Ruthenium-Red-Induced Cell Agglutination and Surface Glycoprotein and Muco-Polysaccharide

1973 ◽  
Vol 13 (3) ◽  
pp. 901-911
Author(s):  
KOZO UTSUMI ◽  
TAKUZO ODA
Keyword(s):  
Sialic Acid ◽  
Heparan Sulphate ◽  
Ruthenium Red ◽  
The Other ◽  
Surface Glycoprotein ◽  
Ascites Hepatoma ◽  
Cell Agglutination ◽  
Acidic Polysaccharides ◽  
Binding Substance ◽  
Papain Digestion

Ruthenium red induces the agglutination of ascites hepatoma cells. The agglutination was inhibited by acidic polysaccharides but not by haptenic inhibitors of some plant agglutinins. The agglutination was also abolished by treatment of the cells with a low concentration of papain. Papain digestion causes a decrease in the number of microvilli on the cell surface and the release of carbohydrate-containing red-binding substance. One of them is a heparan sulphate-like mucopolysaccharide and the other is a glycoprotein which contains sialic acid. The ruthenium red-binding fractions inhibit the cell agglutination induced by ruthenium red.

Sendai virus maturation in actinomycin D pretreated and glutamine starved 37 RC and vero cells

1978 ◽  
Vol 36 (2) ◽  
pp. 372-373
Author(s):  
Wiktor Djaczenko ◽  
Arrigo Benedetto ◽  
Maria Stefania Zaniratti
Keyword(s):  
Actinomycin D ◽  
Sendai Virus ◽  
Vero Cells ◽  
Ruthenium Red ◽  
The Other ◽  
Surface Glycoprotein ◽  
Cell Replication ◽  
Virus Maturation ◽  
Glass Substrates ◽  
Metabolic Conditions

Actinomycin D (AMD) pretreatment (0. 5 μg/ml, 72h) causes surface glycoprotein accumulation in 37 RC and Vero cells (submitted for publication). On the other hand glutamine starvation in these cells does not inhibit the cell replication but reduces the cell adhesiveness to the plastic and glass substrates probably by depletion of glucosamine pool. In such metabolic conditions the modifications of plasma membrane flexibility could be expected. Since such a flexibility influences Sendai virus maturation we decided to study the replication of this virus in AMD pretreated and glutamine starved cells.Three groups of 37 RC and Vero cells grown in monolayers, one untreated control, one AMD pretreated and one glutamine (G) starved were infected with Sendai virus. At 3. 5h and 24 h of infection corresponding cultures were fixed by: (a) classical methods, (b) TAPO techniqe and (c) ruthenium red method.

Studies on the к-casein complex: II. The isolation of a sialic acid-containing fraction by disrupting the complex at low pH in the presence of sodium chloride

1965 ◽  
Vol 32 (1) ◽  
pp. 57-63 ◽  
Author(s):  
R. Beeby
Keyword(s):  
Sialic Acid ◽  
Sodium Chloride ◽  
Deae Cellulose ◽  
Low Ph ◽  
The Other ◽  
Trichloracetic Acid ◽  
Complex Ii ◽  
Total Sialic Acid

SummaryWhen crude к-casein was precipitated at pH 3 in the presence of 0·4m-NaCl the supernatant contained up to 80% of the total sialic acid but no detectable cystine or cysteine. Two fractions were obtained from this supernatant by chromatography on DEAE cellulose; one containing 4–6% sialic acid and the other only onetenth of this amount.Most of the sialic acid of the sialic acid-rich fraction was soluble in 12% trichloracetic acid following treatment with rennin. It is suggested that the glycopeptide released by the action of the enzyme on casein originates from this fraction.

scholarly journals Platelet and Immune Characteristics of Patients with Immune Thrombocytopaenia Non Responders to Therapeutic Treatments

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1089-1089
Author(s):  
Elena Monzón Manzano ◽  
Raul Justo Sanz ◽  
Diana Hernández ◽  
Teresa Álvarez Roman ◽  
Ihosvany Fernandez-Bello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Sialic Acid ◽  
Research Funding ◽  
Caspase 3 ◽  
Mononuclear Cells ◽  
Ricinus Communis ◽  
The Other ◽  
Control Group ◽  
Platelet Surface

