Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.     

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.     

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.     

Ultrastructure of cell surface coat (glycocalyx) in rat urinary bladder epithelium

Earlier statements to the contrary, the present study demonstrates the presence of a cell surface coat (glycocalyx) on the luminal plasma membrane of the superficial transitional epithelial cells lining the urinary bladder of male Buffalo rats. This coat was demonstrated with ruthenium red, an electron dense stain, which revealed a surface layer, 60-80 A thick, separated from the outer leaflet of the plasma membrane by an electron lucent layer, approximately 30 A thick. The structure of the glycocalyx was not affected by 12 weeks of treatment with dibutylnitrosamine, a known bladder carcinogen.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Localization of pyroantimonate-precipitable cation and surface coat anionic binding sites in developing erythrocytic cells and macrophages in normal human bone marrow

Summary The surfaces of developing erythrocytic cells and macrophages have been examined in normal human bone marrow by means of the pyroantimonate-osmium, ruthenium red and Thorotrast techniques for inorganic cations, surface glycoprotein-phospholipid complexes and surface anionic binding sites, respectively. No differences in the degree of surface coat reactivity were noted in the erythrocytic cells at different stages of maturation while pyroantimonate binding to the plasmalemma was not evident developmentally until the final stages of erythrocytic development. Rhopheocytotic invaginations proved to be chemically distinct from the remainder of the cell surface since they did not bind Thorotrast or pyroantimonate and gave more staining with ruthenium red. Pyroantimonate does not bind to the surface of macrophages and the binding of Thorotrast by these cells is less. Macrophage-erythrocytic cell contact zones did not stain with Thorotrast but stained with ruthenium red. The significance of these observations is discussed.Supported by Grant No. AM-HE-12084-14 from the National Institutes of Health, Bethesda, Maryland. — Appreciation is expressed to Anita Topson and Marjorie Griffith for their technical assistance and to Dr. Robert Hilberg for performing the bone marrow aspirations.     

Oxidation of ruthenium red for use as an intercellular tracer

Summary When ruthenium red (RR) is combined with OsO₄, an electronopaque complex forms which readily binds to the cell surface coat. However, the RR-OsO₄ complex is often excluded from intercellular spaces in many cell types, and thus is not dependable as a tracer of regions continuous with the extracellular space. Postfixation of erythrocytes agglutinated by the lectin helix (Helix promatia) and intact carotid artery endothelium with a freshly prepared mixture of 1% OsO₄ containing 0.1% ruthenium red (RR) resulted in a dense surface deposit of these cells, but intercellular regions were penetrated to a minimal degree by the stain. When a similar mixture of RR-OsO₄ was allowed to stand 3 h before use, RR is oxidized by OsO₄ to yield a ruthenium compound that has a spectrophotometric absorbance maximum at 365 nm. This RR molecule has a reduced number of cationic sites due to binding with osmium dioxide OsO ₂ ⁼ . Postfixation of agglutinated RBCs and carotid artery endothelium with this oxidized ruthenium-OsO₄ mixture resulted in a 50–80% decrease in surface deposition but markedly enhanced penetration into intercellular regions. The enhanced penetration is attributed to decreased binding affinity of the oxidized ruthenium for anionic surface membrane components, permitting effective stain penetration into regions of cell-to-cell contact rather than extensive surface deposition. These studies indicate that the ruthenium compound formed by OsO₄ oxidation of ruthenium red may be a useful tracer for ultrastructural visualization of intercellular spaces and junctions.     

Ultrastructural characterization by ruthenium red of the surface of the fat globule membrane of human and rat milk with data on carbohydrates of fractions of rat milk

The fat globules of the cream fractions of human and rat milk were stained with ruthenium red. Under the electron microscope, discrete granules and an amorphous coat of lesser density are seen at the surface of the milk fat globules. Since ruthenium red binds anionic groups selectively, it is probable that the granules contain the greatest concentration of these groups. The cream fraction of rat milk contains hexoses, hexosamines, methylpentoses and sialic acid. Methylpentoses and hexosamines are significantly enriched in the cream fraction. It is concluded that the finding of a surface coat in milk fat globules is in keeping with the Bargmann-Knoop model and suggests a distinct mechanism for carrying certain complex carbohydrates in milk. The role of the negative charges at the outer surface of the membrane coat is maintaining fat globules in suspension and in binding certain cations such as calcium is suggested.     

