scholarly journals Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label.

1986 ◽  
Vol 102 (2) ◽  
pp. 576-586 ◽  
Author(s):  
F W Kan ◽  
P P da Silva
Keyword(s):  
Concanavalin A ◽  
Colloidal Gold ◽  
Freeze Fracture ◽  
Ultrastructural Localization ◽  
Automated Image Analysis ◽  
Cell Nuclei ◽  
Con A ◽  
Gold Particles ◽  
Ulex Europaeus ◽  
Original Label

We used fracture-label to establish ultrastructural localization of glycoproteins in cross-fractured nuclei of duodenal columnar and exocrine pancreatic cells. Mannose residues were detected in cell nuclei by labeling freeze-fractured tissues with concanavalin A-horseradish peroxidase X colloidal gold (Con A-HRP X CG) or direct concanavalin A X colloidal gold (Con A X CG); fucose residues were detected with Ulex Europaeus I X colloidal gold (UEA I X CG) markers. Areas of the three main intranuclear compartments (euchromatin, heterochromatin, and nucleolus) exposed by freeze-fracture were determined by automated image analysis. Colloidal gold particles bound to each nuclear subcompartment were counted and the results expressed in number of colloidal gold particles per square micrometer +/- SEM. Duodenal and pancreatic tissues fractured and labeled with Con A-HRP X CG complex or direct Con A X CG conjugates showed that the vast majority of Con A binding sites was confined to euchromatin regions with only sparse labeling of the heterochromatin and nucleolus. UEA I labeling of duodenal columnar cells showed that colloidal gold particles were almost exclusively confined to cross-fractured areas where euchromatin is exposed. Trypsinization of the fractured tissues before labeling with Con A and UEA I abolished 95-100% of the original label. Our results show that, within the nucleoplasm, mannose and fucose are residues of glycoproteins preferentially located within the regions of euchromatin.

scholarly journals A Universal Photochemical Method to Prepare Carbohydrate Sensors Based on Perfluorophenylazide Modified Polydopamine for Study of Carbohydrate-Lectin Interactions by QCM Biosensor

Polymers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 1023
Author(s):  
Lili Guo ◽  
Shuang Chao ◽  
Pei Huang ◽  
Xiukai Lv ◽  
Quanquan Song ◽  
...  
Keyword(s):  
Schiff Base ◽  
Quartz Crystal Microbalance ◽  
Concanavalin A ◽  
Quartz Crystal ◽  
Sensor Surface ◽  
Con A ◽  
Ulex Europaeus ◽  
Plant Lectins ◽  
Photochemical Method ◽  
Ulex Europaeus Agglutinin I

A universal photochemical method to prepare carbohydrate sensors based on perfluorophenylazide (PFPA) modified polydopamine (PDA) for the study of carbohydrate–lectin interactions by a quartz crystal microbalance (QCM) biosensor was developed. The PFPA was immobilized on PDA-coated gold sensors via Schiff base reactions. Upon light irradiation, the underivatized carbohydrates were inserted into the sensor surface, including mannose, galactose, fucose and N-acetylglucosamine (GlcNAc). Carbohydrate sensors were evaluated for the binding to a series of plant lectins. A kinetic study of the interactions between mannose and concanavalin A (Con A), fucose and Ulex europaeus agglutinin I (UEA-I) were performed. This method can eliminate the tedious modification of carbohydrates, improve the experimental efficiency, and reduce the experimental cost, which is of great significance for the development of QCM biosensors and the study of biomolecular interactions.

scholarly journals Immunoelectron microscopic demonstration of S-100b protein-like in centriole, cilia, and basal body.

1988 ◽  
Vol 36 (6) ◽  
pp. 693-696 ◽  
Author(s):  
T Uchida ◽  
T Endo
Keyword(s):  
Basal Body ◽  
Colloidal Gold ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Basal Bodies ◽  
Cell Organelles ◽  
Gold Particles ◽  
Microscopic Demonstration ◽  
S 100 ◽  
And Function

We report here the ultrastructural localization of S-100b protein-like immunoreactivity in the centriole, cilia, and basal body. Duodenum and trachea of guinea pigs and rats were fixed and immunostained by the protein A-gold method. All centrioles, cilia, and basal bodies observed showed clear S-100b protein-like immunoreactivity. Specific colloidal gold particles were located over the microtubules in these cell organelles. However, other microtubules scattered throughout the cytoplasm were devoid of immunoreactivity. Although the functional significance of S-100b protein-like immunoreactivity in the centriole, cilia, and basal bodies remains to be elucidated, the present results introduce new perspectives into the investigation of localization and function of S-100 proteins.

scholarly journals Ultrastructural localization of concanavalin A and wheat germ agglutinin binding sites in adult and embryonic mouse myocardium.

