes platelets to aggregate. A Ca++-independent effect of CON A could be separated from a main effect which depends on Ca++. The main effect probably is a consequence of the CON A-induced platelet release reaction and therefore is platelet-specific. The weak residual effect observed in the presence of Na2EDTA may be due to a similar mechanism as has been demonstrated for CON A-induced aggregations of several other normal and malignant transformed animal cells.Na2EDTA did not inhibit the carbohydrate-specific binding capacity of CON A. Therefore, Na2EDTA appears not to demineralize the CON A molecules under these experimental conditions.α-methyl-D-glucoside inhibits the Ca++-independent as well as the Ca++-dependent effect of CON A.Pretreatment by neuraminidase stimulated the platelet aggregation induced by CON A. It is possible that removal of terminal sialic acid residues makes additional receptors accessible for the binding of CON A.

scholarly journals The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations.

1991 ◽  
Vol 113 (4) ◽  
pp. 731-741 ◽  
Author(s):  
S H Hansen ◽  
K Sandvig ◽  
B van Deurs
Keyword(s):  
Cell Surface ◽  
Coated Vesicles ◽  
Minor Role ◽  
Specific Marker ◽  
Con A ◽  
Early Endosomes ◽  
Gold Conjugate ◽  
Entire Cell ◽  
A Minor ◽  
Endocytic Vesicles

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.

scholarly journals Visualization of intracellular concanavalin A binding sites in retinal photoreceptors.

1978 ◽  
Vol 26 (10) ◽  
pp. 822-828 ◽  
Author(s):  
I Nir
Keyword(s):  
Concanavalin A ◽  
Binding Sites ◽  
Photoreceptor Cells ◽  
Con A ◽  
Thin Sections ◽  
Glycol Methacrylate ◽  
Outer Segments ◽  
Retinal Photoreceptors ◽  
Carbohydrate Components ◽  
The Relationship

Localization of carbohydrate components in retinal photoreceptor cells and membranes was studied. Frog and rat retinas were fixed with glutaraldehyde and embedded in glycol methacrylate or in a mixture of glycol methacrylate, glutaraldehyde and urea. Thin sections were incubated with ferritin-labeled concanavalin A (F-Con A) and stained with osmium vapors. Intensive binding was observed in both rod and cone outer segments. In the rod inner segment, differential binding of F-Con A was demonstrated. While numerous ferritin granules were observed in the myoid zone, only a few were seen in the ellipsoid zone, except for a local accumulation along the plasma membrane. In the rod outer segment, Con A binding sites were closely associated with the disk membranes. Ferritin granules were observed on both sides of the membranes. The relationship between the localization of Con A binding sites and the orientation of visual pigment molecules within the rod outer segments disk membranes was discussed.

Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

1991 ◽  
Vol 11 (8) ◽  
pp. 4128-4134
Author(s):  
J Venema ◽  
A van Hoffen ◽  
V Karcagi ◽  
A T Natarajan ◽  
A A van Zeeland ◽  
...  
Keyword(s):  
Xeroderma Pigmentosum ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Complementation Group ◽  
C Cells ◽  
Dhfr Gene ◽  
Normal Cells ◽  
Pyrimidine Dimers ◽  
Normal Human ◽  
Active Genes

We have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

Factors Influencing the Reaction of α1-Fetoprotein with Concanavalin A and Lens Culinaris Agglutinin in Crossed Affinoimmunoelectrophoresis

1992 ◽  
Vol 38 (8) ◽  
pp. 1418-1424 ◽  
Author(s):  
D Magne ◽  
N Seta ◽  
D Lebrun ◽  
G Durand ◽  
D Durand
Keyword(s):  
Concanavalin A ◽  
Lens Culinaris ◽  
Biological Fluids ◽  
Con A ◽  
Cellular Origin ◽  
Lens Culinaris Agglutinin ◽  
Lentil Lectin ◽  
Wide Range ◽  
Minimal Quantity ◽  
Antibody Ratio

Abstract Concanavalin A (Con A) and lentil lectin (LCA) analysis of alpha-fetoprotein (AFP) glycosylation heterogeneity is used in a variety of clinical situations. We studied the influence of analytical conditions on the separation of AFP glycoforms by using lectin-crossed affinoimmunoelectrophoresis, regardless of the AFP concentration, which can vary over a wide range in biological fluids. We defined the optimal concentration of Con A (2 g/L) and LCA (0.35 g/L) in the first-dimension gel, together with the optimum antigen (AFP)/antibody ratio in the second-dimension gel. The presence of protein in the diluent used for AFP samples was found to change the shape of crossed affinoimmunoelectrophoresis patterns without changing the percentage composition of AFP fractions. The within-run CV was less than 4% for both lectins, and the between-run CV was less than 6.3%. The minimal quantity of AFP that provided a visible pattern with both lectins was 4 ng, corresponding to 50 microL of an 80 micrograms/L AFP sample. These technical conditions allow the cellular origin of AFP to be determined, regardless of the concentration in the sample. Typical AFP lectin patterns of secreting tumors are compared with fetal and cord serum AFP.