Digitonin induced rearrangement of membrane constituents of aldehyde fixed erythrocytes

The present study demonstrates an altered distribution pattern of anionic sites of the human erythrocyte glycocalyx. The findings prove a significant rearrangement of membrane constituents induced by a digitonin treatment of aldehyde fixed red blood cells. The effect is not confined to lipids and, presumably, it causes the loss of lipidic and nonlipidic membrane components. It can be deduced that the fixation with a mixture of formaldehyde and glutaraldehyde insufficiently stabilizes the membrane structure.     

Improved staining of negative binding sites with ruthenium red on cryosections of frozen cells

Hexavalent cationic dye ruthenium red (RR) binds to anionic sites of cellular components, predominantly to the surface coat rich in glycoconjugates, and can be used as a marker of negative binding sites. Due to limited penetration of RR only superficial layers of cells are stained satisfactorily. To improve RR staining of L1210 leukemic cells isolated from culture and concentrated by centrifugation, cryosections of frozen cells were treated by RR to expose simultaneously all the cells and their components to the dye treatment. Cells were fixed with 2% glutaraldehyde in cacodylate buffer (CB), soaked in 2.2 mol/l sucrose and frozen by plunging into liquid nitrogen. Ultrathin cryosections were cut at a temperature of -90 degrees C, transferred to Formvar coated copper grids, postfixed with 1% OsO4 and stained with 0.05% RR in CB for 60-120 min. After removing RR solution with filter the grids were dried and examined electron microscopically. The resulting staining was a combination of a negative contrast (the plasma membrane and membranes of intracellular organelles) and of a positive contrast (cytoplasmic matrix and the extracellular coat). RR staining of negative binding sites on cryosections has proved useful for uniform exposure of all cells and cellular compartments to the dye and especially of external coat containing glycoconjugates.     

Ruthenium red binding to cell coat in neoplastic neurogenic cell lines in culture.

The cell coat of cultivated fetal rat brain cells as well as malignant rat neurogenic cell lines in culture were studied by transmission electron microscopy with the ruthenium red staining technique. Some of the transformed cell lines demonstrated alteration in the bindng properties of ruthenium red to the cell surface. Otherwise no significant correlation between the visualized cell coat thickness and neoplastic transformation was noted.     

Schistosoma mansoni: topochemical features of cercariae, schistosomula, and adults

The teguments of developing and mature cercariae, recently transformed, and 1-wk-old schistosomula and adult worms were examined for the ultrastructural location of macromolecular carbohydrates and polyelectrolytes. The surface of mature cercariae within sporocysts and cercariae released from the snail is covered by a filamentous coat which reacts with cytochemical reagents for the demonstration of vicinal glycols, but neither the coat nor the surface of the tegument plasmalemma binds cationic colloidal iron at low pH.Upon penetrating mammalian skin, the cercaria sheds its surface coat; the tegument surface of newly transformed schistosomula, older schistosomula and adult worms stains en bloc with acidic colloidal iron, as does the tegument plasmalemma of mature cercariae if the overlying filamentous coat is first removed by physicochemical means. The cercarial coat thus serves to mask anionic groups at the surface of the tegument plasmalemma which become functionally exposed after penetration of the mammalian host. The distribution of colloidal iron binding sites coincides with those for the carbohydrate-complexing phytohemmagglutnin, concanavalin A, which suggests that these membrane-fixed anions are acid mucopolysaccharides, glycoproteins or glycolipids. Carbohydrate-containing material was also localized within membrane-bound vesicles of the tegument matrix and perikarya of developing cercariae and postcercarial schistosomes, suggesting that surface mucosubstances contributing to the tegument glycocalyx of these worms are elaborated, at least in part, by the tegument itself.     

Toxocara canis: a labile antigenic surface coat overlying the epicuticle of infective larvae.

