Ultrastructural demonstration of cell coat on the cell surfaces of normal human oesophageal epithelium

Synopsis The cell coat in human oesophageal biopsies was studied with Alcian Blue, Ruthenium Red, Safranin O, colloidal iron and the ferrocyanide-osmium tetroxide techniques. Alcianophilic material was found on the cell surface of the basal, prickle cell and functional layers, being most abundant on the superficial cells where it appeared as a continuous coat. In the deeper layers, it tended to have a particulate distribution. Some membrane-coating granules were alcianophilic. Ruthenium Red had a particulate distribution over all cell surfaces. Intercellular debris was also stained. Safranin O produced no staining. Colloidal iron stained the cell coat in a particulate manner. The ferrocyanide-osmium technique showed a uniform filamentous cell coat. The oesophageal epithelial cell coats are, in part, acid mucosubstances which, on the surface cells, may have a protective function.     

Calcium metabolism in intact isolated thymocytes.

Isolated rat thymocytes incubated under proper metabolic conditions extrude Ca2+ previously taken up under metabolically unfavourable conditions. The extrusion can be supported by both respiratory and glycolytic energy but glycolysis seems to be more efficient for this purpose. La3+ (50--200 micron) and the ionophore A 23187 inhibit cell Ca2+ extrusion. Ruthenium Red (1--100 micron) does not influence cell Ca2+ extrusion while it inhibits the in situ mitochondrial cation uptake. All the results are consistent with a cell regulation model of Ca2+ content in which both plasma membrane and mitochondria co-operate, acting in opposite directions, in order to decrease cytosolic Ca2+ concentration. The possibility of Na+-Ca2+ hetero-exchange participation to cell Ca2+ homeostasis regulation is also discussed.     

Partial Characterization of the Cuticle Surface of Meloidogyne javanica Females

Negative charges on the outer cuticular surface of Meloidogyne javanica females were visualized with electron microscope labelling techniques. Evidence is presented that the electronegative charge is not borne on neuraminic acid. Ruthenium red staining indicated acid mucopolysaccharides on the outer surface. A surface coat, or glycocalyx, external to the outer cuticle membrane was demonstrated.     

Ruthenium red adsorption method for measurement of extracellular polysaccharides in sludge flocs

A nondestructive method of measuring extracellular polysaccharides (ECP) in activated sludge floes using Ruthenium Red dye adsorption was developed at the Environmental Engineering Laboratory at the University of Colorado at Boulder. The effects of pH, buffer solution, dye concentration, sludge mass, temperature, and incubation time on dye adsorption was determined. Ruthenium Red dye adsorption to bacterial floes was found to fit a Brunauer-Emmett-Teller (BET) isotherm model. Of the other environmental conditions in the system, pH was found to have the strongest effect on dye adsorption to bacterial flocs. The amount of extra cellular polysaccharides (ECP) measured by Ruthenium Red adsorption was compared with extracellular polysaccharides measured by two chemical extraction methods. Of all methods considered Ruthenium Red dye adsorption measured the highest amount of extracellular polysaccharide with the lowest amount of bacterial cell disruption. Thus, Ruthenium Red dye adsorption was more effective than extraction procedures for measurement of extracellular polysaccharides in activated sludge flocs.     

Peculiar behaviour of rabbit thymocytes in interaction with liposomes of different compositions shown by fluorescence polarization studies,lipid analysis,and uptake of vesicle-entrapped carboxyfluorescein

In order to obtain more information on membrane phenomena occurring at the cell surface of rabbit thymocytes we have performed experiments aimed at altering the lipid composition of the plasma membrane. Thymocytes were incubated at 37°C with phospholipid vesicles of different compositions. Vesicle-cell interaction was followed by measuring the degree of fluorescence polarization and the uptake of vesicle-entrapped carboxyfluorescein. Neutral and negatively charged liposomes prepared from egg phosphatidylcholine are currently used in investigations of vesicle-cell interaction. In this report we show that these liposomes do not interact with rabbit thymocytes as is evident from unaltered lipid fluidity measured in whole cells and in isolated plasma membranes. This was confirmed by experiments with vesicle-entrapped carboxyfluorescein showing hardly any uptake of the fluorophor from neutral and negatively charged egg phosphatidylcholine liposomes. Using both techniques substantial interaction was found with positively charged egg phosphatidylcholine liposomes and with liposomes prepared from soybean lecithin which is composed of a variety of phospholipids. The results of these experiments were supported by lipid analysis of cells treated with soybean lecithin liposomes. Increase in phosphatidylcholine contents of mixed phospholipid vesicles was further shown to result in decreased vesicle-cell interaction. From measurements of the quantity of carboxyfluorescein inside cells and the total amount of cell-associated carboxyfluorescein it is concluded that adsorption plays a prominent role in interaction between liposomes and rabbit lymphocytes. The grade of maturation of lymphocytes was also found to affect vesicle-cell interaction. The more mature thymocytes took up more vesicle-entrapped carboxyfluorescein from soybean liposomes than immature thymocytes. Mesenteric lymph node cells exhibited a still stronger interaction. The role of vesicle and cell surface charge and membrane fluidity of both vesicles and cells in interaction between liposomes and rabbit thymocytes is discussed.     

