Effect on glutaraldehyde fixation on lectin-mediated agglutination of mouse leukaemia cells

1976 ◽  
Vol 21 (3) ◽  
pp. 579-594
Author(s):  
W.J. Van Blitterswijk ◽  
E.F. Walborg ◽  
C.A. Feltkamp ◽  
H.A. Hilkmann ◽  
P. Emmelot
Keyword(s):  
Ricinus Communis ◽  
Single Cells ◽  
Cell Aggregation ◽  
Cell Counting ◽  
Cell Aggregates ◽  
Glutaraldehyde Fixation ◽  
Shear Forces ◽  
Con A ◽  
Lectin Receptors ◽  
Free Surfaces

The effect of glutaraldehyde fixation on lectin-mediated agglutination of murine leukaemia (GRSL) cells was investigated using 2 assay methods which differed in the shear forces to which the agglutinated cells were subjected. First, lectin and cells were allowed to interact under conditions in which shear forces were minimized and the degree of agglutination was evaluated microscopically by the appearance and size of the cell aggregates. This assay demonstrated that concanavalin A (con A)-, wheat germ agglutinin (WGA)- or Ricinus communis agglutinin I (RCAI)-mediated cytoagglutination was unaffected (WGA and RCAI) or somewhat enhanced (con A) by prior fixation of the cells with glutaraldehyde. Secondly, an electronic particle counter was used to measure the disappearance of single cells and concomitant appearance of cell aggregates as a function of the lectin concentration. In this assay, in which the aggregated cells are subjected to significant shear forces during dilution and cell counting, agglutination of GRSL cells by each of the 3 lectins was drastically inhibited by prior fixation of the cells with glutaraldehyde. This assay also demonstrated enhanced nonlectin-induced cell aggregation after fixation. In both cytoagglutination assays about the same lectin concentration was required for threshold agglutination of unfixed cells. Comparatively, the results of the 2 cytoagglutination assays indicate that a fraction of the lectin-mediated bonds between unfixed cells is shear resistant and that fixation of the cells either weakens these bonds or inhibits their formation. Morphologically, cells prefixed with glutaraldehyde were sperical at all lectin concentrations, with a continuous dense distribution of cell surface-bound con A, labelled directly with haemocyanin or indirectly using the peroxidase-diaminobenzidine reaction. Unfixed cells showed angular and toadstool-shaped deformations, especially at the highest lectin concentrations, the agglutinating surfaces being flattened against each other over extended areas. The distribution of con A label was continuous and dense between the apposed surfaces and discontinuous on free surfaces. In the presence of con A the free surfaces of prefixed cells exhibited more microvilli than the surfaces of non-prefixed cells. These results favour the view that fixation prevents the formation of shear-resistant, lectin-mediated bonds between cells, not by restricting the lateral mobility of lectin receptors, but by impairing the apposition of rigid cell surfaces.

scholarly journals Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

1977 ◽  
Vol 74 (3) ◽  
pp. 950-962 ◽  
Author(s):  
GL Nicolson ◽  
N Usui ◽  
R Yanagimachi ◽  
H Yanagimachi ◽  
Smith JR
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Similar Degree ◽  
Con A ◽  
Lectin Receptors ◽  
Surface Receptors ◽  
Epididymal Spermatozoa ◽  
Lectin Binding Sites

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.

A scanning electron microscope study of lectin receptors on neuroblastoma cells

1978 ◽  
Vol 36 (2) ◽  
pp. 302-303
Author(s):  
P. Maher ◽  
R.S. Molday
Keyword(s):  
Ricinus Communis ◽  
Neuroblastoma Cells ◽  
Fluorescent Light ◽  
Con A ◽  
Polymeric Microspheres ◽  
Lectin Receptors ◽  
Scanning Electron Microscope Study ◽  
Visual Markers ◽  
Uniform Labeling ◽  
Scanning Electron

The organization and redistribution of concanavalin A (Con A), Ricinus communis agglutinin I (RCA I) and wheat germ agglutinin (WGA) receptors on mouse neuroblastoma cells was analyzed using lectins conjugated to fluorescent polymeric microspheres as visual markers for scanning electron microscopy (SEM) and fluorescent light microscopys. Results indicate that all three lectins are internalized by these cells but the patterns of redistribution for Con A differ from those of RCA I and WGA.Labeling of prefixed undifferentiated and differentiated neuroblastoma cells indicated that Con A, WGA and RCA I receptors were densely and uniformly distributed over the cell surface and neurites. Cells treated with lectin for 5-10 minutes at 37°C, washed free of excess reagent and maintained for up to 15 minutes in buffer exhibited uniform labeling patterns.