An electron-dense coat covering the surface of Toxocara canis infective-stage larvae is described. This coat readily binds to cationized ferritin and ruthenium red, indicating a net negative charge and mucopolysaccharide content, and can be visualized by immuno-electron microscopy only if cryosectioning is employed. Monoclonal antibodies reactive to the surface of live larvae bind the surface coat but not the underlying cuticle in ultrathin cryosections. The surface coat is dissipated on exposure to ethanol, explaining the lack of surface reactivity of conventionally prepared immunoelectron microscopy sections of T. canis. Differential ethanol extraction of surface-iodinated larvae demonstrates that the major component associated with the coat is TES-120, a 120-kDa glycoprotein previously identified by surface iodination, which is also a dominant secreted product. The surface-labeled TES-70 glycoprotein is linked with a more hydrophobic stratum at the surface, while a prominent 32-kDa glycoprotein, TES-32, is more strongly represented within the cuticle itself. Antibody binding to the coat under physiological conditions results in the loss of the surface coat, but this process is arrested at 4 degrees C. This result gives a physical basis to earlier observations on the shedding of surface-bound antibodies by this parasite. An extracuticular surface coat has been demonstrated on Toxocara larvae prior to hatching from the egg and during all stages of in vitro culture, suggesting that it may play a role both in protecting the parasite on hatching in the gastrointestinal tract and on subsequent tissue invasion in evading host immune responses directed at surface antigens.     

An electron microscopic investigation of the surface coat of the electrocyte of Electrophorus electricus

Summary The surface coat of the electrocyte of the main electric organ of Electrophorus electricus was studied using cytochemical methods (periodic acid-silver methenamine, periodic acid-chromic acid-silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, Concanavalin A — horseradish peroxidase, ruthenium red, Alcian-blue lanthanum nitrate, colloidal iron hydroxide and cationized ferritin). The surface of the electrocyte presents perpendicularly oriented tubular invaginations of the cell membrane. The fibrous coat 50–100 nm thick, penetrates into the lumen of the invaginations. It is also observed in the synaptic clefts existent in the posterior face of the electrocyte. The coating of the surface membrane gives a positive reaction with all techniques used. Binding of colloidal iron hydroxide particles was observed only in the outer layer of the coat. With the Alcian-blue lanthanum nitrate technique, microtubules were observed in the cytoplasm of the electrocyte.The results indicate that the surface coat of the electrocyte contains mucopolysaccharides, glycoproteins, acid mucopolysaccharides and anionic sites detected at low (colloidal iron hydroxyde) and neutral (cationized ferritin) pH.This work has been supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Conselho de Ensino e Pesquisa da UFRJ (CEPG) and Banco Nacional de Desenvolvimento Econômico     

Ruthenium Red and the Bacterial Glycocaly

Ruthenium red, a promising cationic reagent for electron microscopy (EM), has long been an important tool in histology. The reagent was initially used by botanists as a semispecific stain for pectic substances, but it has gradually been embraced by investigators in microbiology and the animal sciences as a stain for anionic glycosylated polymeric substances. Luft developed a reliable method and demonstrated that ruthenium red was a useful reagent for visualizing ultrastructural detail. Many investigators, using modifications of Luft's approach, have identified numerous applications for this important reagent. Ruthenium red has been used to show the ultrastructural detail of bacterial glycocalyces. Strong, sharp and consistent observations of this ultrastructural component of the bacterial cell have given a better understanding its fibrous anionic matrix. Any variations in staining owing to artifactual alteration of the fine delicate ultrastructural features have been overcome by incorporation of diamine lysine into ruthenium red methods, thus providing flexible processing times under less than ideal laboratory sampling conditions. Ruthenium red has broad utility in the biological sciences, and in combination with lysine, it is an excellent EM stain for enhanced visualization of bacterial glycocalyx from culture or from clinical specimens.     

Retention of the glycocalyx after cell detachment by EGTA.

Chinese hamster ovary cells were examined ultrastructurally following several detachment procedures. Alterations in the surface glycoproteins were observed by using ruthenium red in the fixation procedure. Trypsin removed a major portion of the cells glycocalyx and formed spherical cellular configurations. EGTA detached cells were also spherical, however, their glycocalyx appeared to remain although redistributed over the cell surface. Kinetic studies showed no alterations in subsequent population doubling times following either detachment procedure. EGTA may thus represent the current method of choice for cell detachment if preservation of the surface glycoproteins is of interest.     

