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

The surface anionic groups of untreated or dimethyl sulfoxide (DMSO)-treatedHerpetomonas samuelpessoai cells were analyzed by cell electrophoresis, ultrastructural cytochemistry, and identification of sialic acids using thin-layer chromatography. Differentiation ofH. 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 ofH. samuelpessoai. Thin-layer chromatography showed thatN-acetyl andN,O-diacylneuraminic acids, in equal proportions, were present inH. samuelpessoai. However,N-acetylneuraminic acid predominates in DMSO-treated cells.     

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 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 .     

Developmentally Regulated Protein Expression Mediated by Dimethylsulfoxide in Herpetomonas samuelpessoai

We have analyzed the total cell extract, cell surface, and secretory protein profiles related to cellular differentiation triggered by dimethylsulfoxide in the insect trypanosomatid Herpetomonas samuelpessoai. The flagellates were cultivated in chemically defined conditions in the absence or in the presence of 4% DMSO, and the resolved protein bands were detected by SDS-PAGE gels and avidin-Western blotting. The cell-associated proteins showed a complex pattern of around 40 silver-staining bands ranging from 15 to 150 kDa. There were generally minor quantitative differences in the protein profile between the non-treated and the DMSO-treated cells. The cell-surface protein profile revealed by the incubation of live parasites with biotin showed a decrease in the expression of the 120 kDa biotinylated polypeptide observed in the DMSO-treated cells when compared with untreated ones. However, control samples of both systems showed an endogenous biotinylated polypeptide of 63 kDa which also presented gelatinolytic activity. The trypanosomatids released at least 10 polypeptides to the culture medium. A low molecular mass exopolypeptide (35 kDa) was found exclusively in untreated cells, whereas a high-molecular-mass exopolypetide (250 kDa) was preferentially found in DMSO-treated cells. Received: 12 July 2000 / Accepted: 14 August 2000     

Sialic acid is a cell surface component of Entamoeba invadens trophozoites

The surface anionic groups of Entamoeba invadens were analysed by cell electrophoresis, by ultrastructural cytochemistry, and by identification of sialic acids using paper and gas-liquid chromatography. Binding of colloidal iron hydroxide (CIH) and of cationized ferritin (CF) particles at pH 1.8 and 7.2, respectively, was observed on the cell surface. E. invadens has a highly negative surface charge (-0.96 microns s-1 V-1 cm). Treatment of the cells with trypsin and neuraminidase significantly reduced the electrophoretic mobility by 24% and 40%, respectively. Treatment of the amoebae with neuraminidase also markedly decreased the binding of CIH to the cell surface. This finding suggests that sialic acid residues are the major anionogenic groups exposed on the surface of E. invadens. Paper and gas-liquid chromatography showed that N-acetylneuraminic acid was the only derivative characterized in E. invadens.     

Disposition of glycoproteins in plasma membrane of cultured rat embryonic fibroblasts

Plasma membrane fractions were isolated from untreated and trypsin- or neuraminidase-treated rat embryo fibroblasts and their sialic acids contents per mg membrane protein were determined. The difference represented enzyme releasable sialic acid exposed on the medium side of the cell mambrane. It was 14 to 23% of the total membrane bound sialic acid. Isolated plasma membrane fraction from entreated and enzyme treated cells were then subjected to trypsin or neuraminidase treatment to obtain enzyme-releasable sialic acid from both faces and from the cytoplasmic face of the membrane respectively. Between 30 and 50% of the total membrane bound sialic was released from both the faces and 14 to 30% from the cytoplasmic face. An average of 59% was insusceptible to these enzymes. As an alternative to a cytoplasmic location of sialic acid containing membrane constituents, inaccessibility of enzymes to some of these constituents present on the surface of intact cells is considered.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of plasma membrane fractions isolated from untreated and trypsin treated cells and of trysinized plasma membrane fraction was carried out to know the number and gel migration of proteins and glycoproteins which are exposed on each of the two faces of the plasma membrane and are sensitive or insensitive to trypsin. The resilts obtained were confirmed by SDS-polyacrylamide gel electrophoresis of untreated and trypsin-treated cells and of isolated plasma membrane fraction after subjecting them to enzymatic radioiodination.     

Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride.

Low concentrations of sodium metaperiodate induce specific oxidative cleavage of sialic acids between carbon 7 and carbon 8 or carbon 8 and carbon 9. The aldehydes formed can easily be reduced with NaB3H4 to tritiated 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid or 5-acetamido-3,5-dideoxy-L-arabino-2-octulosonic acid. At 0 degrees, the periodate anion penetrates the cell plasma membrane very slowly and only externally exposed sialic acids are oxidized. This was shown by (a) limited labeling of the sialoglycoproteins in a preparation of inside-out erythrocyte vesicles; (b) trapping 14C-labeled fetuin within resealed erythrocyte ghosts; fetuin was then poorly labeled, whereas the erythrocyte sialoglycoproteins were highly labeled; (c) comparison of labeled glycoproteins of mouse lymphoid cells before and after treatment with neuraminidase. This simple method of specifically introducing a radioactive label into cell surface sialic acids is useful in the study of cell surface sialic acid-containing glycoproteins.     

