Surface Anionic Groups in Symbiote-Bearing and Symbiote-Free Strains of Crithidia deanei1

The surface anionic groups of symbiote-bearing and symbiote-free strains of Crithidia deanei were compared by determining cellular electrophoretic mobility, by ultrastructural cytochemistry, and by identification of sialic acids by thin-layer and gasliquid chromatography. Symbiote-free Crithidia deanei has a highly negative surface charge (-0.9984 μm?s^-1? V^-1? cm), which is slightly reduced (-0.8527 μm?s^-1? V^-1? cm) by the presence of the endosymbiote. Treatment of both strains of C. deanei with neuraminidase decreased significantly the electrophoretic mobility of cells toward the cathodic pole, indicating the existence of exposed sialic acid residues responsible for the negative charge on the protozoan cell surface. Thin-layer and gas-liquid chromatography showed that N-glycolyl- and N-acetylneuraminic acids were present in both strains of C. deanei.     

Changes in cell surface anionogenic groups induced by propranolol in Herpetomonas muscarum muscarum

The effects of propranolol (10(-3) mM) on the surface anionic groups of Herpetomonas muscarum muscarum were analysed by cell electrophoresis, by ultrastructural cytochemistry and by identification of sialic acids using paper chromatography. Differentiation of H. muscarum muscarum induced by propranolol treatment caused a significant increase in the net negative surface charge. Binding of cationized ferritin (CF) and colloidal iron hydroxide particles was observed at the cell surface of both untreated and propranolol-treated cells. In cells incubated in the presence of the drug the CF particles were distributed in all membrane regions. However, there were small areas where the particles were absent. In H. muscarum muscarum exposed to propranolol the density of residues of sialic acid per cell was higher, and the agglutinating activity with Sendai virus was more intense. However, the pattern of sialic acid, characterized by the presence of N-acetylneuraminic acid derivative, was not modified upon cell interaction with the drug. Treatment of both control and propranolol-treated protozoa with neuraminidase significantly reduced the surface charge. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. muscarum muscarum.     

Changes in cell surface anionogenic groups during differentiation of Herpetomonas samuelpessoai mediated by dimethylsulfoxide

The surface anionic groups of untreated or dimethyl sulfoxide (DMSO)-treated Herpetomonas samuelpessoai cells were analyzed by cell electrophoresis, ultrastructural cytochemistry, and identification of sialic acids using thin-layer chromatography. Differentiation of H. samuelpessoai induced by DMSO treatment caused a significant increase in the net negative surface charge. In flagellates exposed to DMSO, more cationized ferritin, colloidal iron hydroxide, and sendai virus particles bound to the cell surface. Treatment of both untreated and DMSO-treated flagellates with neuraminidase decreased markedly the EPM of cells to the cathodic pole. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. samuelpessoai. Thin-layer chromatography showed that N-acetyl and N,O-diacylneuraminic acids, in equal proportions, were present in H. samuelpessoai. However, N-acetylneuraminic acid predominates in DMSO-treated cells.     

Relative susceptibilities to neuraminidase of the glycoproteins of the human erythrorcyte

Release of sialic acid from the glycoproteins of the normal human erythrocyte surface by neuraminidase was investigated. The glycoproteins of the membrane were separated by electrophoresis in sodium dodecylsulfate polyacrylamide gels. Sialic acid was determined in the sliced gel by a modification of the 2-thiobarbituric acid method, revealing three sialic acid-containing glycoproteins. Treatment of intact erythrocytes with neuraminidase to remove varying amounts of sialic acid indicates that all the glycoproteins are essentially equally accessible to the neuraminidase when 20%–60% of the sialic acid is removed. Similar but not quite identical results were obtained with isolated erythrocyte membranes.Treatment of intact cells with the lectins concanavalin A or phytohemagglutinin-P resulted in shielding of about 25% and 50%, respectively, of the sialic acid from neuraminidase. Concanavalin A blocked sialic acid release over long time periods and with high concentrations of neuraminidase. In contrast, the sialic acid shielding by phytohemagglutinin-P can be overcome by high concentrations of neuraminidase. Both lectins were found to shield the various glycoproteins selectively, with different patterns of shielding. Wheat germ agglutinin exhibited no detectable effect on the susceptibility of the erythrocyte sialic acid to neuraminidase.     