Lectin-mediated agglutination of amphibian embryonic cells

1977 ◽  
Vol 27 (1) ◽  
pp. 227-243
Author(s):  
B.R. Fraser ◽  
S.E. Zalik
Keyword(s):  
Concanavalin A ◽  
Wheat Germ ◽  
Cytochalasin B ◽  
Ricinus Communis ◽  
Soya Bean ◽  
Embryonic Cells ◽  
Glutaraldehyde Fixation ◽  
Con A ◽  
Neuraminidase Treatment ◽  
Ricinus Communis Agglutinin

Dissociated blastula cells of Xenopus laevis are agglutinated with wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), concanavalin A (Con A) and, to a lesser extent with soya bean agglutinin (SBA). They are not agglutinated with fucose-binding protein. Neuraminidase treatment of cells enhances their agglutinability with RCA and SBA, but has no effect on Con A- and WGA-mediated agglutinability. Treatment of cells with procaine, or xylocaine, has no effect on the cells' agglutinability or on the extrusion of lobopodia. Treatment with colchicine or cytochalasin B either separately or simultaneously has no effect on lectin-mediated agglutinability. Cells treated with cytochalasin B or colchicine and cytochalasin B simultaneously lack lobopodial extensions, while colchicine alone has no effect on these structures. Phenothiazine tranquillizers inhibit agglutination mediated by all of the above mentioned lectins. Lobopodial extensions are absent in cells treated with these compounds. Glutaraldehyde fixation inhibits RCA and WGA mediated agglutinability and reduces the Con A-mediated agglutinability. Results suggest that in this system microtubules and microfilaments are not involved in lectin-mediated agglutination.

scholarly journals Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels.

1986 ◽  
Vol 34 (2) ◽  
pp. 251-261 ◽  
Author(s):  
A W Vorbrodt ◽  
D H Dobrogowska ◽  
A S Lossinsky ◽  
H M Wisniewski
Keyword(s):  
Blood Vessels ◽  
Ricinus Communis ◽  
Helix Pomatia ◽  
Ultrastructural Localization ◽  
Con A ◽  
Gold Complexes ◽  
Entire Cross Section ◽  
Lectin Receptors ◽  
Helix Pomatia Agglutinin ◽  
Lowicryl K4m

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.

scholarly journals Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system.

1976 ◽  
Vol 71 (2) ◽  
pp. 487-496 ◽  
Author(s):  
P Kelly ◽  
C W Cotman ◽  
C Gentry ◽  
G L Nicolson
Keyword(s):  
Granule Cells ◽  
Ricinus Communis ◽  
Postsynaptic Membrane ◽  
Synaptic Membranes ◽  
Con A ◽  
Lectin Receptors ◽  
Mature Neuron ◽  
The Central Nervous System ◽  
Restricted Mobility ◽  
Topographic Distribution

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and -Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.

Effects of carbamylcholine on chick embryo aggregate retina cell cultures

1988 ◽  
Vol 46 ◽  
pp. 350-351
Author(s):  
Glenn M. Cohen ◽  
Radharaman Ray
Keyword(s):  
Cell Cultures ◽  
Single Cells ◽  
Retinal Cell ◽  
Cell Aggregates ◽  
High Concentration ◽  
In Ovo ◽  
Sterile Culture ◽  
Retinal Tissue ◽  
Synaptic Junctions ◽  
And Control

Retinal,cell aggregates develop in culture in a pattern similar to the in ovo retina, forming neurites first and then synapses. In the present study, we continuously exposed chick retinal cell aggregates to a high concentration (1 mM) of carbamylcholine (carbachol), an acetylcholine (ACh) analog that resists hydrolysis by acetylcholinesterase (AChE). This situation is similar to organophosphorus anticholinesterase poisoning in which the ACh level is elevated at synaptic junctions due to inhibition of AChE, Our objective was to determine whether continuous carbachol exposure either damaged cholino- ceptive neurites, cell bodies, and synaptic elements of the aggregates or influenced (hastened or retarded) their development.The retinal tissue was isolated aseptically from 11 day embryonic White Leghorn chicks and then enzymatically (trypsin) and mechanically (trituration) dissociated into single cells. After washing the cells by repeated suspension and low (about 200 x G) centrifugation twice, aggregate cell cultures (about l0 cells/culture) were initiated in 1.5 ml medium (BME, GIBCO) in 35 mm sterile culture dishes and maintained as experimental (containing 10-3 M carbachol) and control specimens.

