scholarly journals Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: The influence of fixation on patterns of lectin binding.

1985 ◽  
Vol 33 (2) ◽  
pp. 110-118 ◽  
Author(s):  
A R Hardham
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Binding Sites ◽  
Phytophthora Cinnamomi ◽  
Specific Binding ◽  
Lectin Binding ◽  
Fluorescein Isothiocyanate ◽  
Soybean Agglutinin ◽  
Intracellular Binding

The study of the surface properties of zoospores and cysts of the fungus Phytophthora cinnamomi required a fixation regime that would preserve the cells adequately and not interfere with binding and detection of probes on the cell surface. When they were fixed in 4% formaldehyde (F), specific binding of concanavalin A-fluorescein isothiocyanate and rhodamine-labeled soybean agglutinin was obtained. However, electron microscopy showed that preservation was so poor that intracellular binding sites had become exposed. By contrast glutaraldehyde (G), even at concentrations as low as 0.05%, gave good preservation of the zoospores but induced high levels of nonspecific fluorescence, making its use impractical for studies using fluorescent probes. Addition of 1-4% F to 0.05-0.8% G reduced the level of G-induced fluorescence while not diminishing the quality of ultrastructural preservation. This effect was evaluated quantitatively and an optimum fixation regime for the fungal cells, namely, 0.2% G and 2-4% F in 50 mM PIPES buffer, was determined. This combined fixative facilities correlated fluorescence and ultrastructural labeling with lectins and immunocytochemical probes.

Glycoconjugates on the surface of spores of the pathogenic fungus Phytophthora cinnamomi studied using fluorescence and electron microscopy and flow cytometry

1990 ◽  
Vol 36 (3) ◽  
pp. 183-192 ◽  
Author(s):  
A. R. Hardham ◽  
E. Suzaki
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Phytophthora Cinnamomi ◽  
Pathogenic Fungus ◽  
Fluorescein Isothiocyanate ◽  
Pathogenic Fungi ◽  
Surface Flow ◽  
Soybean Agglutinin ◽  
Binding Material

Glycoconjugates on the surface of zoospores and cysts of the pathogenic fungus Phytophthora cinnamomi have been studied using fluorescein isothiocyanate labelled lectins for fluorescence microscopy and flow cytometry, and ferritin- and gold-labelled lectins for ultrastructural analysis. Of the five lectins used, only concanavalin A (ConA) binds to the surface of the zoospores, including the flagella and water expulsion vacuole. This suggests that of accessible saccharides, glucosyl or mannosyl residues predominate on the outer surface of the zoospore plasma membrane. Early in encystment, a system of flat disc-like cisternae, which underlie the zoospore plasma membrane, vesiculate. These and other small peripheral vesicles quickly disappear. After the induction of encystment, ConA is no longer localised close to the plasma membrane but binds to material loosely associated with the cell surface. Quantitative measurements by flow cytometry indicate that the ConA-binding material is gradually lost from the cell surface. The cyst wall is weakly labelled, but the site of germ tube emergence stains intensely. During the first 2 min after the induction of encystment, material that binds soybean agglutinin, Helix pommatia agglutinin, and peanut agglutinin appears on the surface of the fungal cells. The distribution of this material, rich in galactosyl or N-acetyl-D-galactosaminosyl residues, is initially patchy, but by 5 min the material evenly coats most of the cell surface. Labelling of zoospores in which intracellular sites are accessible indicates that the soybean agglutinin binding material is stored in vesicles that lie beneath the plasma membrane. Quantitation of soybean agglutinin labelling shows that maximum binding occurs 2–3 min after the induction of encystment. Key words: cell surface, flow cytometry, lectins, pathogenic fungi, Phytophthora cinnamomi.

scholarly journals Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: nature of the surface saccharides as determined by quantitative lectin binding studies.

1985 ◽  
Vol 33 (5) ◽  
pp. 384-388 ◽  
Author(s):  
A Bacic ◽  
M L Williams ◽  
A E Clarke
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Phytophthora Cinnamomi ◽  
Lectin Binding ◽  
Soybean Agglutinin ◽  
Binding Studies ◽  
Con A

The nature of the surface saccharides of zoospores, "partially encysted zoospores" and cysts of the root-rotting fungus Phytophthora cinnamomi, has been examined by quantitative lectin binding studies. Zoospores bound concanavalin A (Con A), but did not bind any of a variety of other lectins tested. In contrast, both cysts and "partially encysted zoospores" bound soybean agglutinin (SBA) as well as Con A. This indicates that accessible alpha-D-glucosyl/alpha-D-mannosyl-containing glycoconjugates predominate at the zoospore surface, whereas both alpha-D-glucosyl/alpha-D-mannosyl and galactosyl and/or N-acetyl-D-galactosaminosyl residues are accessible at the surface of cysts and "partially encysted zoospores." Neither Ulex europeus lectin nor wheat germ agglutinin (WGA) bound to any of the three cell preparations, indicating the absence of accessible alpha-L-fucosyl and N-acetyl-D-glucosaminosyl residues.