Characterization of a carbohydrate epitope defined by the monoclonal antibody H185: sialic acid O-acetylation on epithelial cell-surface mucins

Sialic acids comprise a large family of derivatives of neuraminic acid containing methyl, acetyl, sulfate, and phosphate among other groups, which confer specific physicochemical properties (e.g., hydrophobicity and resistance to hydrolases) to the molecules carrying them. Several years ago, a monoclonal antibody, designated H185, was developed, which binds to cell membranes of human corneal, conjunctival, laryngeal, and vaginal epithelia and whose distribution is altered on the ocular surface of patients with keratinizing disease. Recent findings using immunoprecipitation and immunodepletion techniques have demonstrated that, in human corneal epithelial cells, the H185 antigen is carried by the membrane-associated mucin MUC16. In this study, we show that the H185 epitope on human corneal cells and in tear fluid is an O-acetylated sialic acid epitope that can be selectively hydrolyzed in an enzyme-concentration-dependent manner by sialidase from Arthrobacter ureafaciens and to a lesser extent by sialidases from Newcastle disease virus, Clostridium perfringens, and Streptococcus pneumoniae. Binding of the H185 antibody was impaired by treatment of tear fluid with a recombinant 9-O-acetylesterase from influenza C virus. Two O-acetyl derivatives, Neu5,7Ac(2) and Neu5,9Ac(2), were identified in human tear fluid by fluorometric high-performance liquid chromatography (HPLC) and electrospray mass spectrometry (MS). Immunoprecipitation of the H185 epitope from human corneal epithelial cells revealed that Neu5,9Ac(2) was the major derivative on the mucin isolate. These results indicate that exposed wet-surfaced epithelia are decorated with O-acetyl sialic acid derivatives on membrane-associated mucins and suggest that O-acetylation on cell surfaces may protect against pathogen infection by preventing degradation of membrane-associated mucins.     

Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai

We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35%) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.     

The appearance of phospholipase activity in the human macrophage-like cell line U937 during dimethyl sulfoxide induced differentiation

The human histiocyte cell line, U937, with monocyte characteristics, can be induced to differentiate into macrophage-like cells when exposed to growth medium containing 1.5% DMSO. Following three days of exposure, DMSO-treated but not control U937 cells can be stimulated to release endogenous arachidonic acid from their phospholipids. Maximum release of the unsaturated fatty acid occurs with 10 microM calcium ionophore in the presence but not in the absence of exogenously added calcium ion. In addition, DMSO-treated but not control U937 cells exhibit phospholipase activity when exposed to human IgG and then anti-human immunoglobulin. These data suggest that with respect to arachidonic acid metabolism U937 cells differentiate into functional macrophage-like cells when exposed to DMSO.     

Intracellular trafficking of cell surface sialoglycoconjugates

Recent reports have suggested that the majority of the molecular traffic through the Golgi apparatus is comprised of recycling, rather than newly synthesized, molecules. To evaluate the importance of this recycling pathway in greater detail, we examined the internalization and recycling of cell surface glycoproteins on EL-4 cells, a murine T-cell lymphoma, using sialic acids as covalent markers. Sialic acids were removed from the surface of living cells by exhaustive treatment with Vibrio cholerae sialidase at 4 degrees C and shown to be derived primarily from glycoproteins (93%), with only a small amount from glycolipids (7%). Cells were recultured at 37 degrees C over time and monitored for the resialylation of the cell surface using a sensitive high pressure liquid chromatography adaptation of the thiobarbituric acid assay for sialic acids. The return of sialic acid to the cell surface was found to be contingent upon de novo protein synthesis indicating that the bulk of plasma membrane sialoglycoconjugates do not recycle to an endogenous sialyltransferase-containing compartment for oligosaccharide reprocessing. Identical results were found for K562 cells, a human erythroleukemia cell line. The movement of specific glycoproteins was followed using the enzyme rat liver alpha 2-6Gal beta 1-4GlcNAc sialyltransferase together with CMP-[3H]NeuAc as an impermeant probe of the cell surface. Surface sialoglycoproteins were internalized slowly, a process unaffected by cycloheximide treatment. Only a few of these internalized glycoproteins were found to return to a trans-Golgi compartment followed by recycling to the cell surface. Taken together, these data indicate that the majority of replacement of sialic acids on the cell surface is due to de novo synthesis of glycoproteins and that only a small number of glycoproteins recycle through a trans-Golgi compartment.     