Effects on Erythrocyte Aggregation and Blood Coagulation from Iohexol Solutions with and without Sodium Chloride

1995 ◽  
Vol 36 (2) ◽  
pp. 204-209
Author(s):  
C.-M. Chai ◽  
T. Almén ◽  
P. Aspelin ◽  
L. Bååth
Keyword(s):  
Sodium Chloride ◽  
Blood Coagulation ◽  
Glucose Solution ◽  
Cell Aggregation ◽  
Test Solution ◽  
Erythrocyte Aggregation ◽  
Cell Aggregates ◽  
Red Cell ◽  
Red Blood Cell Aggregation ◽  
Osmotic Effects

Solutions of the nonionic monomeric contrast medium iohexol (300 mg I/ml) with and without added NaCl were investigated for effects on red blood cell aggregation and blood coagulation. Three volumes of a test solution were mixed in test tubes with one volume of human blood. During 30 min samples of the mixture were taken for investigation. Six test solutions were used: 1) iohexol, 2) iohexol+glucose 280 mM, 3) iohexol+NaCl 150 mM, 4) glucose 280 mM, 5) glucose 140 mM+NaCl 75 mM, 6) NaCl 150 mM. Test solutions with NaCl caused no aggregation. Test solutions without NaCl always caused macroscopic red cell aggregates. These aggregates always disappeared when saline was added to the sample. The macroscopic red cell aggregates could be dispersed to microscopic aggregates by shaking the test tubes. During the next 30 min macroscopic aggregates returned in the glucose solution but not in the iohexol solutions. In 30 min, blood mixed with iohexol solutions never coagulated while blood layered on top of the same iohexol solutions always coagulated. Blood mixed with solutions 5 and 6, both without iohexol, always coagulated. It is concluded that adding 150 mM NaCl to iohexol did not eliminate its ability to antico-agulate whole blood, but inhibited its ability to aggregate red cells. This inhibition was not caused by the osmotic effects of the added NaCl.

Modulation by Heparin of the Interaction of the A1 Domain of von Willebrand Factor With Glycoprotein Ib

Blood ◽  
1999 ◽  
Vol 94 (12) ◽  
pp. 4186-4194 ◽  
Author(s):  
Christelle Perrault ◽  
Nadine Ajzenberg ◽  
Paulette Legendre ◽  
Ghassem Rastegar-Lari ◽  
Dominique Meyer ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Cell Aggregation ◽  
Cell Aggregates ◽  
Critical Determinant ◽  
Amino Terminal ◽  
A Cell ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Amino Terminal Fragment

Abstract The conformation of the A1 domain of von Willebrand factor (vWF) is a critical determinant of its interaction with the glycoprotein (GP) Ib/V/IX complex. To better define the regulatory mechanisms of vWF A1 domain binding to the GPIb/V/IX complex, we studied vWF-dependent aggregation properties of a cell line overexpressing the GPIb, GPIbβ, and GPIX subunits (CHO-GPIbβ/IX cells). We found that CHO-GPIbβ/IX cell aggregation required the presence of both soluble vWF and ristocetin. Ristocetin-induced CHO-GPIbβ/IX cell aggregation was completely inhibited by the recombinant VCL fragment of vWF that contains the A1 domain. Surprisingly, the substitution of heparin for ristocetin resulted in the formation of CHO-GPIbβ/IX cell aggregates. Using monoclonal antibodies blocking vWF interaction with GPIb/V/IX or mocarhagin, a venom metalloproteinase that removes the amino-terminal fragment of GPIb extending from aa 1 to 282, we demonstrated that both ristocetin- and heparin-induced aggregations involved an interaction between the A1 domain of vWF and the GPIb subunit of the GPIb/V/IX complex. The involvement of heparin in cell aggregation was also demonstrated after treatment of heparin with heparinase that abolished CHO-GPIbβ/IX cell aggregation. These results indicated that heparin was able to induce vWF-dependent CHO-GPIbβ/IX cell aggregation. In conclusion, we demonstrated that heparin is capable of positively modulating the vWF interaction with the GPIb/V/IX complex.

scholarly journals An analysis of lectin-initiated cell agglutination in a series of CHO subclones which respond morphologically to growth in dibutyryl cyclic AMP.

