Glycosaminoglycan expression in intestinal epithelial skin-fibroblastic cell cocultures. Fibroblastic cell-mediated effects of glucocorticoids

1989 ◽  
Vol 92 (4) ◽  
pp. 679-685
Author(s):  
F. Bouziges ◽  
P. Simon-Assmann ◽  
C. Leberquier ◽  
K. Haffen ◽  
M. Kedinger
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Cell Cultures ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Cell Types ◽  
Molecular Forms ◽  
Fibroblastic Cell ◽  
Endodermal Cells ◽  
Molecular Weight Form

The nature and distribution of newly synthesized glycosaminoglycans (GAGs) were studied in foetal rat skin fibroblasts, in rat intestinal endodermal cells and in cocultures of both cell types. The data show that fibroblasts synthesize and secrete hyaluronic acid (HA), heparan sulphate (HS) and chondroitin sulphate molecules (CS). Our data focus on HA, which is found as two different molecular forms, the smallest hydrodynamic-sized species being mostly recovered within the cell or associated with the cell surface, and the largest one secreted into the medium, whatever the cell type. Endodermal cells synthesize only two types of GAGs: the low molecular weight form of HA and HS. Cocultures of rat intestinal endodermal and skin fibroblastic cells in the presence of dexamethasone (Dx), allow optimal epithelial cytodifferentiation (Kedinger et al. 1987a). The main changes in the GAGs synthesized under these conditions as compared to skin fibroblastic cell cultures concern: (1) the enhancement of the lowest molecular weight form of HA to the detriment of the highest form in the cellular, pericellular and extracellular compartments; (2) the increase in the proportion of HS molecules associated with the cell surface. Interestingly, similar modifications are obtained by addition of Dx to the skin fibroblastic cell cultures. The data are discussed with reference to the constitution of a basement membrane at the epithelial-fibroblast interface in the cocultures, to the fibroblastic-dependent induction of epithelial differentiation and to the glucocorticoid response.

Widespread use of Lactobacillus OppA, a surface located protein, as an adhesin that recognises epithelial cell surface glycosaminoglycans

2019 ◽  
Vol 10 (4) ◽  
pp. 463-472 ◽  
Author(s):  
C. Martín ◽  
S. Escobedo ◽  
J.E. Suárez ◽  
L.M. Quirós
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Cell Cultures ◽  
Lactobacillus Reuteri ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Surface Protein ◽  
Epithelial Cell Surface ◽  
Surface Proteome ◽  
Reduced Adherence

Specific adherence is the first requisite that a microorganism has to fulfil to become established onto a mucosal surface. It was previously shown that the OppA surface protein of Lactobacillus salivarius Lv72 bound HeLa cell cultures through interaction with glycosaminoglycans (GAGs). To determine whether this is a peculiarity of that strain or whether it can be extended to other lactobacilli, 12 strains, belonging to six species, were confronted with HeLa-cell cultures in the presence of soluble GAGs. Interference was observed to six of them, heparan sulphate and chondroitin sulphate C being more interfering than chondroitin sulphate A or chondroitin sulphate B. Furthermore, inhibition of the biosynthesis of GAGs or their elimination from the cell surface with specific enzymes also resulted in reduced adherence. Analysis of the surface proteome of Lactobacillus crispatus Lv25 and of Lactobacillus reuteri RC14 revealed single proteins that immunoreacted with antibodies raised against OppA, the main adhesin of L. salivarius Lv72. Upon MALDI-TOF-TOF analysis, they were identified as OppA-like proteins, thus indicating that these proteins participate as adhesins in attachment of diverse lactobacilli to the surface of human epithelial cells.