The cell surface of Phytomonas davidi

Carbohydrates were located on the surface of Phytomonas davidi using ultrastructural cytochemistry, and agglutination induced by lectins which bind to residues of mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. The surface charge of the cells was analysed by the binding of cationic particles (colloidal iron and cationized ferritin) to the cell surface and by cell electrophoretic mobility (EPM). Based on observations of binding of cationic particles to the cell surface; a decrease in the binding of these particles to the cell surface; a decrease in the mean EPM of the cells after their incubation in the presence of neuraminidase; and detection of N-acetylneuraminic acid by paper and gas-liquid chromatography, it was concluded that sialic acid residues are exposed on the surface of P. davidi. These residues may be glycolipids or are masked on the cell surface since only after brief trypsinization were the cells agglutinated by the lectin from Limulus polyphemus.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Sialoglycolipids in Trypanosoma cruzi

Mild oxidation of epimastigote forms of T.cruzi followed by sodium borotritide reduction incorporates radioactivity into glycolipid fractions. Column chromatography on silica gel of the chloroform:methanol (2:1) extract separated two main peaks of radioactivity. Treatment with neuraminidase released 30% and 18% of the radioactivity, respectively. Paper chromatography showed peaks of radioactivity with relative migration to NANA7 of 1.33 in fraction A and 1.33 and 1.51 in fraction B. When unlabeled cells were submitted to a Folch extraction, thin layer chromatography of the upper phase showed at least two components detected with the resorcinol-copper reagent. Enzymatic and mild acid hydrolysis released a sialic acid with a migration relative to NANA of 1.22. These results suggest that a substituted sialic acid is present in glycolipids of the epimastigote form of T.cruzi.     

Changes in Cell Surface Anionogenic Groups Induced by Propranolol in Herpetomonas muscarum muscarum

The effects of propranolol (10⁻³ mM) on the surface anionic groups of Herpetomonas muscarum muscarum were analysed by cell electrophoresis, by ultrastructural cytochemistry and by identification of sialic acids using paper chromatography. Differentiation of H. muscarum muscarum induced by propranolol treatment caused a significant increase in the net negative surface charge. Binding of cationized ferritin (CF) and colloidal iron hydroxide particles was observed at the cell surface of both untreated and propranolol-treated cells. In cells incubated in the presence of the drug the CF particles were distributed in all membrane regions. However, there were small areas where the particles were absent. In H. muscarum muscarum exposed to propranolol the density of residues of sialic acid per cell was higher, and the agglutinating activity with Sendai virus was more intense. However, the pattern of sialic acid, characterized by the presence of N-acetylneuraminic acid derivative, was not modified upon cell interaction with the drug. Treatment of both control and propranolol-treated protozoa with neuraminidase significantly reduced the surface charge. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. muscarum muscarum .     

Humoral immunostimulation. VII. Sialic acid masks antigenic sites on an antibody-selected bariant cell line.

Variant cell lines (LC1, LC2) obtained by growth of mouse L cells (L) in cytostimulatory and cytotoxic doses, respectively, of rabbit anti-L cell antiserum AL) were found previously to be altered in many ways relative to the parent cell line. A major change was the reduction of those surface membrane antigens that AL recognizes. These cell variants have now been found to have increased membrane sialic acid relative to L. Treatment of intact variant cells with neuraminidase (50 units/ml, 37 degrees C, 1 hr) greatly restored the susceptibility of LC1 to lysis with AL. In the presence of 1/100 dilution of AL and 5% complement the viability indices (1.00 = no cell kill) of untreated and neuraminidase-treated cells were respectively: L 0.10 and 0.03 and LC1 0.91 and 0.40. Neuraminidase-treated LC2 cells retained their resistance to AL. Parallel studies with 125I-ALIgG showed increased binding to neuraminidase-treated LC1 relative to native LC1. These findings suggest that the altered membrane sialic acid content affects the immunologic behavior of this cell variant by masking the original cell surface antibody-binding sites. This represents a possible mechanism for tumors to escape immunologic control.     

Mediator-induced macrophage activation, as shown by enhanced cytotoxicity for tumor, requires macrophage surface fucose and sialic acid

Macrophage-activating factor (MAF) activates macrophages so that their cytotoxic capacity is enhanced. This effect of MAF is inhibited by removing fucose from the macrophage cell surface by incubation with fucosidase, or by removing sialic acid by treatment with neuraminidase. After incubation with fucosidase or neuraminidase the average inhibition of cytotoxicity was 92 and 73%, respectively. β-Galactosidase had no effect. Addition of the specific products, fucose or sialic acid, to the incubation mixture of macrophages and enzyme blocked the effect of the enzymes. Taken together these observations indicate that macrophage surface fucose and sialic acid are essential for the interaction of MAF with macrophages which results in enhanced cytotoxicity for tumor cells.     

