Effect of the Removal of Cell Surface Sialic Acids on Cell Aggregation in vitro

Nature ◽  
1968 ◽  
Vol 218 (5148) ◽  
pp. 1255-1256 ◽  
Author(s):  
R. B. KEMP
Keyword(s):  
Cell Surface ◽  
Cell Aggregation ◽  
Sialic Acids

The Effect of Neuraminidase (3:2:1:18) on the Aggregation of Cells Dissociated from Embryonic Chick Muscle Tissue

1970 ◽  
Vol 6 (3) ◽  
pp. 751-766
Author(s):  
R. B. KEMP
Keyword(s):  
Cell Surface ◽  
Muscle Tissue ◽  
Muscle Cells ◽  
Cell Aggregation ◽  
Inhibitory Effect ◽  
Sialic Acids ◽  
Embryonic Chick ◽  
Experimental Conditions ◽  
Cell Counts

Embryonic chick muscle cells were used to investigate the effect of removing cell-surface sialic acids on cell aggregation in vitro. Single cell suspensions were prepared by dissociating skeletal muscle tissue of 9-day-old chick embryos with either crystalline or crude trypsin. Cell aggregation was quantitatively estimated by turbidimetric and gyratory shaker methods. Cells dissociated with crude trypsin and suspended in Hanks's balanced salts solution (BSS) containing 25u./ml neuraminidase (NANase) only aggregated for 2h when rotated in an absorptiometer. The inhibitory effect of the enzyme was more pronounced with increasing concentration up to 25u./ml. Cells dissociated with crystalline trypsin and treated with 100u./ml NANase immediately exhibited a reduced aggregative competence when gyrated in Eagle's minimum essential medium (MEM) containing 25u./ml NANase, compared with the controls which were not exposed to NANase. The aggregation rate of muscle cells pretreated with 100u./ml NANase and suspended in Eagle's MEM was similar to that of the untreated controls. Cell counts showed that under all three experimental conditions cells were not added to aggregates after the 12-h stage. Aggregates formed in Eagle's MEM (the controls) joined together to form larger aggregates after 12 h, but those rotating in the presence of NANase did not display this property. Lissamine green viability tests showed that cells remained alive throughout the 24-h period in the presence of NANase. Determinations of oxygen uptake, protein synthesis and mitotic index confirmed that general cellular viability was not affected by NANase. Fluorescent-labelled NANase was not taken up by the cells. Treatment of crystalline trypsin-dissociated muscle cells with 100u./ml NANase for 30 min at 37°C significantly reduced their negative electrophoretic mobility. This diminution closely corresponded to the removal of cell-surface sialic acids, as measured by colorimetric tests. Interpretation of the results in the light of current theories of cell adhesion failed to give support to the concept of adhesion by physical forces. The mechanism by which cellular deformability could influence cellular adhesiveness is modified in the knowledge of the present results.

scholarly journals A STUDY ON THE MECHANISM OF INTERCELLULAR ADHESION

1974 ◽  
Vol 60 (3) ◽  
pp. 641-652 ◽  
Author(s):  
Joris J. Deman ◽  
Erik A. Bruyneel ◽  
Marc M. Mareel
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Calcium Ions ◽  
Hela Cells ◽  
Previous Treatment ◽  
Cell Aggregation ◽  
Sialic Acids ◽  
Magnesium Ions ◽  
Trypsin Treatment ◽  
Calcium Effect

Aggregation of suspended HeLa cells is increased on removal of cell surface sialic acid. Calcium ions promote aggregation whereas magnesium ions have no effect. The calcium effect is abolished by previous treatment of the cells with neuraminidase. Trypsinization of the HeLa cells followed by thorough washing diminishes the rate of mutual cell aggregation. Subsequent incubation with neuraminidase restores the aggregation rate to the original value before trypsin treatment. Cells which had acquired a greater tendency for aggregation after removal of peripheral sialic acid lose this property when subsequently treated with trypsin. Calcium ions have no aggregative effect on trypsinized cells. In contrast to HeLa cells, aggregation of human erythrocytes was not increased after treatment with neuraminidase or on addition of calcium. The results with HeLa cells are interpreted as follows: (a) Trypsin-releasable material confers adhesiveness upon the cells. (b) The adhesive property of this material is counteracted by the presence of cell surface sialic acids. (c) Calcium ions exert their effect by attenuating the adverse effect of sialic acid.