Two alkaline motifs in the Lactobacillus salivarius Lv72 OppA surface are important to its adhesin function

2019 ◽  
Vol 10 (1) ◽  
pp. 101-109 ◽  
Author(s):  
C. Martín ◽  
S. Escobedo ◽  
G. Pérez-Martínez ◽  
J.M. Coll-Marqués ◽  
R. Martín ◽  
...  
Keyword(s):  
Cell Surface ◽  
Hela Cell ◽  
Cell Cultures ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Eukaryotic Cell ◽  
Cloning And Expression ◽  
Lactobacillus Salivarius ◽  
Normal Microbiota

Glycosaminoglycans are involved in the attachment of Lactobacillus salivarius Lv72, a strain of vaginal origin, to HeLa cell cultures, indicating that they play a fundamental role in the attachment of mutualistic bacteria to the epithelium lining cavities where the normal microbiota thrives. The bacterial OppA protein has been proposed as an adhesin involved in this adherence since, once purified, it significantly interferes with attachment of the lactobacilli to HeLa cell cultures. In this article, the role of OppA is confirmed through the determination of its location at the cell surface and its ability to promote Lactobacillus casei and Lactococcus lactis adherence to eukaryotic cell cultures upon cloning and expression of oppA in these bacteria. The OppA sequence showed five potential domains for glycosaminoglycan-binding, and structural modelling of the protein showed that two of them were located in the vicinity of an OppA superficial groove whose width approached the diameter of the helical form of heparin in solution. Their involvement in the binding was demonstrated through substitution of critical basic amino acids by acidic ones, which resulted in loss of affinity for heparan sulphate and chondroitin sulphate depending on the domain mutated, suggesting that there might be a certain degree of specialisation. In addition, circular dichroism analysis showed that the spectrum changes induced by OppA-heparan sulphate binding were attenuated by the variant proteins, indicating that these motifs are the OppA recognition domains for the eukaryotic cell surface.

scholarly journals Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1

1987 ◽  
Vol 245 (2) ◽  
pp. 543-550 ◽  
Author(s):  
D H Farrell ◽  
D D Cunningham
Keyword(s):  
Complex Formation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Order Rate Constant ◽  
Subsequent Treatment ◽  
Protease Nexin ◽  
Inhibition Of Thrombin

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

Polylactosaminoglycan modification of a small integral membrane glycoprotein, influenza B virus NB

1988 ◽  
Vol 8 (3) ◽  
pp. 1186-1196
Author(s):  
M A Williams ◽  
R A Lamb
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Gel Filtration ◽  
Cell Types ◽  
Influenza B Virus ◽  
Influenza B ◽  
Membrane Glycoprotein ◽  
Intact Cells ◽  
B Virus ◽  
Carbohydrate Chains

The structure of the carbohydrate components of NB, the small integral membrane glycoprotein of influenza B virus, was investigated. The carbohydrate chains of NB are processed from the high-mannose form (NB18) to a heterogeneous form of much higher molecular weight, designated NBp. Selection of this carbohydrate-containing form of NB with Datura stramonium lectin, its susceptibility to digestion by endo-beta-galactosidase, and determination of the size of NBp glycopeptides by gel filtration chromatography suggested that the increase in molecular weight is due to processing to polylactosaminoglycan. Investigation of the polypeptides produced by influenza B/Lee/40 virus infection of several cell types and another strain of influenza B virus suggested that the signal for modification to polylactosaminoglycan is contained in NB. Expression of mutants of NB lacking either one or both of the normal N-terminal sites of asparagine-linked glycosylation indicated that both carbohydrate chains are modified to contain polylactosaminoglycan. NBp and a small amount of unprocessed NB18 are expressed at the infected-cell surface, as determined by digestion of the surfaces of intact cells with various endoglycosidases. Unglycosylated NB, expressed either in influenza B virus-infected cells treated with tunicamycin or in cells expressing the NB mutant lacking both N-linked glycosylation sites, was expressed at the cell surface, indicating that NB does not require carbohydrate addition for transport.

