Ferritin Labeling Research Articles

Permeability of frog mesenteric capillaries after partial pronase digestion of the endothelial glycocalyx.

1. The proteolytic enzyme pronase, which degrades the endothelial cell glycocalyx, was perfused through single capillaries of frog mesentery. Hydraulic conductivity (Lp) of each vessel was determined before and after pronase perfusion. In three vessels in which Lp increased, the ultrastructure of interendothelial clefts was examined. In a separate group of frogs the effect of pronase on the endothelial glycocalyx was assessed by using cationized ferritin to label the capillary luminal surface. 2. Control Lp was 2.0 x 10(-7) cm s-1 cmH2O-1 (10 mg ml-1 bovine serum albumin, BSA, in frog Ringer solution). Vessels were then perfused with a solution containing 0.1 mg ml-1 pronase and 10 mg ml-1 BSA for 1 min. Lp measured in the same eleven vessels increased to 4.9 x 10(-7) cm s-1 cmH2O-1 (P less than 0.005). 3. Transverse sections of three of these vessels were examined by transmission electron microscopy at eight sites along each vessel. In these sections a total of 156 interendothelial cell clefts were found and photographed. No morphological features, such as fenestrations, transendothelial channels, or intercellular gaps associated with inflammation, were found which might account for the increases in Lp. 4. Measurement of cleft dimensions yielded a harmonic mean cleft depth (delta x) of 0.32 microns and an arithmetic mean cleft depth of 0.64 microns. Mean width (w) of the clefts outside the tight regions was 0.012 microns and the cleft length per unit area (L) was 1330 cm-1. The mean fractional pore area of vessel wall per unit cleft depth, Ap/delta x, calculated as Lw/delta x, was 48.7 cm-1. 5. There was less cationic ferritin (CF) labelling of the luminal glycocalyx in pronase-perfused than in control capillaries. On average, the proportion of the luminal surface covered by CF was 85% in controls and 42% in pronase-treated capillaries (P less than 0.01). In some vessels the CF pattern was greatly disrupted, indicating large changes in the glycocalyx structure. 6. It is concluded that the moderate increases in Lp induced by pronase perfusion are associated with partial digestion of the endothelial glycocalyx but are not accompanied by changes in the dimensions of the intercellular cleft. These observations support the fibre matrix hypothesis of capillary permeability and suggest that the endothelial glycocalyx contributes as much as 60% of the hydraulic resistance of the capillary wall.

Ultrastructural study of surface components of Streptococcus suis

The presence of capsular material on cells of nine reference strains of Streptococcus suis representing serotypes 1 to 8 and 1/2 was determined by transmission electron microscopy after polycationic ferritin labeling, immunostabilization, or fixation with a combination of glutaraldehyde and lysine. All the cells of the reference strains examined were covered with a layer of capsular material whose thickness varied between 20 to 30 nm and 350 to 375 nm when examined by immunostabilization. Capsular material from cells exposed to homologous antiserum was usually thicker than that from polycationic ferritin-labeled cells or cells fixed with glutaraldehyde-lysine. Negative staining revealed detectable surface structures on S. suis strains. All strains carried peritichous, thin, and flexible fimbriae with a diameter of approximately 2 nm and a length of up to 250 nm. This study indicated that morphological differences of surface structure exist among S. suis reference strains.

Differentiation of trophoblast of the baboon blastocyst.

The structure of trophoblast of the baboon blastocyst undergoes a number of maturational changes from the early blastocyst to the late blastocyst stage. The striking expansion of the blastocyst that occurs during the preimplantation period is accompanied by the development of an extensive endocytic apparatus. Cationized ferritin labels coated depressions and vesicles near the apical cell surface, numerous uncoated tubules and larger apical vesicles, and multivesicular bodies within trophoblast cells. Basally and laterally the labeled components are primarily small uncoated vesicles and tubules. Small, discrete clusters of ferritin particles were seen within the basolateral compartment between trophoblast and its basal lamina and beneath trophoblast cells that do not have a basal lamina. the results indicate that ingested materials may be directed in two pathways, one involving breakdown within the lysosomal system and one involving transcytosis. The zona pellucida is a trilaminar structure consisting of a fibrillar outer layer that often contains spermatozoa, an intermediate zone, and an inner layer containing columns of dense zonal material. Loss of the zona occurs after expansion of the blastocyst and development of the endocytic organelles. During the late blastocyst stage, syncytial trophoblast differentiates at the margin of the polar trophoblast. Because blastocysts were flushed from the uterus, it could not be determined whether azonal blastocysts had been adherent to the uterine surface prior to collection.

Lectin teratogenesis. II: Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the teratogenically sensitive period.

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats.