scholarly journals Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri

Microbiology ◽  
2010 ◽  
Vol 156 (11) ◽  
pp. 3368-3378 ◽  
Author(s):  
Donald A. MacKenzie ◽  
Faye Jeffers ◽  
Mary L. Parker ◽  
Amandine Vibert-Vallet ◽  
Roy J. Bongaerts ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Binding Proteins ◽  
Lactobacillus Reuteri ◽  
Surface Protein ◽  
Cell Aggregation ◽  
Spontaneous Mutant ◽  
Vertebrate Hosts

Mucus-binding proteins (MUBs) have been revealed as one of the effector molecules involved in mechanisms of the adherence of lactobacilli to the host; mub, or mub-like, genes are found in all of the six genomes of Lactobacillus reuteri that are available. We recently reported the crystal structure of a Mub repeat from L. reuteri ATCC 53608 (also designated strain 1063), revealing an unexpected recognition of immunoglobulins. In the current study, we explored the diversity of the ATCC 53608 mub gene, and MUB expression levels in a large collection of L. reuteri strains isolated from a range of vertebrate hosts. This analysis revealed that the MUB was only detectable on the cell surface of two highly related isolates when using antibodies that were raised against the protein. There was considerable variation in quantitative mucus adhesion in vitro among L. reuteri strains, and mucus binding showed excellent correlation with the presence of cell-surface ATCC 53608 MUB. ATCC 53608 MUB presence was further highly associated with the autoaggregation of L. reuteri strains in washed cell suspensions, suggesting a novel role of this surface protein in cell aggregation. We also characterized MUB expression in representative L. reuteri strains. This analysis revealed that one derivative of strain 1063 was a spontaneous mutant that expressed a C-terminally truncated version of MUB. This frameshift mutation was caused by the insertion of a duplicated 13 nt sequence at position 4867 nt in the mub gene, producing a truncated MUB also lacking the C-terminal LPxTG region, and thus unable to anchor to the cell wall. This mutant, designated 1063N (mub-4867i), displayed low mucus-binding and aggregation capacities, further providing evidence for the contribution of cell-wall-anchored MUB to such phenotypes. In conclusion, this study provided novel information on the functional attributes of MUB in L. reuteri, and further demonstrated that MUB and MUB-like proteins, although present in many L. reuteri isolates, show a high genetic heterogeneity among strains.

Cell surface carbohydrates modulate neutrophil adherence to alveolar type II cells in vitro

1993 ◽  
Vol 264 (4) ◽  
pp. L391-L400 ◽  
Author(s):  
B. Crestani ◽  
C. Rolland ◽  
A. Petiet ◽  
N. Colas-Linhart ◽  
M. Aubier
Keyword(s):  
Cell Surface ◽  
A549 Cells ◽  
Sialic Acids ◽  
Alveolar Type ◽  
Type Ii ◽  
Adhesive Interaction ◽  
Human Polymorphonuclear Leukocyte ◽  
Type Ii Cell ◽  
Surface Carbohydrates