scholarly journals Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules

2005 ◽  
Vol 390 (2) ◽  
pp. 407-418 ◽  
Author(s):  
Catherine de Coupade ◽  
Antonio Fittipaldi ◽  
Vanessa Chagnas ◽  
Matthieu Michel ◽  
Sophie Carlier ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Mammalian Cells ◽  
Heparan Sulphate ◽  
Intracellular Delivery ◽  
Membrane Lipid ◽  
Cell Penetrating Peptides ◽  
Heparin Binding ◽  
Independent Manner ◽  
Cell Penetrating

Short peptide sequences that are able to transport molecules across the cell membrane have been developed as tools for intracellular delivery of therapeutic molecules. This work describes a novel family of cell-penetrating peptides named Vectocell® peptides [also termed DPVs (Diatos peptide vectors)]. These peptides, originating from human heparin binding proteins and/or anti-DNA antibodies, once conjugated to a therapeutic molecule, can deliver the molecule to either the cytoplasm or the nucleus of mammalian cells. Vectocell® peptides can drive intracellular delivery of molecules of varying molecular mass, including full-length active immunoglobulins, with efficiency often greater than that of the well-characterized cell-penetrating peptide Tat. The internalization of Vectocell® peptides has been demonstrated to occur in both adherent and suspension cell lines as well as in primary cells through an energy-dependent endocytosis process, involving cell-membrane lipid rafts. This endocytosis occurs after binding of the cell-penetrating peptides to extracellular heparan sulphate proteoglycans, except for one particular peptide (DPV1047) that partially originates from an anti-DNA antibody and is internalized in a caveolar independent manner. These new therapeutic tools are currently being developed for intracellular delivery of a number of active molecules and their potentiality for in vivo transduction investigated.

scholarly journals Pathogenesis of shigella diarrhea. VII. Evidence for a cell membrane toxin receptor involving β1 {arrow} 4-linked N-acetyl-D-glucosamine oligomers

1977 ◽  
Vol 146 (2) ◽  
pp. 535-546 ◽  
Author(s):  
GT Keusch ◽  
M Jacewicz
Keyword(s):  
Cell Membrane ◽  
Cholera Toxin ◽  
Liver Cell ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Cell Membranes ◽  
Proteolytic Enzymes ◽  
Cell Monolayer ◽  
Toxin Receptor ◽  
Toxin Uptake

The binding of ShigeUa dysenteriae 1 cytotoxin to HeLa cells in culture and to isolated rat liver cell membranes was studied by means of an indirect consumption assay of toxicity from the medium, or by determination of cytotoxicity to the HeLa cell monolayer. Both liver cell membranes and HeLa cells removed toxicity from the medium during incubation, in contrast to WI-38 and Y-1 mouse adrenal tumor cells, both of which neither bound nor were affected by the toxin. Uptake of toxin was directly related to concentration of membranes added, time,and temperature, and indirectly related to the ionic strength of the buffer used. The chemical nature of the membrane receptor was characterized by using three principal approaches: (a) enzymatic sensitivity; (b) competitive inhibition and (c) receptor blockade studies. The receptor was destroyed by proteolytic enzymes, phospholipases (which markedly altered the gross appearance of the membrane preparation) and by lysozyme, but not by a variety of other enzymes. Of 28 carbohydrate and glycoprotein haptens studied, including cholera toxin and ganglioside, only the chitin oligosaccharide lysozyme substrates, per N-acetylated chitotriose, chitotetraose, and chitopentaose were effective competitive inhibitors. Greatest inhibition was found with the trimer, N, N', N" triacetyl chitotriose. Of three lectins studied as possible receptor blockers, including phytohemagglutinin, concanavalin A, and wheat germ agglutinin, only the latter, which is known to possess specific binding affinity for N, N', N" triacetyl chitotriose, was able to block toxin uptake. Evidence from all three approaches indicate, therefore, existence of a glycoprotein toxin receptor on mammalian cells, with involvement of oligomeric β1{arrow}4-1inked N-acetyl glucosamine in the receptor. This receptor is clearly distinct from the G(M1) ganglioside thought to be involved in the binding of cholera toxin to the cell membrane of a variety of cell types susceptible to its action.

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