The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry. Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.

Electron microscopic examination of capsular material from various serotypes of Actinobacillus pleuropneumoniae.

The capsular material on PPLO broth-grown cells of Actinobacillus pleuropneumoniae representing serotypes 1 to 10 was visualized by transmission electron microscopy after polycationic ferritin labeling and also after stabilization with specific antibodies. All the isolates examined were covered with a layer of capsular material whose thickness varied between 80 to 90 nm and 210 to 230 nm when examined by immunostabilization. We were also able to visualize A. pleuropneumoniae in lungs of infected pigs and to estimate the amount of capsular material covering the cells. Our results indicate that differences in capsular structure exist among the different A. pleuropneumoniae serotypes, and this result may explain in part why the serotypes are not equally virulent.

Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts.

We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.

Lambert-Eaton myasthenic syndrome: II. Immunoelectron microscopy localization of IgG at the mouse motor end-plate.

The autoimmune origin of the Lambert-Eaton myasthenic syndrome (LEMS) was documented by passive transfer of its electrophysiological features from humans to mice with IgG. Freeze-fracture electron microscopy has demonstrated a loss of active-zone particles in human LEMS and in its mouse passive transfer model. These data imply that the active zones are targets of the pathogenic LEMS autoantibodies. Immunolocalization of the antibodies has been hindered, however, by a paucity of active-zone particles (about 50/micron2 normally and still lower in LEMS) and by diffusion artifacts in the immunoperoxidase method. To obviate these problems, we employed sensitive avidin-biotin detection systems, both peroxidase and ferritin labels, and quantitative immunoelectron microscopy and end-plate morphometry. We compared mice treated with LEMS IgG, control IgG, and no IgG. In all mice, nonspecific background staining was found in the basal lamina covering the muscle fibers and Schwann cells. When a single 10-mg dose of IgG was injected intravenously, IgG samples from 12 patients produced significant immunostaining of the mouse active zones; from 7 patients they did not. Higher doses of intraperitoneally injected IgG (20 mg, three times a day for 2 days, or 10 mg/day for 15 days) from each of 4 patients (3 of whose IgG previously transferred LEMS to mice) caused significant immunostaining of mouse active zones: (1) the mean density (no./micron presynaptic membrane length) of positive active zones was 0.91 in the immunoferritin study and 0.72 in the immunoperoxidase study (control values, 0.12 and 0.02); and (2) 43% of the ferritin particles in the primary cleft were concentrated at the active zones and the rest were scattered randomly (control value, 5.3%). The findings indicate that LEMS IgG binds to the active zones of the presynaptic membrane.

Electron microscopic visualization of capsular material of Pasteurella multocida types A and D labeled with polycationic ferritin.

The presence of capsular material on cells of Pasteurella multocida types A and D was determined by transmission electron microscopy after polycationic ferritin labeling. The capsule of agar-grown isolates of P. multocida type A was thick (70 to 90 nm) and regular, whereas that of type D isolates was thinner (20 to 30 nm) and irregular. Such layers were seen on cells from 4- to 6-h broth cultures, but cells from older cultures (12 to 18 h) had very little cell-associated capsular material. Our data indicate that the capsular material of P. multocida types A and D is morphologically different and that capsule production in broth culture is maximal during early log phase.

Ultrastructural Localization of Cell Surface Immunospecificity in Anabaena azollae Using Indirect Fluorescent Antibody Staining

Antisera prepared against whole cells were used to differentiate between freshly isolated and cultured cyanobacteria from the nitrogen-fixing fern Azolla filiculoides using both light and transmission electron microscopy (TEM). TEM suggested that the differentiating antigens are evenly distributed on the cell wall outer membrane of the cyanobacteria. The fluorescein isothiocyanate (FITC)-conjugated secondary antibody accumulation was sufficiently electron-dense to be detected without the need for either gold or ferritin labelling. Relative amounts of fluorescence which occurred on specimens viewed with fluorescent light microscopy were comparable to the thickness of electron dense material on the surfaces of these cells. Thus, it is possible to use the same immunological procedure to prepare cells for both light and TEM study.

ULTRASTRUCTURAL LOCALIZATION OF CELL SURFACE IMMUNOSPECIFICITY IN ANABAENA AZOLLAE USING INDIRECT FLUORESCENT ANTIBODY STAINING

Antisera prepared against whole cells were used to differentiate between freshly isolated and cultured cyanobacteria from the nitrogen‐fixing fern Azolla filiculoides using both light and transmission electron microscopy (TEM). TEM suggested that the differentiating antigens are evenly distributed on the cell wall outer membrane of the cyanobacteria. The fluorescein isothiocyanate (FITC)‐conjugated secondary antibody accumulation was sufficiently electron‐dense to be detected without the need for either gold or ferritin labelling. Relative amounts of fluorescence which occurred on specimens viewed with fluorescent light microscopy were comparable to the thickness of electron dense material on the surfaces of these cells. Thus, it is possible to use the same immunological procedure to prepare cells for both light and TEM study.