332. GYCOMIC ANALYSES OF THE GRANULOSA AND THECA OF BOVINE OVARIAN FOLLICLES

2010 ◽  
Vol 22 (9) ◽  
pp. 132
Author(s):  
N. Hatzirodos ◽  
J. Nigro ◽  
A. V. Vashi ◽  
H. F. Irving-Rodgers ◽  
B. Caterson ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Cell Function ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Cell Types ◽  
Ovarian Follicles ◽  
Cell Type ◽  
Thecal Cells ◽  
Theca Externa

Development of ovarian follicles involves changes in cell function and remodelling of the follicular wall. Remodelling necessitates changes in the extracellular matrix, including proteoglycans (PGs). PGs contain glycosaminoglycans (GAGs) covalently bound to a protein core. The length of GAG chains in PGs and the degree and pattern of sulphation differs between cell types and change as cells alter their phenotype. PGs that have been identified in follicles include the chondroitin sulphate (CS) PG, versican and inter-a trypsin inhibitor, and the heparan sulphate (HS) PGs, perlecan and type XVIII collagen. The latter two are found in focimatrix, the follicular and the thecal subendothelial basal laminas. To examine GAGs composition in follicles, bovine antral follicles of various sizes were collected. Follicles were dissected and a biopsy taken for histological classification of health. Theca layers and granulosa cells were collected separately and analysed by fluorophore-assisted carbohydrate (FACE) analysis of GAGs following digestion to disaccharides with chondroitinase ABC, hyaluronidase, heparinase, and heparitinases I and II. Four non GAG sugars and 12 different GAG derived disaccharides were identified and quantitated on a per DNA basis. Healthy versus atretic follicles for each cell type were compared and correlation analyses were also undertaken. Immunohistochemistry using CS specific antibodies was also conducted. There was no effect of size on the GAG content for either granulosa or theca cells. The 4- and 6- sulphated CS sugars were the most abundant following digestion in all tissues. Theca had higher levels than granulosa cells of HS derived disaccharides and also of un- or 4- or 6- N-sulphated CS derived disaccharides. Some sulphated CS moieties localised uniquely to the stroma surrounding blood vessels in the theca externa. Atretic follicles had lower amounts of disaccharides derived from HS in both granulosa and thecal cells, suggesting that heparanase may be activated upon atresia.

The production and effect of interferon on influenza virus growth in chick embryo lung epithelial and fibroblast cell cultures

1969 ◽  
Vol 15 (3) ◽  
pp. 273-277 ◽  
Author(s):  
Sunidhkumar S. Gandhi ◽  
Robert B. Stewart
Keyword(s):  
Epithelial Cells ◽  
Chick Embryo ◽  
Cell Cultures ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Fibroblastic Cell ◽  
Virus Growth ◽  
Lung Epithelial ◽  
Fibroblastic Cells ◽  
Virus Strains

Cultures of fibroblastic cells prepared from chick embryo lung infected with low multiplicities of influenza type A virus strains were found to produce more interferon than did cultures of epithelial cells prepared from the same organ. Fibroblastic cell cultures were also found to be more sensitive to the action of interferon than were epithelial cells with respect to the levels of infectious virus produced and the duration of interferon action. Cultures of the two cell types treated with interferon did not differ with respect to the number of cells involved in virus synthesis.

scholarly journals Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs

1984 ◽  
Vol 217 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Y Ohhashi ◽  
F Hasumi ◽  
Y Mori
Keyword(s):  
Cell Surface ◽  
Guinea Pigs ◽  
Lysosomal Enzymes ◽  
Culture Medium ◽  
Chondroitin Sulphate ◽  
Alkali Treatment ◽  
Heparan Sulphate ◽  
Polymorphonuclear Leucocytes ◽  
Dermatan Sulphate ◽  
Cell Cell

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.

scholarly journals Proteoglycans synthesized by an osteoblast-like cell line (UMR 106-01)