Metabolic labeling of sialic acids in tissue culture cell lines: methods to identify substituted and modified radioactive neuraminic acids

The parent sialic acid N-acetylneuraminic acid can be modified or substituted in various ways, giving rise to a family of more than 25 compounds. The definitive identification of these compounds has previously required isolation of nanomole amounts for mass spectrometry or NMR. We have explored the possibility of using the known metabolic precursors of the sialic acids, particularly N-acetyl-[6-3H]mannosamine, to label and identify various forms of sialic acids in tissue culture cells. Firstly, we defined several variables that affect the labeling of sialic acids with N-acetyl-[6-3H]mannosamine. Secondly, we have devised a simple screening method to identify cell lines that synthesize substituted or modified sialic acids. We next demonstrate that it is possible to definitively identify the natures of the various labeled sialic acids without the use of mass spectrometry, even though they are present only in tracer amounts. The methods used include paper chromatography, analytical de-O-acetylation, periodate release of the 9-3H as [3H]formaldehyde (which is subsequently converted to a specific 3H-labeled chromophore), acylneuraminate pyruvate lyase treatment with identification of [3H]acylmannosamines, gas-liquid chromatography with radioactive detection, and two new high-pressure liquid chromatography methods utilizing the amine-adsorption:ion suppression and ion-pair principles. The use of an internal N-acetyl-[4-14C]neuraminic acid standard in each of these methods assures precision and accuracy. The combined use of these methods now allows the identification of radioactive tracer amounts of the various types of sialic acids in well-defined populations of tissue culture cells; it may also allow the identification of hitherto unknown forms of sialic acids.     

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.     

Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions

Human leukaemic HL-60 cells are widely used for studying interactions involving adhesion molecules [e.g. P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1)] since their rolling behaviour has been shown to mimic the dynamics of leucocyte rolling in vitro. HL-60 cells are neutrophilic promyelocytes that can undergo granulocytic differentiation upon exposure to compounds such as DMSO (dimethylsulfoxide). Using a parallel plate flow chamber functionalized with recombinant P-selectin-Fc chimaera, undifferentiated and DMSO-induced (48, 72 and 96?h) HL-60 cells were assayed for rolling behaviour. We found that depending on P-selectin incubation concentration, undifferentiated cells incurred up to a 6-fold increase in rolling velocity while subjected to an approximately 10-fold increase in biologically relevant shear stress. HL-60 cells exposed to DMSO for up to 72?h incurred up to a 3-fold increase in rolling velocity over the same shear stress range. Significantly, cells exposed for up to 96?h incurred up to a 9-fold decrease in rolling velocity, compared with undifferentiated HL-60 cells. Although cell surface and nuclear morphological changes were evident upon exposure to DMSO, flow cytometric analysis revealed that PSGL-1 expression was unchanged, irrespective of treatment duration. The results suggest that DMSO-treated HL-60 cells may be problematic as a substitute for neutrophils for trafficking studies during advanced stages of the LAC (leucocyte adhesion cascade). We suggest that remodelling of the cell surface during differentiation may affect rolling behaviour and that DMSO-treated HL-60 cells would behave differently from the normal leucocytes during inflammatory response in vivo.     

Stimulation of sialyltransferase activity of melanoma cells by retinoic acid

Retinoic acid (RA) treatment of murine S91-C2 melanoma cells has been found to augment the activity of glycoprotein: sialyltransferase in a dose-dependent and time-dependent process. The enzymatic activity in cells treated with 10 microM RA reached a maximal level, 3-fold higher than in untreated cells, 72 h after initiation of treatment. In contrast, the addition of RA directly into the reaction mixture had no stimulatory effect on sialyltransferase. The endogenous glycoproteins to which sialic acid is transferred from cytidine monophosphate (CMP)-[14C] sialic acid by the action of sialyltransferase have been identified by fluorography after polyacrylamide gel electrophoresis. One of these acceptors, a glycoprotein of Mr 160 000, comigrated in gel electrophoresis with a cell surface sialoglycoprotein that can be labeled by the periodate-tritiated borohydrate procedure more intensely on intact RA-treated than on untreated cells. Removal of sialic acid residues exposed on the surface of either control or RA-treated cells enhanced 2- to 3-fold the transfer of sialic acid to endogenous acceptors. These results suggest that the increased sialyltransferase activity in RA-treated melanoma cells may be responsible for the enhanced sialylation of certain cell surface glycoproteins. RA treatment of several other tumor cell lines also resulted in stimulation of sialyltransferase activity indicating that this effect of RA is not limited to the S91-C2 melanoma cells.     