Characterization of the exposed carbohydrates on the surface membrane of adult Schistosoma mansoni by analysis of lectin binding

Parasitology ◽  
1980 ◽  
Vol 81 (1) ◽  
pp. 1-15 ◽  
Author(s):  
A. J. G. Simpson ◽  
S. R. Smithers
Keyword(s):  
Schistosoma Mansoni ◽  
Adult Male ◽  
Wheat Germ ◽  
Ricinus Communis ◽  
Specific Binding ◽  
Lectin Binding ◽  
Surface Membrane ◽  
Soybean Agglutinin ◽  
Peanut Agglutinin

SUMMARYThe surface architecture of adult male Schistosoma mansoni was explored using a range of lectins with differing carbohydrate specificities. Highest specific binding was achieved with concanavalin A and the agglutinin of molecular weight 60000 from Ricinus communis; the binding of wheat germ agglutinin was mostly non-specific. Small amounts of peanut agglutinin and soybean agglutinin binding were observed and the binding of these lectins was increased by pre-treating the parasite with neuraminidase. The fucose binding protein of Lotus tetragonolobus failed to bind. These results indicate that mannose and/or glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine and sialic acid are exposed on the surface of the adult male schistosome.

scholarly journals Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

1975 ◽  
Vol 23 (8) ◽  
pp. 607-617 ◽  
Author(s):  
T Amakawa ◽  
T Barka
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Concanavalin A ◽  
Binding Sites ◽  
Lectin Binding ◽  
Single Cells ◽  
Apical Cell ◽  
Acinar Cells ◽  
Con A ◽  
Dissociated Cells

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.

scholarly journals Reversibility of cell surface label rearrangement.

1976 ◽  
Vol 68 (3) ◽  
pp. 629-641 ◽  
Author(s):  
S S Brown ◽  
J P Revel
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Binding Sites ◽  
Random Distribution ◽  
Cytochalasin B ◽  
Con A ◽  
Original Distribution ◽  
Sensitive Structure ◽  
Membrane Components ◽  
Scanning Electron

Cell surface labeling can cause rearrangements of randomly distributed membrane components. Removal of the label bound to the cell surface allows the membrane components to return to their original random distribution, demonstrating that label is necessary to maintain as well as to induce rearrangements. With scanning electron microscopy, the rearrangement of concanavalin A (con A) and ricin binding sites on LA-9 cells has been followed by means of hemocyanin, a visual label. The removal of con A from its binding sites at the cell surface with alpha-methyl mannoside, and the return of these sites to their original distribution are also followed in this manner. There are labeling differences with con A and ricin. Under some conditions, however, the same rearrangements are seen with both lectins. The disappearance of labeled sites from areas of ruffling activity is a major feature of the rearrangements seen. Both this ruffling activity and the rearrangement of label are sensitive to cytochalasin B, and ruffling activity, perhaps along with other cytochalasin-sensitive structure, may play a role in the rearrangements of labeled sites.

Surface structure of the golgi complex and identification of concanavalin-A binding sites

1978 ◽  
Vol 36 (2) ◽  
pp. 246-247
Author(s):  
J.M. Sturgess ◽  
M. Teitelman ◽  
M.A. Moscarello
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Golgi Complex ◽  
Concanavalin A ◽  
Binding Sites ◽  
Lectin Binding ◽  
Bicarbonate Buffer ◽  
Sodium Phosphate Buffer ◽  
Scanning Electron ◽  
Lectin Binding Sites

Scanning electron microscopy has been applied to study the surface ultrastructure of the Golgi complex and labelling techniques have been developed to investigate the distribution of lectin-binding sites on the membrane surfaces. The study is based on the examination of Golgi-rich fractions, isolated by homogenisation and differential centrifugation of rat liver. The membranes are fixed in suspension with 1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4 for 60 mins and then rinsed in distilled water. For scanning electron microscopy, a thin film of membrane is frozen rapidly on coverglasses using liquid Freon 22, cooled by liquid nitrogen and dried in vacuo at -60°C. Membranes are coated with approximately 100 Å gold in a sputter coater and examined at 20 kV in a JEOL JSM-35U scanning electron microscope. For transmission electron microscopy, membranes are processed as described previously. For examination of lectin binding sites, isolated Golgi membranes are washed in sodium bicarbonate buffer, fixed in glutaraldehyde, incubated with concanavalin A (Con A), rinsed in buffer and then incubated with hemocyanin1.

scholarly journals Lectin binding in normal donkey eyeball

2013 ◽  
Vol 3 (1) ◽  
pp. 7
Author(s):  
Khaled Aly
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Lectin Binding ◽  
Fluorescein Isothiocyanate ◽  
Con A ◽  
Ocular Tissues ◽  
Cone Cells ◽  
Wide Range ◽  
Sexually Mature

In the present study, the distribution of various sugar residues in the eyeball tissues of sexually mature donkey was examined by employing fluorescein isothiocyanate-conjugated lectins. Our results revealed the presence of mannose (labeled by lectins ConA), galactose (labeled by PNA, GSAI, ECA), GalNAc (labeled by SBA, VVA), and GlcNAc (labeled by WGA) residues in the donkey ocular tissues. The epithelium and stroma of the ocular tissues were labeled with mannose (ConA) and GlcNAc (WGA) binding lectins. Binding sites for WGA and PNA to the rod and cone cells of the retina were evident. The lectins Con A, WGA and GSAI are bound strongly to the endothelium of blood vessels and to smooth muscle cells of the iris. In conclusion, the findings of the present study clearly indicate that the donkey eyeball contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosly residues), and it lacks fucosyl residues.

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