1991 ◽  
Vol 277 (1) ◽  
pp. 199-206 ◽  
Author(s):  
D J McQuillan ◽  
D M Findlay ◽  
A M Hocking ◽  
M Yanagishita ◽  
R J Midura ◽  
...  
Keyword(s):  
Cell Line ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Cell Types ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Umr 106

The proteoglycans synthesized by an osteoblast-like cell line of rat origin (UMR 106-01) were defined after biosynthetic labelling with [35S]sulphate and [3H]glucosamine. Newly synthesized labelled proteoglycans were characterized by differential enzymic digestion in combination with analytical gel filtration and SDS/PAGE. UMR 106-01 cells were found to synthesize three major species of proteoglycan: a large chondroitin sulphate proteoglycan of Mr approximately 1 x 10(6), with a core protein of Mr approximately 350,000-400,000; a small chondroitin sulphate-containing species of Mr approximately 120,000 with a core protein of Mr 43,000; and a heparan sulphate proteoglycan of Mr approximately 150,000, with a core protein of Mr approximately 80,000. Over 70% of the newly synthesized intact proteoglycan species are associated with the cell layer of near-confluent cells; however, accessibility to trypsin digestion suggests an extracellular location. Chemical characteristics of the proteoglycans and preliminary mRNA hybridization indicate that the small chondroitin sulphate proteoglycan is probably PG II (decorin). The large chondroitin sulphate proteoglycan is most likely related to a hyaluronate-aggregating species from fibroblasts (versican), and the heparan sulphate proteoglycan bears striking similarities to cell-membrane-intercalated species described for a number of cell types.

scholarly journals Schwann cell proliferation in vitro is under negative autocrine control.

1990 ◽  
Vol 111 (6) ◽  
pp. 2663-2671 ◽  
Author(s):  
D Muir ◽  
S Varon ◽  
M Manthorpe
Keyword(s):  
Molecular Weight ◽  
Schwann Cell ◽  
Molecular Mass ◽  
Conditioned Medium ◽  
Schwann Cells ◽  
Cell Cultures ◽  
Antiproliferative Activity ◽  
Gel Filtration ◽  
Molecular Weight Form

In healthy adult peripheral nerve, Schwann cells are believed to be generally quiescent. Similarly, cultures of isolated rat sciatic nerve Schwann cells hardly proliferate in serum-supplemented medium. The possibility that Schwann cells negatively regulate their own proliferation was supported by the demonstration that conditioned media from Schwann cell cultures inhibited the proliferation of mitogen-stimulated test cultures. The inhibition could be complete, was dose dependent, and was exhibited when the test Schwann cells were under the influence of different types of mitogens such as cholera toxin, laminin, and living neurons. The inhibition of proliferation was completely reversible and a rapid doubling of cell number resulted when treatment with conditioned medium was withdrawn from mitogen-stimulated Schwann cells. Conditioned medium from cholera toxin-stimulated and immortalized Schwann cell cultures contained less antiproliferative activity than that found in medium from quiescent Schwann cell cultures. However, media conditioned by two actively proliferating rat Schwannoma cell lines were rich sources of antiproliferative activity for Schwann cells. Unlike the mitogen-stimulated Schwann cells, whose proliferation could be inhibited completely, the immortalized and transformed Schwann cell types were nearly unresponsive to the antiproliferative activity. The antiproliferative activity in Schwann and Schwannoma cell conditioned media was submitted to gel filtration and SDS-PAGE. The activity exists in at least two distinct forms: (a) a high molecular weight complex with an apparent molecular mass greater than 1,000 kD, and (b) a lower molecular weight form having a molecular mass of 55 kD. The active 55-kD form could be derived from the high molecular weight form by gel filtration performed under dissociating conditions. The 55-kD form was further purified to electrophoretic homogeneity. These results suggest that Schwann cells produce an autocrine factor, which we designate as a "neural antiproliferative protein," which completely inhibits the in vitro proliferation of Schwann cells but not that of immortalized Schwann cells or Schwannoma lines.

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