Introduction: Mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) appear to be multifactorial: autoantibodies, autoreactive CD8+ cytotoxic T cells, enhanced apoptosis and loss of sialic acid which mediates platelet clearance through the Ashwell-Morell receptors present in hepatocytes. Differential involvement of each of them might condition the ability of patients with ITP to respond to treatments. We aimed to examine platelet features and the immunological state of patients with ITP who do not respond to any treatment to detect the unique characteristics of this group. Methods: This was an observational, prospective and transversal study. Patients with chronic primary ITP were included: 28 ITP patients without treatment for at least 6 months (UT-ITP); 36 responders to agonists of thrombopoietin receptors (TPO-RA); and 14 ITP patients who did not respond to first- and second-line treatments (NR-ITP). A healthy control group (n=104) was also included in the study. Active caspase-3, -7, -8 or -9 were determined by flow cytometry using CaspaTag kits (Millipore, Madrid, Spain) in PRP diluted with HEPES-buffer containing 2 mM Ca2+ and 2 mM Gly-Pro-Arg-Pro (Sigma-Aldrich, Madrid, Spain) to prevent fibrin formation . Platelet surface glycan exposure was analysed by determining the binding of lectins by flow cytometry. To do so, washed platelets were incubated with 1 μg/ml Alexa fluor 488-conjugated wheat germ agglutinin lectin (WGA, Invitrogen, Spain) or with 1 μg/ml FITC-conjugated Ricinus communis agglutinin (RCA, Vector Labs, UK). WGA binds to sialic acid and N-acetylglucosaminyl residues, and RCA is a galactose-specific legume lectin which binding serves as an indirect measurement of the loss of sialic acid. Peripheral blood mononuclear cells (PBMCs) subsets were analysed by flow cytometry using specific antibodies. Experimental data was analysed using SPSS 9.0 software (SPSS Inc., Chicago, IL). Results: Platelets from TPO-RA treated and from NR-ITP patients had increased caspase-3, -7, -8 and -9 activities (Figure 1A). Platelets from NR-ITP patients exposed less sialic acid and more N-acetylglucosaminyl residues than the other groups (Figure 1B). Binding of WGA and RCA correlated with caspase activities (Table 1). Distribution of lymphocytes, monocytes and natural killer cells is shown in Table 1. NR-ITP patients had an increased proportion of B lymphocyte (LB), maybe due to a significant rise in the fraction of naive LB cells, and a diminution in LTreg subset. Whereas classical monocytes was increased, nonclassical monocyte fraction was decreased in the UT-ITP and NR-ITP groups. NR-ITP patients also presented an increased CD16+CD56bright cells fraction and a diminished NK CD16+CD56dim subset. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls (monocyte and NK cells subset distribution and LTreg count similar to control group). It is of interest to note the relationship between loss of sialic acid from platelet surface glycans and Tregs count: the most reduced surface exposure of sialic acid, the less Treg count (Figure 2). Conclusions: Platelets from NR-ITP patients had more signs of apoptosis and a different composition of surface glycans, accompanied by a diminished LTreg population, a higher LB naïve percentage, and an increased CD16+CD56bright cells fraction in circulation, indicating a severe deregulation of the immune system. Since an inverse correlation was observed between loss of sialic acid and LTreg count, a potential relationship between glycan composition on the platelet surface and immune response is suggested, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP. On the other hand, TPO-RA appears to have a beneficial effect on immune response. Nevertheless, one of the limitations of our study was that patients were recruited once the response to TPO-RA was achieved; therefore, a longitudinal study would provide more information regarding TPO-RA effects. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Álvarez Roman: Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau. Fernandez-Bello:Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Martín:SOBI: Research Funding; Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. Canales:Novartis: Honoraria; Takeda: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Celgene: Honoraria; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Janssen: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

Two deoxyribonucleases from Novikoff ascites hepatoma cells

1968 ◽  
Vol 46 (9) ◽  
pp. 1121-1129 ◽  
Author(s):  
Peter M. K. Ip ◽  
Shan-Ching Sung
Keyword(s):  
Actinomycin D ◽  
Early Stage ◽  
Maximum Activity ◽  
The Other ◽  
Average Chain Length ◽  
Ascites Hepatoma ◽  
Dnase Digestion ◽  
Other Hand ◽  
Acid Dnase ◽  
Sulfhydryl Compounds

Two DNases have been isolated and separated from Novikoff ascites hepatoma by ammonium sulfate fractionation and have been further purified by chromatography on ion-exchange columns of DEAE types.The partially purified acid DNase is free of any measurable RNase activity, while the partially purified alkaline DNase preparation still exhibits RNase activity.The alkaline DNase requires sulfhydryl compounds for maximum activity, whereas the acid DNase does not. Both DNases require Mg2+ ions for maximum activity. EDTA strongly inhibits the alkaline DNase activity and the inhibition can be reversed by the addition of Mg2+ ions. On the other hand, EDTA activates the acid DNase either in the presence or in the absence of Mg2+.Sarkomycin inhibits the alkaline DNase but does not inhibit the acid DNase. Actinomycin D and heparin inhibit both DNase activities.The products of the alkaline DNase digestion consist of four deoxymononucleotides as well as higher oligonucleotides, all terminating in 5′-phosphate. The alkaline DNase seems to exhibit an endonucleolytic mode of attack in the early stage of hydrolysis with a subsequent exonucleolytic action. However, the possibility of contamination by an unknown exonuclease cannot be ruled out. On the other hand, the products of the acid DNase digestion consist mainly of oligonucleotides with average chain length larger than 8 units all terminating in 3′-phosphate. No mononucleotides can be detected. This suggests that the acid DNase is a typical endonuclease and possesses no detectable exonuclease activity.The acid DNase preferentially attacks linkages of the type dPupGp, whereas the preferential linkage(s) for the alkaline DNase has not been established.