Electron-microscopic studies on location of SH-groups in mitochondrial F 1-ATPase using a ferritin label

A new approach has been suggested for electron-microscopic study of the structure of mitochondrial F 1-ATPase based on ferritin labeling. By means of sequential treatment with 2-iminothiolane and Nbs 2 we obtained a modified ferritin (NbsSPrCNH-Ft) able to react with SH-groups of proteins and to form conjugates in which the protein and ferritin are bound by disulfide bonds. An electron-microscopic investigation of the negatively stained preparations of mitochondrial F 1-ATPase, preincubated with modified ferritin, revealed such enzyme-ferritin conjugates. In case of modified ferritin, containing 360 mol SH-groups per mol protein, and F 1-ATPase, pretreated with N-ethylmaleimide and then with dithiothreitol, conjugates were obtained in which ferritin molecules are bound to several (as many as four) of the six protein masses, comprising a bilayer molecule of the enzyme. Taking into consideration the biochemical data on the location of accessible SH-groups (only in α, γ or ε subunits), it is inferred from the results obtained that one of the protein masses is a complex between β subunit and at least one of the minor subunits located partially on the molecule's external side. This indicates the nonequivalence of different copies of the major subunits. Averaged images of the particles of the F 1-F 0 complex from bovine heart mitochondria and bacteria Micrococcus lysodeicticus were obtained. It was found that F 0 component is bound to two adjacent protein masses of the F 1-ATPase molecule. It is suggested that this binding may be due the nonequivalency of single-type major subunits.

Intracellular localization of thymosin alpha 1 by immunoelectron microscopy using a monoclonal antibody.

Distribution of thymosin alpha 1 in normal mice (OF1) or autoimmune mice (NZB) was investigated using immunocytochemical techniques on sections of GMA- and Epon-embedded mouse thymuses. A monoclonal antibody directed against synthetic thymosin alpha 1 was used. With the immunofluorescence assay, patchy staining of thymosin alpha 1 was found in the cytoplasm of epithelial cells of the subcapsullary and medullary zones of OF1 thymus. In NZB thymus, the fluorescent pattern was less precisely localized. At the electron microscopic level, immunolabeling of Epon-embedded ultra-thin sections revealed ferritin in some vacuoles of epithelial cells. Ferritin labeling in OF1 thymus was found in several small vacuoles of the same cell, but was present in large, dense vacuoles in NZB thymus. These differences might reflect differences in the secretory process of thymic hormone.

Qualitative and quantitative analysis of erythrocyte surface membrane sialyl residues using affinity cytochemistry with special reference to diabetic patients.

Erythrocyte surface membrane sialyl residues were investigated by means of affinity cytochemistry using the avidin-biotin complex technique. Mild oxidation with the periodate (MO)-biotin hydrazide (BHZ)-ferritin avidin conjugate (FAv) sequence revealed numerous ferritin particles on erythrocytes from healthy donors. The ferritin particles attached on the perpendicularly sectioned membrane were seen at an average distance of 10 to 12 nm from the outer dense leaflet of the cell membrane. Pretreatment with neuraminidase followed by the MO-BHZ-FAv sequence almost eliminated erythrocyte ferritin labeling. Erythrocytes from diabetic patients showed less dense ferritin labeling compared with those from healthy donors. Quantiative analysis of sialyl residues demonstrated a marked reduction in ferritin labeling of erythrocytes from diabetic patients which was significantly less (p less than 0.01) than that of erythrocytes from healthy donors. This observation supports previous biochemical data demonstrating lower levels of surface membrane negative charge and sialyl residues on erythrocytes from patients with diabetes mellitus.

Effects of Hybridoma Antibodies on Invasion of Cultured Cells by Sporozoites of Eimeria