Electron microscopic observations on pulmonary connective tissue stained by Ruthenium Red

Summary Ultrastructural studies on human lung were performed with special attention to the interstitial acid mucopolysaccharides by Ruthenium Red staining and several enzyme digetion tests withStreptomyces hyaluronidase, chondroitinase ABC, chondroitinase AC, heparinase, trypsin and collagenase.Periodic lateral granules on the major cross bands of collagen fibrils and amorphous coats on them became visible by Ruthenium Red staining. The surface of elastic fibres, associated microfibrils, and some fine fibrils 10–20 nm in diameter were stained. Ruthenium Red also stained the surface of fibroblast and smooth muscle cells, basement membrane and filamentous long segments. In the interstructural space, granular substances 10–80 nm in diameter and fine filaments 3–4 nm thick, which formed a fine reticular network, were clearly observed. They were not visible on the usual thin section. The granular substances were located on the cross points of the fine filaments. They spread continuously and connected with each of the cells and extracellular structures in the pulmonary interstitium. The results of the enzyme digestion tests on the Ruthenium Red-positive material are discussed.     

Polysaccharides connected with microtubules in the lipotubuloids of Ornithogalum umbellatum L.

Summary The presence of polysaccharides connected with microtubules in the lipotubuloids of Ornithogalum umbellatum L. was detected by the silver hexamine method and Ruthenium Red staining. Polysaccharides are localized on the outer surface of microtubules. Staining of polysaccharides by Ruthenium Red causes an increase in the microtubule walls thickness by 2.5 nm and in their diameter by 4.5 nm.     

Electron Microscopic Observations on the Cortical Reaction of the Brittle-Star Amphipholis kochii Lütken, with Special Reference to its Vitelline Coat Modification as Revealed by the Surface Replica Method

This paper describes the fine structural changes of the egg of the brittle-star Amphipholis kochii Lütken during the cortical reaction. The vitelline coat is 20 nm thick, when Ruthenium Red stain is used, and consists of a dense network of fibers. The cortical granules are large, 1.5–2.0 μm in diameter, and exist in several layers in the egg cortex, unlike the monolayer arrangement found in many other animals. The contents of the cortical granules are clearly distinguished into two components: peripheral fibrous (PF) material and central fibrous (CF) material that consists of two components differing in electron density. The PF material is densely stained by periodic acid-chromic acid-silver methenamine stain, while the CF material is stained little if at all by this technique. The vitelline coat and some PF materials form the fertilization membrane, which is about 40 nm thick and consists of three layers; the outer and the inner layer of the fertilization membrane each have a trilaminated structure. The vitelline coat substances are probably located in the upper part of the fertilization membrane. The hyaline layer, 7–8 μm thick, consists mainly of CF materials. These observations on the morphology of the ophiuroid egg are discussed in comparison with those on other echinoderms, especially echinoids and asteroids.     

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry.

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.     

Surface and intracellular localization of concanavalin A in human lymphocytes

Normal human lymphocytes were treated at +4 °C with concanavalin A and subsequently washed. The cells were then incubated for various periods at +37 °C in a culture medium and the fate of the agglutinin was followed by an electron microscopic stain specific for concanavalin A. At zero incubation time, a positive reaction was noted on the entire cell membrane and on numerous pinocytic vesicles; 80% of the lymphocytes were stained. Within 15 min of incubation, the cell surface appeared discontinuously labelled while all the intracytoplasmic vesicles were strongly positive. After 2 h of incubation, the cell surface was almost free of label and positive vesicles were found to be concentrated at one pole of the cell. After 3 h of incubation, no label was found on the cell membrane; instead, large vesicles communicating with the cell membrane contained labelled material.     