Cell surface components and growth regulation in cultivated arterial smooth muscle cells

1983 ◽  
Vol 64 (1) ◽  
pp. 107-121
Author(s):  
J. Nilsson ◽  
T. Ksiazek ◽  
J. Thyberg ◽  
A. Wasteson
Keyword(s):  
Smooth Muscle ◽  
Sialic Acid ◽  
Cell Surface ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Growth Regulation ◽  
Heparan Sulphate ◽  
Carboxyl Groups ◽  
Anionic Sites ◽  
Arterial Smooth Muscle Cells

The surface of rat arterial smooth muscle cells was characterized with respect to some of its chemical and functional properties. The effects of selective enzymic degradations (hyaluronidase, chondroitinases, heparitinase or neuraminidase) on [35S]sulphate-prelabelled cells and on binding sites for cationized ferritin (CF) were examined to assess the presence and relative importance of individual species of macromolecules on the cell surface. The results indicate that about half of the strongly anionic sites on the cell surface (binding CF at pH 2.0) could be ascribed to sulphate groups of glycosaminoglycans and about half to carboxyl groups of sialic acid residues in glycoproteins and/or glycolipids. Weaker anionic sites (binding CF at pH 7.0) largely originated from carboxyl groups of glycosaminoglycans. Chondroitin sulphate and heparan sulphate were the main glycosaminoglycans. The surface of cells from young animals showed a higher glycosaminoglycan and a lower sialic acid content than that of cells from adult animals. Continuous treatment of the cultures with neuraminidase stimulated serum-induced initiation of DNA synthesis, while treatment with hyaluronidase or heparitinase inhibited it. Addition of hyaluronic acid, heparin or heparan sulphate to the culture medium inhibited initiation of DNA synthesis as well as cell proliferation. The effect was more marked in cultures of cells from young animals than from adults, although the latter cells were found to grow at a higher rate and to higher densities. These results suggest a role for cell-surface and pericellular glycoconjugates in growth regulation. A possible mechanism of action is that these molecules, due to their anionic charge or by steric exclusion, interfere with the binding of platelet-derived growth factor, a highly cationic polypeptide, to its cell-surface receptor.

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

1975 ◽  
Vol 19 (3) ◽  
pp. 621-644
Author(s):  
D.M. Dwyer
Keyword(s):  
Fine Structure ◽  
Cell Surface ◽  
Alcian Blue ◽  
Chondroitin Sulphate ◽  
Surface Membrane ◽  
Ruthenium Red ◽  
Surface Coat ◽  
Cell Agglutination ◽  
Trypanosoma Lewisi ◽  
Cationic Compounds

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.

Cell surface heparan sulphate and adhesive property of sublines of rat ascites hepatoma AH7974

1988 ◽  
Vol 90 (4) ◽  
pp. 683-689 ◽  
Author(s):  
A. Kimura ◽  
T. Kawaguchi ◽  
T. Ono ◽  
A. Sakuma ◽  
Y. Yokoya ◽  
...  
Keyword(s):  
Cell Surface ◽  
Culture Medium ◽  
Heparan Sulphate ◽  
Soluble Form ◽  
Foetal Calf Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Ascites Hepatoma ◽  
Serum Free ◽  
Rat Ascites Hepatoma

Two variants (74AD and 74FL) established from rat ascites hepatoma AH7974 were examined for the production of glycosaminoglycans in culture. There was no difference between the adhesive (74AD) and the floating (74FL) variants in quantity of glycosaminoglycans produced by their cultivation in minimum essential medium supplemented with 10% foetal calf serum. However, they were distinctly different in the distribution patterns of heparan sulphate. In 74FL, about 70% of total heparan sulphate was found in the culture medium in soluble form, whereas in 74AD, only 7% was found in the medium and the rest was in the cell-substratum complex. In a serum-free medium, 74AD cells grew without adhering to the substratum. After cultivation, more than 90% of total heparan sulphate was found in the cell-associated fractions and the rest in the substratum fractions. No heparan sulphate was detected in the culture medium. On the other hand, 74FL cells released heparan sulphate to the serum-free medium as much as to the serum-containing medium. The increase in amount of heparan sulphate in the culture medium of 74FL cells was supposed to be caused by failure of the cells to deposit heparan sulphate at the cell surface and not caused by increased production. Cell-substratum adhesion mechanisms involving cell surface heparan sulphate (heparan sulphate proteoglycan) and some serum intermediate(s) are discussed for 74AD cells.