We characterized the influence of phosphorylated sugars and cell surface sialic acids on the adherence of human polymorphonuclear leukocyte (PMN) to rat alveolar type II cell (ATII cells) and human-derived A549 cell monolayers in vitro. Percent adherence of radiolabeled polymorphonuclear leukocytes was assessed after incubating cells with the carbohydrates, enzymes, or lectins to be tested. Lactose-1-phosphate (Lact1P) and maltose-1-phosphate (Malt1P) (10 mM) inhibited adherence of PMN to ATII cells and A549 cells. Maximal inhibition followed treatment of both PMN and rat ATII cells and amounted to 85 +/- 7% with Lact1P and 92 +/- 3% with Malt1P. Inhibition was concentration dependent. Incubation of PMN with mannose-6-phosphate reduced adherence to rat ATII cells and A549 cells by 36 +/- 11 and 39 +/- 8%, respectively. Maximal concentrations of sugars did not alter cellular viability. Neuraminidase-induced desialilation of ATII cells increased adherence of PMN by 36 +/- 7% to rat ATII cells and by 86 +/- 18% to A549 cells. Masking of terminal sialic acids on rat ATH cells with Limulus polyphemus agglutinin (100 micrograms/ml) increased adherence by 50 +/- 2%. These results indicate that cell surface carbohydrates are involved in the regulation of the adhesive interaction between PMN and ATII cells in vitro.

Aggregation of mononucleated precursors triggers cell surface expression of alphavbeta3 integrin, essential to formation of osteoclast-like multinucleated cells

1998 ◽  
Vol 111 (17) ◽  
pp. 2563-2574 ◽  
Author(s):  
P. Boissy ◽  
I. Machuca ◽  
M. Pfaff ◽  
D. Ficheux ◽  
P. Jurdic
Keyword(s):  
Cell Surface ◽  
De Novo ◽  
Osteoclast Differentiation ◽  
Cell Aggregation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Dihydroxyvitamin D3 ◽  
Osteoclast Precursors ◽  
Integrin Alphavbeta3

Alphavbeta3 is a key integrin mediating adhesion of multinucleated osteoclasts during bone resorption. 1, 25-dihydroxyvitamin D3 upregulates alphavbeta3 integrin expression in mononucleated osteoclast precursors and concomitantly stimulates their differentiation into osteoclasts. This suggests that this integrin could play a major role during osteoclast differentiation.We have developed an in vitro model, in which 1, 25-dihydroxyvitamin D3 sequentially modifies the behavior of macrophages: It first induces rounding up of these cells, then their subsequent aggregation and spreading, which finally leads to cell fusion and the formation of osteoclast-like multinucleated giant cells. We show that, while 1,25-dihydroxyvitamin D3 stimulates the de novo synthesis of alphavbeta3 in macrophages early in this process, its accumulation on the surface is triggered by cell aggregation. A high level of integrin alphavbeta3 cell surface expression correlates with macrophage spreading preceding fusion. This was confirmed by means of novel cell permeable peptides containing the C-terminal sequence of the integrin beta3 tail to specifically block (alphavbeta3 function. Although this peptide has no effect on the aggregation step, it disrupts the spreading of osteoclast precursors and consequently inhibits their fusion. These findings suggest a novel role of the integrin alphavbeta3 in a discrete step of osteoclast differentiation.

In Vitro Sialylation of Recombinant Alpha-1-Antitrypsin using α2,6 Sialyltransferase from Photobacterium Damselae Produces Di-sialyl Galactose Motifs in N-Glycans

2019 ◽  
Author(s):  
Edward Pallister ◽  
Matthew Choo ◽  
jien nee tai ◽  
Dawn leong ◽  
Wen-Qin Tang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Surface ◽  
Detailed Analysis ◽  
Sialic Acids ◽  
Complex Mixtures ◽  
Photobacterium Damselae ◽  
Alpha 1 Antitrypsin

Sialic acids are cell surface sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where lack of sialylation can reduce bioactivity. Here we describe how α-2,6-sialyltransferase from <i>Photobacterium Damselae</i> can be used to markedly increase sialylation of CHO produced alpha-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second di-sialyl galactose motif was produced, which had never been recorded on a mammalian glycoprotein. The influence of this unique di-sialylation on the <i>in vitro</i> activity of alpha-1-antitrypsin was studied and a toolkit of mass spectrometry methods to identify this new di-sialyl galactose motif in complex mixtures was developed