Effect of modification of synaptosomal membrane glycoproteins on adenylate cyclase activity

The effect of the modification of synaptosomal membrane glycoproteins on the activity of adenylate cyclase was studied. It was found that the binding of concanavalin A to unmodified guinea pig cerebral cortex synaptosomal membrane did not change adenylate cyclase activity. Concanavalin A binding to synaptosomal membrane of hypoxic brain cortex resulted in no decrease of enzyme activity. The level of protein-bound sialic acid in these synaptosomal fractions was 20% lower than in the control. Treatment of synaptosomal membranes with neuraminidase resulted in a decrease of sialic acid content by about 70%, but it had no significant effect on adenylate cyclase activity. The modification with concanvalin A of sugar end groups exposed by neuraminidase treatment resulted in significant decrease of both basal and fluoride-stimulated adenylate cyclase activity. These results seem to indicate that some component of the adenylate cyclase complex of brain synaptosomal membranes is closely interacting with a carbohydrate-containing macromolecule on the cell surface.This work was supported by, the Polish Academy of Sciences within the project 10.4.     

The effects of cell membrane alteration on glucocorticoid uptake by the AtT-20/D-1 target cell

The glucocorticoid-sensitive AtT-20/D-1 cell line was used to study cellular uptake of glucocorticoids. A previous observation that glucocorticoid uptake by these cells was temperature dependent had prompted us to postulate that glucocorticoids entered the cell by a temperature-sensitive transport process located in the cell membrane. Attempts were then made to perturb the membrane mechanism. In some of these experiments, intact cells were treated with neuraminidase or pronase. The release of sialic acid in the case of neuraminidase treatmentand of sialic acid and cell surface peptides in the case of pronase treatment demonstrated that the enzymes were effective. Approx. 60% of total cellular sialic acid was released by a 15 min incubation with 20 μg/ml neuraminidase at 25°C. The treated cells appeared to be viable, in that they continued to produce corticotropin at a normal rate, yet intact cell glucocorticoid binding at both 4 and 25°C was only 20–30% of that of untreated cells. Treatment with pronase also caused steroid uptake at 4 and 25°C to be reduced, although the extent of reduction was less than that seen following neuraminidase treatment.In other experiments, the effect of exposure of AtT-20/D-1 cells to ethanol or dimethyl sulfoxide was determined. The solvent concentrations used (0.5–10%) did not alter cell viability significantly, and the ability of the cytosol receptor to bind steroid in a cell-free preparation was unimpaired. However, incubation of intact cells with 10% (v/v) dimethyl sulfoxide or ethanol resulted in an 80–90% decrease in steroid uptake at 25°C.We conclude that steroid uptake by the intact cell can be perturbed by treatments which do not affect the cytosol receptor or alter cell viability. These results support the postulate that glucocorticoids enter the AtT-20/D-1 cell by a specific membrane-associated mechanism.     

Cell surface sialic acid and the regulation of immune cell interactions: the neuraminidase effect reconsidered

It has been known for over a decade that sialidase (neuraminidase) treatment could substantially enhance the capacity of resting B cells to stimulate the proliferation of allogeneic and antigen specific, syngeneic T cells. Thus, cell-surface sialic acid was implicated as a potential modulator of immune cell interaction. However, little progress has been made in either identifying explicit roles for sialic acid in this system or in hypothesizing mechanisms to explain the "neuraminidase effect." Here we show for the first time that cell surface sialic acid on medium incubated B cells blocks access to costimulatory molecules on the B cell surface, and that this is the most likely explanation for the neuraminidase effect. Further, we show that it is likely to be upregulation of ICAM-1 and its subsequent engagement of LFA-1 rather than loss of cell surface sialic acid that in part regulates access to CD86 and other costimulatory molecules. However, we cannot exclude a role for CD86-bound sialic acid on the B cell in modulating binding to T cell CD28. Because sialidase treatment of resting B cells but not resting T cells enables T cell activation, we suggest that sialidase treatment may still be an analogue for an authentic step in B cell activation, and show that for highly activated B cells (activated with polyclonal anti-IgM plus INF-gamma) there is specific loss 2, 6-linked sialic acid. Potential roles for sialic acid in modulating B cell/T cell collaboration are discussed.     

A neuraminidase from Streptococcus sanguis that can release O-acetylated sialic acids

The naturally occurring sialic acids can have different types of N- and O-substitutions, resulting in more than 20 known isomers and compounds. Most methods for the detailed study of these various sialic acids require that the molecules be first released from their alpha-glycosidic linkage. When mild acid hydrolysis is used for this purpose, significant destruction of O-substituent groups occur. On the other hand, the presence of O-substituent groups renders the sialic acid molecule partially or completely resistant to the action of the currently known neuraminidase. To circumvent this problem, we searched for a neuraminidase whose activity is not affected by O-substitution. We reasoned that because Streptococcus sanguis from the human oral cavity is continually exposed to O-substituted sialic acids, its extracellular neuraminidase might not be blocked by O-substitution. We therefore purified this enzyme 3100-fold (56% yield) using ammonium sulfate precipitation, N-(p-aminophenyl)oxamic acid-agarose affinity chromatography, and chromatography on quaternary aminoethyl (QAE)-Sephadex, sulfopropyl (SP)-Sephadex, and Sephacryl S-200. The purified preparation is free of other significant glycosidase activities and proteolytic activities. It is capable of quantitatively releasing all the O-acetylated sialic acids that we studied with the single exception of the 4-O-acetylated sialic acid of equine submaxillary mucin. The activity of the enzyme is also not restricted by the type pf sialic acid linkage or the nature of the underlying oligosaccharide. However, it has maximal activity on gangliosides only in the presence of detergents. The general properties of this enzyme are described and its substrate specificities are contrasted with those of the commonly used neuraminidase from Vibrio cholerae.     

Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay

Summary MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.     

Some electrical properties of the surfaces of murine B- and T-lymphocytes and thymocytes. II. Effects of neuraminidase and ribonuclease.

The electrical properties of the peripheries of murine thymocytes, B-lymphocytes and T-lymphocytes were studied by measurement of electrophoretic mobilities and electron microscopic quantitation of adsorbed, positively charged CIH particles. The effects of neuraminidase and/or ribonuclease treatment upon these parameters were examined. Neuraminidase-susceptible groups accounted for 17%, 13% and 21% of the net surface negativity of T-lymphocytes, B-lymphocytes and thymocytes, respectively, and 28%, 63% and 78% respectively of particle binding. The calculated numbers of charges at the cellular electrokinetic surface per observed CIH particle were similar in control T-cells and thymocytes and higher than in B-lymphocytes. In neuraminidase-treated T- and B-lymphocytes the calculated charges per CIH particle were much lower than in thymocytes. These results may well indicate heterogeneities in the distribution of groups susceptible to neuraminidase, and also in the distribution and/or chemical nature of anionic groups susceptible to neither neuraminidase nor ribonuclease at the peripheries of different murine lymphocyte populations; however, at present we cannot discriminate between these and other possibilities.     

Differential exposure of the major gangliosides in rabbit thymocytes to Vibrio cholerae neuraminidase

Neuraminidase treatment of lymphocytes is known to cause changes of cellular responses in several biological phenomena, but the molecules modified on the cell surface by neuraminidase are not known in detail. Rabbit thymocytes, which contain tissue-characteristic gangliosides, were treated with Vibrio cholerae neuraminidase, and the susceptibility of the cell surface sialic acid residues was examined. The amount of sialic acid released from the thymocytes at the highest level was 42.4 nmol per 1 X 10(9) cells, among which 26.5% was from gangliosides. Ninety-three percent of the VI3NeuGc-nLc6Cer, 84% of the IV3NeuGc-nLc4Cer, and 50% of the II3NA2-LacCer in the thymocytes was hydrolyzed to nLc6Cer, nLc4Cer, and LacCer, respectively, but II3NA-LacCer was completely cryptic. Also, among the molecular species of II3NA2-LacCer, C20:0- to C24:0-containing, but not C16:0- to C18:0-containing molecules, were susceptible to neuraminidase. After neuraminidase treatment, nLc4Cer and nLc6Cer became the major glycosphingolipids, and a 15-fold increase of radioactivity incorporated into the glycosphingolipids was observed by the galactose oxidase-sodium borotritide procedure, suggesting that the beta-galactose of the glycosphingolipids produced by neuraminidase treatment is accessibly to the several ligands which are functionally associated with lymphocytes.     

Histochemical investigations on sialic acid containing compounds in the CNS of teleosts (author's transl)]

By means of histochemical experiments the qualitative (microphotographs) and quantitative (cytophotometry) distribution of sialic acid containing compounds was investigated in the mesencephalon and cerebellum of Carassius carassius. 1. The perikarya of nerve cells are especially stained by means of Azur A. 2. Nerve fibres, on the other hand, showed positive reactions with colloidal ironhydroxyd (CIH). 3. In different structures of the CNS the intensity of CIH-staining is decreased up to about 55% following treatment with neuraminidase. 4. There are no differences in the reactions of 7 degrees C as compared to 20 degrees C adapted animals.     

Histochemical study of the distribution of sialic acid on the surface of HeLa cells in culture

Summary The distribution of sialic acid on the surface of HeLa cells is studied using Hale's staining technique. Treatment of the cells with neuraminidase before staining, indicates that the staining technique is specific for the demonstration of sialic acid.HeLa cell monolayers, grown in Leighton tubes, are treated with a solution of E.D.T.A. During separation and rounding up, cells are fixed in calcium-formalin and stained. We found a gradual increase of the Hale's positivity during treatment with E.D.T.A.HeLa cells from suspension cultures are grown in Rose-chambers. They are fixed and stained after various periods of incubation. We found a decrease of Hale's positivy during spreading out of cells and monolayer formation.These findings are discussed in terms of surface charge density and formation of stable cell contacts.The authors thank Mr. C. Dragonetti for technical assistance.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.