1982 ◽  
Vol 30 (3) ◽  
pp. 193-200 ◽  
Author(s):  
D Gros ◽  
B Bruce ◽  
C E Challice ◽  
J Schrevel
Keyword(s):  
Concanavalin A ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Ultrastructural Localization ◽  
Embryonic Cells ◽  
Con A ◽  
Embryonic Mouse ◽  
Transverse Tubular System ◽  
Receptor Sites ◽  
Ultrathin Sections

The lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA), have been used to localize with precision glycosyl residues in adult and embryonic mouse myocardium. They were detected by means of an affinity method using peroxidase and chitobiosylperoxidase, respectively, which then were revealed with 3,3'-diaminobenzidine and H2O2. Exhaustive controls have shown that the binding of Con A and WGA is reversible when experiments are performed with adult specimens (tissue blocks or ultrathin sections of glycol methacrylate-embedded material) or with isolated embryonic cells. Experiments carried out with tissue blocks from embryonic hearts have shown peroxidase binding. This finding is discussed on the basis of the presence of the endogenous lectin-like components in embryonic hearts. Results show that the surface of adult and embryonic myocardial cells specifically bind both Con A and WGA, thus indicating the presence of glycosyl residues similar to alpha-methyl-D-mannoside and N-acetyl-D-glucosamine. In adult heart the transverse tubular system was also labeled. The absence of Con A and WGA receptor sites in the gap junction regions was demonstrated by means of an electron microscope postembedding staining method.

Ferritin-Conjugated Plant Agglutinins as Specific Stains for Cell Surface Saccharides

1971 ◽  
Vol 29 ◽  
pp. 536-537
Author(s):  
G. L. Nicolson ◽  
S. J. Singer
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Room Temperature ◽  
Ultrastructural Localization ◽  
Final Concentration ◽  
Alkaline Ph ◽  
Con A ◽  
Specific Sugar

Plant agglutinins are proteins which bind to specific sugar ligands. For the ultrastructural localization of specific saccharides, we have now conjugated two different plant agglutinins with ferritin (Fer): concanavalin A (Con A), which binds specifically to sugars sterically related to D-glucose and D-mannose, and ricin (Ric), specific for sugars related to D-galactose and L-rhamnose.The conjugates Fer-Con A and Fer-Ric are prepared as follows. To a solution containing 8% Fer and 2.5% purified agglutinin in a buffer (SPB) containing 0.5M NaCl, 0.05M Na phosphate, pH 6.8, is added with stirring freshly distilled glutaraldehyde to a final concentration of 0.05%. Alkaline pH must be avoided to prevent aggregation of the agglutinins. After 45 min. at room temperature the mixture is dialyzed against SPB containing 0.1M NH4Cl. The Fer-agglutinins are centrifuged to remove large aggregates and finally chromatographically purified on Agarose A-1.5m (Fer-ConA) or Biogel P300 (Fer-Ric).

scholarly journals Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling.

1987 ◽  
Vol 35 (2) ◽  
pp. 229-231 ◽  
Author(s):  
H Morioka ◽  
M Tachibana ◽  
M Machino ◽  
A Suganuma
Keyword(s):  
Escherichia Coli ◽  
Bovine Serum Albumin ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Ultrastructural Localization ◽  
Polymyxin B ◽  
Thin Sections ◽  
Gold Particles ◽  
E Coli ◽  
Escherichia Coli Cells

A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells. Gold particles were found on the outer membrane of E. coli, which is consistent with reported biochemical findings. We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.

scholarly journals Membrane differentiation of developing hemic cells of the bone marrow demonstrated by changes in concanavalin A surface labeling.

1979 ◽  
Vol 27 (11) ◽  
pp. 1413-1423 ◽  
Author(s):  
G A Ackerman ◽  
W H Freeman
Keyword(s):  
Bone Marrow ◽  
Concanavalin A ◽  
Bone Marrow Cells ◽  
Ultrastructural Localization ◽  
Spatial Arrangement ◽  
Membrane Glycoproteins ◽  
Con A ◽  
Monocytic Cell ◽  
Receptor Sites ◽  
Cell Series