1976 ◽  
Vol 70 (1) ◽  
pp. 204-216 ◽  
Author(s):  
J van Veen ◽  
R M Roberts ◽  
K D Noonan
Keyword(s):  
Surface Membrane ◽  
Con A ◽  
Cell Agglutination ◽  
Lectin Receptors ◽  
Lectin Receptor ◽  
Free Mobility ◽  
Receptor Sites ◽  
Protein And Rna Synthesis ◽  
A Cell ◽  
Membrane Components

We have investigated the molecular basis of the agglutinability of CHO subclones which respond differentially in terms of morphology and surface architecture in the presence of dB-cAMP in the medium. We have demonstrated that the agglutinability of these subclones with both wheat germ agglutinin (WGA) and concanavalin A (Con A) probably depends on the free lateral mobility of the lectin receptor sites in the plane of the membrane. The nonagglutinable surface architecture seems to depend on the presence in the membrane of a protease-labile peptide(s), which appears to be distinct from the lectin receptors, as well as on continuous protein and RNA synthesis. This dependence on continuous transcription and translation may be related to the maintenance of the protease-labile peptide(s) in such a state as to restrict mobility of the lectin receptors. The surface architecture defined as nonagglutinable also depends on the state of polymerization of the intracellular microtubules and microfilaments. It is suggested that these microskeletal elements serve to anchor the lectin receptors in such a manner as to restrict their mobility and thereby reduce the relative agglutinability of a cell line. We suggest that control of the free mobility of both the Con A and WGA receptor sites is dependent on two constraints, one applied by protease-labile ("surface") membrane components and the other by components of the intracellular microskeletal system.

Hydra regeneration from recombined ectodermal and endodermal tissue. I. Epibolic ectodermal spreading is driven by cell intercalation

1996 ◽  
Vol 109 (4) ◽  
pp. 763-772 ◽  
Author(s):  
Y. Kishimoto ◽  
M. Murate ◽  
T. Sugiyama
Keyword(s):  
Contact Surface ◽  
Single Cells ◽  
Cell Interaction ◽  
Inhibitory Effect ◽  
Cell Aggregates ◽  
Signaling Mechanism ◽  
Initial Adhesion ◽  
Radial Cell ◽  
Epithelial Sheet ◽  
Cell Intercalation

Cell-cell interaction and cell rearrangement were examined in the process of epithelial sheet formation during regeneration from hydra cell aggregates. The ectodermal and endodermal epithelial cell layers of Hydra magnipapillata were separated by procaine treatment. Each of the separated layers was then dissociated into single cells and reaggregated to produce ectodermal or endodermal cell aggregates. When the two aggregate types were recombined, a firm adhesion was quickly established between them. This was followed by a vigorous spreading of the ectodermal epithelial cells as a thin layer over the endoderm in a manner similar to the ‘epiboly’ in some developing embryos. Cell movement in this spreading process was examined using fluorescent dyestaining. It revealed that cells initially located in the inside of the aggregate migrated to intercalate themselves among the cells originally present in the contact surface. This radial cell intercalation took place continuously in the contact surface of both the ectodermal and endodermal aggregates, and produced a rapid growth of the contact surface, eventually leading to complete envelopment of the entire endoderm by the ectoderm. The resulting structure was a small sphere having a two-layered epithelial organization as in normal hydra. This sphere regenerated into a complete hydra a few days later. A tryptic extract of hydra membrane fraction specifically inhibited the ectodermal spreading over the endoderm, but not the initial adhesion or the later regeneration processes. These observations suggest that radial cell intercalation at the contact surface plays a crucial role in producing ectodermal spreading and establishing epithelial sheet organization in the recombined aggregates. The intercalation is presumably activated by a signal exchange through the contact surface. The inhibitory effect of the membrane extract suggests that it contains a factor that is involved in some way in this signaling mechanism.

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