Hybridoma antibodies (Hab) produced against sporozoites or merozoites of four species of Eimeria were tested for the ability to inhibit the invasion of cultured primary avian kidney cells by sporozoites of Eimeria. Five of 16 Hab that were tested showed inhibitory activity. All five of these Hab were produced against sporozoites and reacted with sporozoite surface antigens or surface/internal antigens. Four Hab produced against merozoites of E. acervulina cross-reacted with sporozoite surface antigens but failed to inhibit invasion. Similarly, Hab reacting with sporozoite anterior tips or refractile bodies had little effect on invasion. Collectively, the data suggest that surface antigens or surface/internal antigens that are unique to the sporozoite stage may influence or be part of the invasion process. Indirect immunofluorescent-antibody tests and ferritin (Fe) labeling combined with electron microscopy indicated differences in binding of two of the Hab to the sporozoite surface membranes. For example, after exposure to Hab 43A6 and a fluorescein-antimouse IgG conjugate, extracellular sporozoites of E. meleagrimitis fluoresced brightly but intracellular sporozoites exhibited little fluorescent label. Sporozoites labeled with Hab 43A6 plus a ferritin-antimouse IgG conjugate that were observed in the process of cell invasion had ferritin on the extracellular portion of the parasite but not on the intracellular portion. Extracellular aggregates of ferritin were observed near the site of invasion. The data suggested that antigens of the sporozoite surface that are recognized by Hab 43A6 are "scraped off" during the invasion of cells. In contrast, after exposure to Hab E5, both extracellular and intracellular sporozoites of E. tenella fluoresced. However, ferritin label was not observed on viable sporozoites, even when they were fixed immediately after the labeling procedure. The antigens recognized by Hab E5 may be associated with parasite secretory products rather than with an integral part of the sporozoite surface membrane.

Electronmicroscopy of the surface of Pasteurella haemolytica.

The surfaces of Pasteurella haemolytica, biotype A, serotypes 1, 2, 6, 7 and 9 and of P. haemolytica, biotype T serotypes 3, 4, 10 and 15 were examined by transmission electronmicroscopy with ruthenium red staining and polycationic ferritin labelling, by scanning electronmicroscopy, and by light microscopy. Electronmicroscopy showed that the surface of strains of P. haemolytica biotype A was covered by irregular protrusions which were probably capsular material. The surface and general morphology of P. haemolytica biotype T were distinct from those of biotype A.

Mechanical Wounding Induces the Formation of Extensive Coated Membranes in Giant Algal Cells

Mechanically wounding giant cells of Boergesenia forbesii induces the formation of bristle-coated, plasma-membrane invaginations (coated pits) and coated vesicles, easily providing a plentiful source of coated membranes in a clean cellular system unencumbered by other tissues. Contractions evoked by wounding partition the cytoplasm into hundreds of spherical protoplasts with approximately 40 percent less total plasma-membrane surface area than the original cell. Ferritin labeling and the appearance of numerous large coated pits and vesicles at the peak period of contraction indicate that these organelles play a role in extensive endocytosis of the plasma membrane.

The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells.

The preservation and contrast of membranous structures in cultured cells using various postfixation procedures prior to embedding have been investigated. These include routine OsO4, ferrocyanide-reduced OsO4, osmium-thiocarbohydrazide-osmium (OTO), and ferrocyanide-reduced osmium-thiocarbohydrazide-ferrocyanide-reduced osmium (R-OTO). With standard ethanol-Epon dehydration/embedding techniques, a dramatic improvement in both membrane contrast and preservation of bilayer membrane structure was achieved using preembedding OTO in cultured cells. R-OTO yielded similar enhanced preservation and contrast of membranes. Both of these methods also resulted in an increase in the contrast of diaminobenzidine reaction product from horseradish peroxidase activity, and of lipid droplets and lipoprotein particles. However, R-OTO did not cause the same increase in the density of proteinaceous elements as was seen with the OTO method. Ferrocyanide-reduced osmium alone showed significant advantages for quantitation of immunocytochemistry using ferritin labels with bismuth subnitrate counterstain. These methods should have general usefulness for the preservation of lipid-containing structures in cultured cells.

Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O2 production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.

Detection of antibodies to adult T-cell leukemia (ATL)-associated virus in ATL patients' sera by immunoelectron microscopy

In an attempt to demonstrate the presence of antibodies to ATL-associated type-C virus particles (ATLV), an indirect immunoferritin method of immunoelectron microscopy was performed on an ATLV-producing human cord T-cell line (MT-2) and short-term cultures of ATL cells. All five sera from ATL patients seropositive to ATL-associated antigens (ATLA) but not three sera from healthy adults were positive for the ferritin labeling of ATLV and plasma membranes in both MT-2 and short-term cultured ATL cells. Sera absorbed with sheep red blood cells or human T-cell acute lymphatic leukemia cells labeled ATLV much more intensely with ferritin than with plasma membranes. These results demonstrated at the ultrastructural level that anti-ATLA-positive sera contained antibodies to surface glycoproteins and/or structural proteins of ATLV and that they differed from anti-Forssman or anti-T-cell antibodies. It was also demonstrated that anti-ATLA-positive sera showed the same reactivity with ATLV from both MT-2 and short-term cultured ATL-cells by the immunoferritin method.