Ultrastructural demonstration of cartilage acid glycosaminoglycans

Synopsis A method for the demonstration of cartilage acid glycosaminoglycans by light and electron microscopy is described. Rabbit ear cartilage was fixed in cacodylate buffered 2.5% methanol-free formaldehyde with 0.001 M Ruthenium Red andp-chloromercuribenzoate (PCMB). Dehydration was carried out in ethylene glycol followed by embedding in the water-soluble glycol methacrylate (GMA). In some experiments unfixed cartilage was rapidly dehydrated. Sections, 1 thick, and ultrathin sections from the same blocks were stained with 0.001 M Ruthenium Red. Semi-thin sections from cartilage fixed without heavy metal additives were, in addition, stained with the acidophilic fluorochrome Berberine sulphate. It was found that Ruthenium Red intensely stained the same pericellular zone that stained metachromatically with Toluidine Blue or fluoresced after staining with Berberine sulphate. Prior treatment with 0.05% cetylpyridinium chloride entirely blocked the three reactions. Previous digestion with 0.2 mg hyaluronidase/ml for 30 min at 37°C led to the abolition of the fluorescence reaction with Berberine sulphate. It is concluded that Ruthenium Red selectively stains cartilage acid glycosaminoglycans. With the electron microscope the pericellular zones were found to be built up of a three-dimensional branched meshwork of fibrils covered with a mantle of electron-dense material, presumably acid glycosaminoglycans bound to Ruthenium Red.     

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Immunoelectron microscopic observations

In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.     

Cell surface in the contact interaction of normal masto- and lymphocytes and those subjected to freezing

On studying the surface of intact and cryogenic masto- and lymphocytes by the scanning electron microscopy the formation of close membrane contacts was detected between thymocytes and mast cells looking in vitro as mastolymphocyte rosettes (ML-rosettes). During ML-rosette formation changes in cell surface organization were observed. Availability of viable thymocytes is necessary for ML-rosette formation. The surface topography of cells constituting the ML-rosettes is seen changing during rosette formation. It is proposed that the mastocyte may function as an acceptor acquiring immunological specificity through the contact with lymphocytes whose membrane responds actively towards the antigen by changing its surface. Reorganization of the lymphocyte plasma membrane may be perceived by mastocytes as a signal of granule secretion.     

Studies on the in vitro effects of insulin on glycogen synthesis and ultrastructure in isolated rat liver hepatocytes

Rat liver hepatocytes were isolated by collagenase $\text{in vitro}$ perfusion technique and effect of insulin on glycogen synthesis and ultra-structure was studied. Addition of insulin stimulated glycogen synthesis and maintained better cellular structure. Synthesis of glycogen was linear in isolated hepatocytes when incubated with various concentrations of glucose (0–800 mg%) reaching initial levels. Concanavaline A inhibited epinephrine stimulated glycogenolysis but had no effect on glucagon stimulated glycogenolysis. These studies indicate that insulin is required for glycogen synthesis and for maintaining hepatocytes ultrastructure. Furthermore, isolated hepatocytes retain various receptors and that different hormones utilize different receptor sites.     

Electronmicroscopic histochemical investigation of the surface of the cell membrane of the renal tubules with the use of ruthenium red and lantanum nitrate.

The renal tubules were investigated with the use of ruthenium red and lanthanum nitrate to titrate the carbohydrate groups which are localized on the differentiated surface of the cell membranes and intercellular spaces. Ruthenium red visualized the surface coat which is tightly bound to the outer lamellae of the cell membrane. Lanthanum nitrate used in this investigations is a valuable marker of the intercellular spaces. The applied markers have visualized in the kidney the canals which are originated from the foldings of membranes of the tubular cells which adhere to the basal membrane. The markers used in combination with the fixative glutaraldehyde-paraformaldehyde give an electrondense envelope of the cell membrane which is more distinct as compared with specimen treated with glutaraldehyde and markers only.     