Structural features of alveolar wall basement membrane in the adult rat lung

The ultrastructural characteristics of alveolar (ABM) and capillary (CBM) basement membranes in the adult rat lung have been defined using tannic acid fixation, ruthenium red staining, or incubation in guanidine HCl. ABM is dense and amorphous, has 3- to 5-nm filaments in the lamina rara externa (facing the alveolus) that run between the lamina densa and the basal cell surface of the epithelium, has an orderly array of ruthenium red-positive anionic sites that appear predominantly (79%) on the lamina rara externa, and has discontinuities beneath alveolar type II cells but not type I cells that allow penetration of type II cytoplasmic processes into the interstitium of the alveolar wall. The CBM is fibrillar and less compact than ABM, has no lamina rara filaments, and has one fifth the number of ruthenium red- positive anionic sites of ABM that appear predominantly (64%) overlying the lamina densa. Incubation of lung tissue with Flavobacterium heparinum enzyme or with chondroitinase has shown that ABM anionic sites represent heparan sulfate proteoglycans, whereas CBM anionic sites contain this and other sulfated proteoglycans. The CBM fuses in a local fashion with ABM, compartmentalizing the alveolar wall into a thick and thin side and establishing a thin, single, basement-membrane gas-exchange surface between alveolar air, and capillary blood. The potential implications of ABM and CBM ultrastructure for permeability, cell differentiation, and repair and morphogenesis of the lung are discussed.     

The glycocalyx of the mouse uterine luminal epithelium during estrus, early pregnancy, the peri-implantation period, and delayed implantation. I. Acquisition of Ricinus communis I binding sites during pregnancy

Mouse uteri were examined during estrus, early pregnancy, the peri-implantation period, and delayed implantation to determine whether changes in the surface coat of the luminal epithelium could be associated with receptivity of the uterus to the presence of blastocyst-stage embryos or blastocyst adhesion. By using alkaline bismuth subnitrate to label periodate-oxidized glycols within the glycocalyx we were able to measure the thickness and examine the morphology of the glycocalyx by electron microscopy. Ferritin-conjugated Ricinus communis agglutinin (RCA-I) demonstrated the presence of D-galactose at terminal, nonreducing positions within the glycocalyx. A relatively thick (0.06-0.1-micron) surface coat was present during estrus, but contained almost no RCA-I binding sites. During Day 3 of pregnancy the surface coat remained up to 0.1 micron thick and RCA-I binding sites were present. At Day 4 and during delay the glycocalyx had a fibrillar appearance, contained RCA-I binding sites, and was reduced to 0.06-0.08 micron in thickness. During Day 5 of pregnancy the thickness of the surface coat was greatly reduced, but there remained uniform lectin binding adjacent to the plasma membrane both at sites of blastocyst attachment and between implantation sites. The results indicate that the luminal epithelium of the mouse uterus acquired RCA-I binding sites during pregnancy and that the thickness of the surface coat was greatly reduced at the time of implantation.     

The role of sialated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium.

Changes in the anionic charge distribution at the luminal face of the endothelium of the sinusoids of the bone marrow have been studied at sites of endocytosis by large bristle coated vesicles and at the sites of molecular permeability through diaphragmed fenestrae. The anionic charge distribution has also been studied at the abluminal aspect of these vessels at sites of transmural blood cell passage. Cationic surface markers such as colloidal iron, native ferritin and polycationic ferritin used at low pH, 1.8, and the use of neuraminidase show that the nonmodified endothelial cell surface has exposed sialic acid groups, which are absent at the sites of these functional specializations. Polycationic ferritin binding over a range of pH levels indicates the prsence of another species of anionic materials present at both the nonmodified cell surface and at the sites of the cell surface modifications. This second group of anionic compounds is neuraminidase resistant and has a pKa higher than that of sialic acid (pKa:2.6).     

Electron microscopic observations of the surface of L-cells in culture

Summary Techniques are described for the preparation of preshadowed replicas of both the upper and lower surfaces of L-cells in culture, and of cross sections of L-cells growing on a cellophane substrate. These revealed long slender microvilli, 800 to 1,100 A in diameter, projecting from both upper and lower surfaces of the cells. These microvilli were frequently observed to contact other cells and substrate, and to leave material behind on the substrate. The plasma membrane of the lower surface was separated from the substrate by an electron-lucent gap 200 to 300 A wide. The surface coat of the L-cell was visualized by staining with colloidal iron and ruthenium. Staining with colloidal iron was most intense on the surface of the microvilli. The gap between cell and substrate was intensely stained with ruthenium red. Enzymatic digestion of living cells revealed that both trypsin and neuraminidase reduced the staining of the cell coat by colloidal iron, whereas only trypsin altered its staining with ruthenium red. After trypsin treatment, fragments of an amorphous material with the staining characteristics of the cell coat were observed between the denuded cells. Treatment with ribonuclease, chymotrypsin or hyaluronidase did not affect the staining of the cell coat.