scholarly journals Collagen binding by vaginal aggregative lactobacilli

2015 ◽  
Vol 46 (No. 4) ◽  
pp. 89-94
Author(s):  
I. Štyriak ◽  
V. Demečková ◽  
B. Žatkovič ◽  
V. Kmeť
Keyword(s):  
Hyaluronic Acid ◽  
Lactobacillus Acidophilus ◽  
Culture Medium ◽  
Heparan Sulphate ◽  
The Other ◽  
Collagen Binding ◽  
Vaginal Lactobacillus ◽  
Gene Coding ◽  
Vaginal Swabs

Ten autoaggregating vaginal Lactobacillus strains (five of these strains were selected among isolates from sows‘ vaginal swabs and the other five among isolates from cows‘ vaginal swabs) were investigated for their ability to bind type Icollagen (Cn-I). All 10 autoaggregating strains in the range of A<sub>570nm</sub> readings 0.118–1.806 bound to immobilised Cn-I (at concentration of 100 μg/ml) in wells of microtitre plates, however, Lactobacillus acidophilus SV31 was much more adherent than the rest of the tested strains. The influence of culture medium on Cn-I binding was confirmed only in 50% of the tested strains when agar-grown cells bound significantly more Cn-I than broth-grown cells. The specificity of the binding was confirmed since the Cn-I binding by lactobacilli was abolished after their preincubation with this protein. The effect of heparan sulphate and hyaluronic acid was tested on 5 vaginal strains displaying the best Cn-I binding in microtitre plates after their cultivationon MRS agar plates. Both selected inhibitors significantly (P &lt; 0.001 or P &lt; 0.01) reduced Cn-I binding by the majority of strains. The presence of the gene coding APF (aggregation-promoting factor) was detected in seven strains (all five sows‘ and two cows‘ Lactobacillus strains) by PCR.

scholarly journals Membrane glycopeptides from old and young human erythrocytes (Short Communication)

1974 ◽  
Vol 140 (3) ◽  
pp. 557-560 ◽  
Author(s):  
Cesare Balduini ◽  
Carlo Luigi Balduini ◽  
Edoardo Ascari
Keyword(s):  
Sialic Acid ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Short Communication ◽  
Chemical Characterization ◽  
Erythrocyte Membranes ◽  
Main Peak ◽  
Human Erythrocyte Membranes ◽  
Papain Digestion

Glycopeptides were extracted by papain digestion from old and young human erythrocyte membranes and fractionated on DEAE-Sephadex A-25. Chemical characterization of the unfractionated samples and of the main peak eluted from the column indicates that glycoproteins of the erythrocyte membrane undergo significant decreases in sialic acid and galactosamine content with aging.

scholarly journals Pathways of glycogen synthesis in Novikoff ascites-hepatoma cells

1969 ◽  
Vol 115 (2) ◽  
pp. 315-322 ◽  
Author(s):  
V. N. Nigam
Keyword(s):  
Intact Cell ◽  
Glycogen Synthesis ◽  
Hepatoma Cells ◽  
Tumour Cells ◽  
The Other ◽  
Ascites Hepatoma ◽  
Saturation Concentration ◽  
Glucan Phosphorylase ◽  
Glycogen Formation

Affinity of glucose, fructose and mannose for tumour hexokinase and their rates of phosphorylation at saturation concentration have been correlated with rates of glycogen synthesis by intact tumour cells at different concentrations of the three substrates. Competition experiments with one sugar labelled and the other sugar unlabelled indicate inhibition of glycogen synthesis by the sugar with a low Km for hexokinase. Glycogen synthesis from glucose 1-phosphate in aged cells and from nucleoside in freshly prepared cells is stimulated by fructose and inhibited by glucose. The decrease in glycogen formation from glucose 1-phosphate by oligomycin is partially overcome by increased fructose concentrations. These results are explained by an activation of α-glucan phosphorylase by fructose and an inhibition of this enzyme by glucose. It is suggested that differences in localization of glucose 6-phosphate, available to the intact cell in various ways, determine its transformation into glycogen by either the UDP-glucose–α-glucan glucosyltransferase reaction or by the α-glucan phosphorylase reaction.

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