The concanavalin A-gold labeled horseradish peroxidase (Con A-HRP-G) method has been employed in the ultrastructural localization of Con A surface receptor sites on glutaraldehyde-fixed normal human and guinea pig bone marrow cells. The number of gold particles per micron of cell surface was counted and data subjected to statistical analysis. All cells of the bone marrow exhibited Con A binding; however, the extent of surface labeling was dependent both on cell type and stage of differentiation. Distinctive modifications in mean surface density correlated with specific periods during the maturation of the erythrocytic, neutrophilic, eosinophilic and monocytic cell series. In several instances, the differentiative changes in surface Con A labeling proved to be species dependent. These observations are discussed in relationship to methodology and to potential changes in number and/or spatial arrangement of Con A receptor sites, primarily attributable to mannosyl and/or glucosyl residues associated with membrane glycoproteins and/or glycolipids of developing neutrophilic and erythrocytic cells.

scholarly journals Microtubule and microfilament rearrangements during capping of concanavalin A receptors on cultured ovarian granulosa cells.

1977 ◽  
Vol 73 (1) ◽  
pp. 111-127 ◽  
Author(s):  
D F Albertini ◽  
E Anderson
Keyword(s):  
Granulosa Cells ◽  
Concanavalin A ◽  
Freeze Fracture ◽  
Con A ◽  
Lectin Receptor ◽  
Receptor Complexes ◽  
Thick Filaments ◽  
Section Electron ◽  
Electron Microscope Analysis ◽  
Endocytic Vesicles

Thin-section electron microscope analysis of rat and rabbit-cultured granulosa cells treated with concanavalin A (Con A) at 37 degrees C revealed coordinated changes in the cytoplasmic disposition of microfilaments, thick filaments, and microtubules during cap formation and internalization of lectin-receptor complexes. Con A-receptor clustering is accompanied by an accumulation of subplasmalemmal microfilaments which assemble into a loosely woven ring as patches of receptor move centrally on the cell surface. Periodic densities appear in the microfilament ring which becomes reduced in diameter as patches coalesce to form a single central cap. Microtubules and thick filaments emerge associated with the capped membrane. Capping is followed by endocytosis of the con A-receptor complexes. During this process, the microfilament ring is displaced basally into the cytoplasm and endocytic vesicles are transported to the paranuclear Golgi complex along microtubules and thick filaments. Eventually, these vesicles aggregate near the cell center where they are embedded in a dense meshwork of thick filaments. Freeze-fracture analysis of Con A-capped granulosa cells revealed no alteration in the arrangement of peripheral intramembrane particles but large, smooth domains were conspicuous in the capped region of the plasma membrane. The data are discussed with reference to the participation of microtubules and microfilaments in the capping process.

scholarly journals Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A.

1982 ◽  
Vol 93 (1) ◽  
pp. 155-163 ◽  
Author(s):  
K A Fisher
Keyword(s):  
Concanavalin A ◽  
Biological Membrane ◽  
Freeze Fracture ◽  
Photographic Emulsion ◽  
Con A ◽  
Cell Monolayers ◽  
New Information ◽  
Freeze Dried ◽  
Iodine 125 ◽  
Silver Grains

The technique of monolayer freeze-fracture autoradiography (MONOFARG) has been developed and the principles, quantitation, and application of the method are described. Cell monolayers attached to polylysine-treated glass were freeze-fractured, shadowed, and coated with dry, Parlodion-supported Ilford L4 photographic emulsion at room temperature. Quantitative aspects of MONOFARG were examined using radioiodinated test systems. Background was routinely less than 2.5 X 10(-4) grains/microns 2/day, the highest overall efficiency was between 25% and 45%, and grain density and efficiency were dependent on radiation dose for iodine-125 and D-19 development. Corrected grain densities were linearly proportional to iodine-125 concentration. The method was applied to an examination of the transmembrane distribution of radioiodinated and fluoresceinated concanavalin A (125I-FITC-Con-A). Human erythrocytes were labeled, column-purified, freeze-dried or freeze-fractured, autoradiographed, and examined by electron microscopy. The number of silver grains per square micrometer of unsplit single membrane was essentially identical to that of split extracellular membrane "halves." These data demonstrate that 125I-FITC-Con-A partitions exclusively with the extracellular "half" of the membrane upon freeze-fracturing and can be used as a quantitative marker for the fraction of extracellular split membrane "halves." This method should be able to provide new information about certain transmembrane properties of biological membrane molecules and probes, as well as about the process of freeze-fracture per se.

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

1987 ◽  
Vol 45 ◽  
pp. 1002-1005
Author(s):  
D. R. Abrahamson ◽  
P. L. St.John ◽  
E. W. Perry
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Light Microscope ◽  
Ultrastructural Localization ◽  
The Other ◽  
Quantitative Studies ◽  
Dense Suspension ◽  
Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

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