Cytolytic effects of glucocorticoid on thymus-medullary lymphocytes incubated in vitro

Thymus cortical and medullary lymphocytes (TCL and TML) were incubated in vitro with or without cortisol. The cytolytic effects of the steroid on both thymocytes were examined in the microscope and by measurements of protein and RNA synthetic activities. There did not appear to be significant differences in the extent of damage by glucocorticoid exposure between both thymocytes, although the reduction of RNA synthesis in TML was less marked as compared with that in TCL. The facts that lymphocytes in thymus medulla were able to survive after cortisone injection and that the cells incubated in vitro were affected by the steroid presented the hypothesis that there might be some particular mechanisms in thymus medulla to protect lymphocytes from the cytolytic action of glucocorticoid.     

Pathway for uncoupler-induced calcium efflux in rat liver mitochondria: inhibition by ruthenium red

The rate of uncoupler-induced Ca2+ efflux from rat liver mitochondria is increased by acetate and decreased by phosphate. This effect depends on a shift of the apparent Km, which is increased by phosphate and decreased by acetate, while the Vmax is not modified. The modification of the apparent Km by permeant anions presumably reflects changes in the concentration of matrix free Ca2+. A major part of uncoupler-induced Ca2+ efflux is sensitive to Ruthenium Red, the specific inhibitor of the Ca2+ uniporter , but an apparent insensitivity is observed when the H+ permeability is rate limiting in the process of Ca2+ efflux. The titer of uncoupler required for maximal stimulation of Ca2+ efflux increases with the Ca2+ load and may be 1-2 orders of magnitude higher than that required for maximal stimulation of respiration. On the other hand, when the uncoupler concentration is raised above 10(-6) M, the process of Ca2+ efflux becomes again Ruthenium Red insensitive. The Ruthenium Red inhibition of uncoupler-induced Ca2+ efflux is time dependent, in that the degree of inhibition exerted by low amounts of Ruthenium Red increases with the incubation time. Since the inhibition of the rate of Ca2+ influx undergoes a parallel relief, it is possible that Ruthenium Red moves from the cytosolic to the matrix side of the inner membrane. It is concluded that, in native mitochondria, uncoupler-induced Ca2+ efflux occurs via reversal of the uniport Ca2+ carrier, and not through activation of an independent pathway.     

Shedding of gangliosides from calf thymocytes

In the course of our work on calf thymus gangliosides [Dyatlovitskaya, Zablotskaya, Azizov & Bergelson (1980) Eur. J. Biochem. 110, 475-483] we studied the gangliosides exfoliated from the cell surface of thymocytes. It was shown that calf thymocytes shed gangliosides both in vivo and in vitro. Various gangliosides were found to be present in high amounts both in extracellular plasma membrane vesicles and in the 64000 X g supernatant. The extracellular membrane fragments were comparatively higher in disialosyllactosylceramide and the 64000 X g supernatant was higher in sialosyllactosylceramide than the cells. Comparison of the ganglioside composition of extracellular membrane fragments, thymocytes and lymphocytes led us to suggest that the shedding of gangliosides from the surface of thymocytes may be involved in the transformation of immunologically incompetent cortical thymocytes into immunocompetent virgin T-cells.     

Relation between the organization of spectrin and of membrane lipids in lymphocytes

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.     

The isolation of lymphocyte mitochondria and their regulation of extramitochondrial free Ca2+ concentration.

1. A method for the isolation of functionally intact mitochondria from lymphocytes is described. It involves digitonin breakage of the plasma membrane, followed by differential centrifugation. The yield was 36 mg of mitochondrial protein/200 g of pig mesenteric lymph node (6 mg of mitochondrial protein/10(9) lymphocytes). The mitochondrial had a respiratory-control ratio of 2--3.5 with succinate as substrate. 2. Ca2+ transport by these mitochondria was investigated. They were able to regulate the extramitochondrial free [Ca2+] very precisely, by buffering any displacements from the steady-state. The exact extramitochondrial free [Ca2+] of this steady-state depended on the conditions of incubation. In a medium designed to resemble the cytoplasmic environment, with added Ca2+, lymphocyte mitochondria maintained a steady-state free [Ca2+] of 0.63 microM (pCa of 6.2). The rates of Ca2+ uptake and efflux under these conditions, with both lymphocyte and liver mitochondria, were very much lower than those in a less complex medium. 3. Lymphocyte mitochondria were shown to possess an Na+-independent Ruthenium Red-insensitive efflux pathway similar to that of liver mitochondria. Ruthenium Red totally inhibited the electrophoretic uniporter. Although Na+ had no effect on the steady-state maintained by lymphocyte mitochondria, they were shown to possess an Na+/H+ antiporter.