In Vitro Sialylation of Recombinant Alpha-1-Antitrypsin using α2,6 Sialyltransferase from Photobacterium Damselae Produces Di-sialyl Galactose Motifs in N-Glycans

2019 ◽  
Author(s):  
Edward Pallister ◽  
Matthew Choo ◽  
jien nee tai ◽  
Dawn leong ◽  
Wen-Qin Tang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Surface ◽  
Detailed Analysis ◽  
Sialic Acids ◽  
Complex Mixtures ◽  
Photobacterium Damselae ◽  
Alpha 1 Antitrypsin

Sialic acids are cell surface sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where lack of sialylation can reduce bioactivity. Here we describe how α-2,6-sialyltransferase from <i>Photobacterium Damselae</i> can be used to markedly increase sialylation of CHO produced alpha-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second di-sialyl galactose motif was produced, which had never been recorded on a mammalian glycoprotein. The influence of this unique di-sialylation on the <i>in vitro</i> activity of alpha-1-antitrypsin was studied and a toolkit of mass spectrometry methods to identify this new di-sialyl galactose motif in complex mixtures was developed

The Mesothelial Cell as a Non-Thrombogenic Surface

1984 ◽  
Vol 52 (02) ◽  
pp. 102-104 ◽  
Author(s):  
L J Nicholson ◽  
J M F Clarke ◽  
R M Pittilo ◽  
S J Machin ◽  
N Woolf
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Blood Flow ◽  
Cell Surface ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Plastic Film ◽  
In Vitro System ◽  
Scanning Electron

SummaryA technique for harvesting mesothelial cells is described. This entails collagenase digestion of omentum after which the cells can be cultured. The technique has been developed using the rat, but has also been successfully applied to human tissue. Cultured rat mesothelial cells obtained in this way have been examined by scanning electron microscopy. Rat mesothelial cells grown on plastic film have been exposed to blood in an in vitro system using a Baumgartner chamber and have been demonstrated to support blood flow. No adhering platelets were observed on the mesothelial cell surface. Fibroblasts similarily exposed to blood as a control were washed off the plastic.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

scholarly journals Cell Surface Expression of Nrg1 Protein in Candida auris

2021 ◽  
Vol 7 (4) ◽  
pp. 262
Author(s):  
Anuja Paudyal ◽  
Govindsamy Vediyappan
Keyword(s):  
Cell Surface ◽  
Growth Conditions ◽  
Zinc Ion ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Unexpected Finding ◽  
Candida Auris ◽  
Cell Surface Proteins ◽  
Biofilm Growth

Candida auris is an emerging antifungal resistant human fungal pathogen increasingly reported in healthcare facilities. It persists in hospital environments, and on skin surfaces, and can form biofilms readily. Here, we investigated the cell surface proteins from C. auris biofilms grown in a synthetic sweat medium mimicking human skin conditions. Cell surface proteins from both biofilm and planktonic control cells were extracted with a buffer containing β-mercaptoethanol and resolved by 2-D gel electrophoresis. Some of the differentially expressed proteins were excised and identified by mass spectrometry. C. albicans orthologs Spe3p, Tdh3p, Sod2p, Ywp1p, and Mdh1p were overexpressed in biofilm cells when compared to the planktonic cells of C. auris. Interestingly, several proteins with zinc ion binding activity were detected. Nrg1p is a zinc-binding transcription factor that negatively regulates hyphal growth in C. albicans. C. auris does not produce true hypha under standard in vitro growth conditions, and the role of Nrg1p in C. auris is currently unknown. Western blot analyses of cell surface and cytosolic proteins of C. auris against anti-CalNrg1 antibody revealed the Nrg1p in both locations. Cell surface localization of Nrg1p in C. auris, an unexpected finding, was further confirmed by immunofluorescence microscopy. Nrg1p expression is uniform across all four clades of C. auris and is dependent on growth conditions. Taken together, the data indicate that C. auris produces several unique proteins during its biofilm growth, which may assist in the skin-colonizing lifestyle of the fungus during its pathogenesis.

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