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.

Location of Radioactively Labelled Extracellular Fluid Indicators in Nervous Tissue by Autoradiography

1969 ◽  
Vol 4 (1) ◽  
pp. 265-288
Author(s):  
D. A. BROWN ◽  
W. E. STUMPF ◽  
L. J. ROTH
Keyword(s):  
Extracellular Fluid ◽  
Radioactive Material ◽  
Photographic Emulsion ◽  
Frozen Sections ◽  
Myelinated Nerve ◽  
Freeze Dried ◽  
Autoradiographic Technique ◽  
Silver Grains

The location of three radioactively labelled extracellular-space indicators ([3H]methoxyinulin, [3H]-D-mannitol and sodium [35S]sulphate) in the superior cervical sympatmhetic and nodose ganglia of cats was studied using an autoradiographic technique. The technique was designed to eliminate movement of soluble, diffusible substances in tissues after excision, and to provide high autoradiographic resolution. Ganglia were rapidly excised after administration of the radioactive compounds in vivo, and frozen in liquid propane. Frozen sections were cut at -60°C at a thickness of 0.7-0. µ. The frozen sections were freeze-dried, dry-mounted on dried, photographic emulsion-coated microscope slides, and exposed at -15°C until development. The highest densities of silver grains in the autoradiographs were associated with regions of the tissue containing the greatest amounts of connective tissue. Lowest densities occurred beneath neurons and myelinated nerve fibres. The silver grain density beneath neuron perikarya was between 10% and 15% of that associated with plasma. Attempts were made to determine the source of these subneuronal silver grains. The results suggested that they could not be ascribed to the following: background; chemographs and pressure artefacts; spread of radiation from radioactive material outside the perikarya; and in vitro translocation of radioactive material into the neurons or into the emulsion beneath the neurons. It was concluded that the subneuronal grains reflected a small amount of intraneuronal penetration of the compounds in vivo. There was very little difference between inulin, mannitol and sulphate with regard to the proportion of intracellular activity. Except for this small amount of intracellular radioactivity, the findings accord with the view that inulin, mannitol and sulphate are confined predominantly to the extracellular fluid.

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 An improved method for freeze-fracture radioautography of tissues and cells, as applied to duodenal epithelium and thymic lymphocytes.

1984 ◽  
Vol 32 (1) ◽  
pp. 17-29 ◽  
Author(s):  
F W Kan ◽  
B M Kopriwa ◽  
C P Leblond
Keyword(s):  
Freeze Fracture ◽  
Electron Microscopic Examination ◽  
Improved Method ◽  
Electron Microscopic ◽  
Carbon Replica ◽  
Radiation Sources ◽  
Freeze Dried ◽  
Duodenal Epithelium ◽  
Tissue Specimens ◽  
Silver Grains

An improved method has been devised for the localization of radioactive substances to either one of the leaflets of cellular membranes. After tissue specimens are freeze-fractured and covered with a platinum-carbon replica, they are freeze-dried to allow coating with radioautographic emulsion at room temperature. After exposure at 4 degrees C and development, the emulsion is protected by layers of carbon and grease before the tissue underlying the replica is dissolved in sodium hypochlorite. The grease is removed in Freon 14 and the replica with its emulsion cover is mounted on a specimen grid for electron microscopic examination. The accuracy of radioactivity localization was demonstrated using 3H-thymidine-labeled liver by finding silver grains over the same sites after freeze-fracture as after thin section radioautography. Tests with 3H-methacrylate revealed that the interposition of a platinum-carbon replica decreased the radioautographic reaction by over 80%; hence, the need for long exposure. Only 67% of the silver grains came from radiation sources located beyond the upper 0.05 micron of the specimen and, therefore, the emulsion could be affected by radiation sources located not only within membrane leaflets but also in nearby cytoplasm. Thus, when 3H-fucose was injected into rats to locate newly formed glycoproteins within intestinal epithelium membranes, some of the silver grains found over E and P faces might be produced by radiation coming from the adjacent cytoplasm. To localize label within membrane leaflets in the absence of radiation sources in the cytoplasm, lymphocyte suspensions were incubated with 3H-concanavalin A at 0 degrees C. The plasmalemma radioactivity was then restricted to the two membrane leaflets, with 87-93% of the silver grains on the E leaflet and 7-13% on the P leaflet. It appears that, under these conditions, the technique provides adequate localization of radioactivity to the leaflets of the cell membrane.

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.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

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.

AUTORADIOGRAPHIC TECHNIQUES FOR THE LOCALIZATION OF HORMONES AND DRUGS AT THE CELLULAR AND SUBCELLULAR LEVEL

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S205-S222 ◽  
Author(s):  
Walter E. Stumpf
Keyword(s):  
Drug Distribution ◽  
Photographic Emulsion ◽  
Frozen Sections ◽  
Hormone Research ◽  
Freeze Dried ◽  
Subcellular Level

ABSTRACT The paper describes four autoradiographic techniques which can be recommended, not without restrictions, for the study of the cellular and subcellular hormone or drug distribution in tissues. In all of the techniques desiccated slides are used which are precoated with photographic emulsion. The techniques are (I) Dry-mounting of freeze-dried sections on emulsion precoated slides; (II) Thaw-mounting of frozen sections on emulsion precoated slides; (III) Smear-mounting on emulsion precoated slides; and (IV) Touch-mounting on emulsion precoated slides. The techniques are designed to avoid or minimize translocation of the labelled molecules during preparation and during the application to photographic emulsion. Cited examples of application of these techniques demonstrate their utility in hormone research.

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.

The Synthesis and Migration of Nuclear Proteins during Mitosis and differentiation of Cells in Rats

1965 ◽  
Vol s3-106 (75) ◽  
pp. 229-240
Author(s):  
R. T. SIMS
Keyword(s):  
Granular Cell ◽  
Nuclear Proteins ◽  
Photographic Emulsion ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei ◽  
Cell Layers ◽  
The Difference ◽  
Mitotic Figures ◽  
And Migration ◽  
Silver Grains

Hooded rats were given an intraperitoneal injection of 3H-tyrosine, and killed in pairs 10 min, 30 min, 12 h, 36 h, 7 days, and 30 days later. A piece of skin with white growing hair, and the tongue, were taken from each animal and radioautographs were prepared. Silver grains were counted over whole nuclei and whole mitotic figures of the germinal cells and whole nuclei of differentiating cells of both tissues. It was found that the interphase nuclei have significantly more silver grains over them than the chromosomes at all stages of mitosis and there are virtually no grains over metaphase, anaphase, and early telophase chromosomes in both tissues of all the animals killed up to 36 h after the injection. The difference between the grain counts over the interphase nuclei and the chromosomes of dividing cells is at least 20-fold at 30 min in the hair matrix, at least 5-fold at 30 min in the tongue and at 36 h in both tissues. It was established that the differences observed between the radioactivities of the nuclei and chromosomes of mitotic figures are real from estimates of: the radioactivity of the cell cytoplasm, volumes of the metaphase chromosomes and interphase nuclei within 1µ of the photographic emulsion, and the volumes of cytoplasm separating the photographic emulsion and these structures. No protein synthesis was demonstrable in the chromosomes during metaphase, anaphase, and early telophase. Nuclear proteins leave the chromosomes during prophase and prometaphase and return to the nucleus during late telophase. The cells in the matrix and upper bulb of the growing hair follicle and those in the germinal, prickle, and granular cell layers of the tongue are in different functional states; 30 min after injection of 3H-tyrosine they have different amounts of it in their nuclear proteins. It is suggested that the amount incorporated into each nucleus is related to the rate at which proteins are being synthesized by the cell.

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.

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