Effects of dimethyl sulphoxide on ATP content and protein synthesis inTetrahymena

Summary The content of ATP in cells exposed for 1 hour to 2.5% and 7.5% dimethyl sulphoxide (DMSO) was 90% and about 80%, respectively, of that in control cells. This difference of about 10% in the ATP content cannot explain the previously reported cessation of food vacuole formation in 7.5% DMSO and the uninhibited function in 2.5% DMSO (Nilsson 1974). However, DMSO had a dose-dependent effect on the rate of turnover of ATP in cell extracts, thus the amount of ATP expended per unit time in 7.5% DMSO is only 60% of that expended by extracts of control cells. The rate of protein synthesis was studied by electron microscope autoradiography which revealed considerably less labelled material in DMSO-treated cells than in control cells. Semi-quantitative estimates showed that cells in 2.5%, 5%, and 7.5% DMSO had a rate of incorporation of the labelled amino acid corresponding to 38%, 31%, and 51%, respectively, of that of control cells; the seemingly high rate of incorporation in 7.5% DMSO may reflect a low internal pool of amino acids in these cells. An additional fine structural detail is the induction of intranuclear fibrous bundles in all concentrations of DMSO. The findings are in accord with a random interference of DMSO, presumably by inducing conformational changes in some macromolecules which affect their cellular function.     

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.     

Alteration of the electrophoretic mobility of human peripheral blood mononuclear cells following treatment with dimethyl sulfoxide

Studies have been conducted to determine the effects of DMSO and freezing on the electrophoretic distribution of peripheral blood mononuclear cells. Sodium [51Cr]chromate was used to label the cells, and the distributions of cell number and cell-associated radioactivity were determined. Cells treated with DMSO had a narrower distribution of electrophoretic mobilities when compared with those not treated. DMSO-treated cells also demonstrated a more homogeneous distribution of radioactivity relative to the cell distribution than did the nontreated cells. The freezing of DMSO-treated cells did not result in any additional alteration of electrophoretic pattern compared to DMSO treatment alone. Analysis by linear categorization techniques indicated that the DMSO-treated and nontreated cells were completely distinguished by their electrophoretic behavior.     

Guinea pig erythrocytes, after their contact with influenza virus, acquire the ability to activate the human alternative complement pathway through virus-induced desialation of the cells

Guinea pig erythrocytes that had been exposed to influenza A virus activated the alternative complement pathway in whole human serum in the absence of natural antibodies. Because all virus particles were eluted from the treated cells, activation was not dependent on antiviral antibodies or on virus particles themselves. The relative capacity of treated erythrocytes to activate the alternative pathway was dependent on the amount of virus to which the cells had been exposed and was directly related to the amount of sialic acid removed from the erythrocyte membrane during incubation with either whole virus particles or purified viral sialidase. C3b bound to cells that had been treated with virus, and P-stabilized amplification convertase sites P,C3b,Bb formed on these cells, exhibited increased resistance to the action of the regulatory proteins beta-1H and C3b Ina compared with C3b and P,C3b,Bb on untreated, nonactivating cells. The acquired resistance of the cell-bound, P-stabilized amplification convertase to decay-dissociation by beta-1H was directly related to the activating capacity of the treated cells in whole serum (r = 0.95) and to the amount of sialic acid removed from the cells by the virus (r = 0.98). Desialation represents a specific alteration of the cell surface by which a nonimmune host, through activation of the alternative pathway, may deposit C3b on a target cell that had been exposed to influenza virus and may lyse virus virus-modified cells during orthomyxovirus infections.     

Effects of dimethyl sulfoxide (DMSO) on microfilament organization, cellular adhesion, and growth of cultured mouse B16 melanoma cells

Cell shape is involved in a variety of cellular activities including proliferation, adhesion, migration, and transformation. Agents known to promote differentiation, such as retinoic acid, butyrate, and dibutyryl cyclic AMP, induce marked alterations in cell shape which are often accompanied by changes in cell functions. In this paper we study the effects of the differentiating polar solvent dimethyl sulfoxide (DMSO) on cytoskeleton, adhesion, and growth properties of cultured mouse B16 melanoma cells. DMSO induced a progressive reorganization of the cytoskeleton which was fully developed in 4 days of continuous exposure to the agent. DMSO-treated cells developed thick and regularly oriented microfilament bundles of the stress fiber type ending at vinculin-rich areas of focal contact between the ventral membrane and the substratum (interference reflection microscopydark adhesion plaques). Such a rearrangement of the cytoskeleton resulted in increased adhesion to the substratum and inhibition of cell growth in comparison to control untreated cells. Cells which became highly flattened and tightly adherent after exposure to DMSO for 4 days progressively reverted their phenotype to that of control untreated cells within 3 days of DMSO withdrawal. Namely, they lost stress fibers and adhesion plaques, became rounded and less adherent, and increased their growth rate. These results indicate that DMSO can change the transformed appearance of B16 mouse melanoma cells to a phenotype which is typical of a variety of nontransformed cells in culture.