scholarly journals Altered glycosaminoglycan production in cultured osteogenesis-imperfecta skin fibroblasts

1983 ◽  
Vol 213 (1) ◽  
pp. 171-178 ◽  
Author(s):  
H Turakainen
Keyword(s):  
Molecular Weight ◽  
Hyaluronic Acid ◽  
Osteogenesis Imperfecta ◽  
Collagen Synthesis ◽  
Gel Filtration ◽  
Exponential Phase ◽  
Skin Fibroblasts ◽  
Collagen Secretion ◽  
Sulphated Glycosaminoglycans ◽  
Sulphated Glycosaminoglycan

Collagen and glycosaminoglycan syntheses were studied in skin fibroblasts cultured from patients with osteogenesis imperfecta (OI) and from age-matched controls. Collagen synthesis (measured as protein-bound [3H]hydroxyproline) was decreased in all four OI cell lines studied in the present experiments, comprising 16-24% of total protein synthesis (40% in normal cells). Hyaluronic acid production in OI skin fibroblasts per cell was higher than in age-matched controls, but the production of sulphated glycosaminoglycans was at the normal level. Thus the ratio of the hyaluronic acid and sulphated-glycosaminoglycan radioactivities was markedly higher in OI cultures than in control cultures, especially at the exponential phase of growth where the synthesis of hyaluronic acid was highest. Hyaluronic acid in OI had a normal molecular weight when determined by gel filtration on Sepharose 2B. The removal of high-molecular-weight hyaluronic acid from the medium by hyaluronidase had no effect on the rate of collagen secretion in OI cell line 1 (A.T.C.C. 1262), in which the rate of collagen secretion was lowest.

scholarly journals Biochemical studies on the sulphated glycosaminoglycan fraction of skin fibroblasts cultured from a patient with the Hurler syndrome

1973 ◽  
Vol 132 (3) ◽  
pp. 395-402 ◽  
Author(s):  
Ralph J. Germinario ◽  
Arthur Kahlenberg ◽  
Leonard Pinsky
Keyword(s):  
Specific Radioactivity ◽  
Cell Types ◽  
Skin Fibroblasts ◽  
Hurler Syndrome ◽  
Normal Cells ◽  
Relative Retention ◽  
Cultured Skin ◽  
Corrective Factor ◽  
Sulphated Glycosaminoglycans ◽  
Sulphated Glycosaminoglycan

1. The metabolism of the sulphated glycosaminoglycan fraction in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was studied. Two labelled precursors, Na235SO4 and d-[2-3H]glucose, were used and their intracellular fates during uptake and `chase' periods were assessed after separation of sulphated glycosaminoglycans from hyaluronic acid. After 4 or 8h of exposure to culture medium containing both labels, [35S]sulphate incorporation into the sulphated glycosaminoglycan fraction was twofold greater in Hurler-syndrome cells than in normal cells. At the same time, the rate of incorporation of [3H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased 35S/3H ratio (nmol of [35S]sulphate incorporated/nmol of [3H]glucose incorporated) was observed for Hurler-syndrome cells compared with normal cells. 2. The results of `chase' experiments revealed that although the expected loss and relative retention of labelled sulphate occurred in the sulphated glycosaminoglycan fraction of normal and Hurler-syndrome cells, both cell types retained all of their radioactivity derived from [3H]glucose. 3. After 34h exposure to a `corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [35S]sulphate) of the Hurler-syndrome cells approached normal values. At the same time, there was an increase in specific radioactivity of `corrected' Hurler-syndrome cells.

scholarly journals 117: DECREASED COLLAGEN SYNTHESIS BY SKIN FIBROBLASTS FROM 31 PATIENTS SUFFERING FROM OSTEOGENESIS IMPERFECTA

1988 ◽  
Vol 24 (2) ◽  
pp. 280-280
Author(s):  
R E Brenner ◽  
U Vetter ◽  
W M Teller ◽  
O Wörsdorfer ◽  
P K Müller
Keyword(s):  
Osteogenesis Imperfecta ◽  
Collagen Synthesis ◽  
Skin Fibroblasts

scholarly journals Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides

1984 ◽  
Vol 221 (3) ◽  
pp. 707-716 ◽  
Author(s):  
M K Cowman ◽  
M F Slahetka ◽  
D M Hittner ◽  
J Kim ◽  
M Forino ◽  
...  
Keyword(s):  
Alcian Blue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Unit Structure ◽  
Electrophoretic Patterns ◽  
Sulphated Glycosaminoglycans ◽  
Sulphated Glycosaminoglycan ◽  
Testicular Hyaluronidase

Oligosaccharide fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. An extensive ladder-like set of bands is observed for partial testicular hyaluronidase digests of chondroitin 4- or 6-sulphate, and for dermatan sulphate. Co-electrophoresis of purified oligosaccharides has established that the major bands of these patterns represent fragments differing in chain length by one disaccharide unit, with the smallest fragments having the greatest mobility. Additional minor bands, representing heterogeneity in the repeating unit structure, are also observed. There are slight differences in the mobilities of oligosaccharides derived from the three major types of sulphated glycosaminoglycans. Alcian Blue is employed for visualization of the digest fragments. Sample loads of 5-10 micrograms per band appear optimum. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. After consideration of this effect, a good correlation is found to exist between densitometric scans of the gel-electrophoretic patterns and gel-filtration chromatographic profiles based on uronic acid concentration.

scholarly journals Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

1980 ◽  
Vol 190 (2) ◽  
pp. 243-254 ◽  
Author(s):  
J T Gallagher ◽  
N Gasiunas ◽  
S L Schor
Keyword(s):  
Hyaluronic Acid ◽  
Salt Concentration ◽  
Heparan Sulphate ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Sulphated Glycosaminoglycans ◽  
Sulphated Glycosaminoglycan ◽  
Spent Media

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.

Antagonistic effects of TGF-beta 1 and MSF on fibroblast migration and hyaluronic acid synthesis. Possible implications for dermal wound healing

1992 ◽  
Vol 102 (3) ◽  
pp. 447-456 ◽  
Author(s):  
I. Ellis ◽  
A.M. Grey ◽  
A.M. Schor ◽  
S.L. Schor
Keyword(s):  
Molecular Weight ◽  
Wound Healing ◽  
Hyaluronic Acid ◽  
High Molecular Weight ◽  
Cell Density ◽  
Elevated Level ◽  
Skin Fibroblasts ◽  
Tgf Beta ◽  
Fibroblast Migration ◽  
Fetal Fibroblasts

The migration of adult skin fibroblasts into three-dimensional collagen gel matrices is differentially affected by cell density, with subconfluent cells displaying a significantly elevated level of migration compared to confluent ones. Fetal fibroblasts differ from adult cells in that they display an elevated level of migration at both subconfluent and confluent cell densities. We have previously reported that this difference in behaviour results from the secretion by fetal fibroblasts of a ‘migration stimulating factor’ (MSF) which is not made by their normal adult counterparts, and that MSF appears to act by stimulating the synthesis of hyaluronic acid (HA). Data presented in this communication indicate that (a) MSF specifically stimulates the synthesis of high molecular weight species of HA, (b) TGF-beta 1 inhibits the elevated migration of adult fibroblasts plated at subconfluent cell density, (c) under these conditions, TGF-beta 1 induces a parallel decrease in the synthesis of high molecular weight HA and increase in the synthesis of low molecular weight HA, (d) TGF-beta 1 is a potent antagonist of MSF, effectively blocking its stimulation of cell migration and synthesis of high molecular weight HA, and (e) the inhibition of fibroblast migration by TGF-beta 1 does not appear to be a chemotactic response dependent upon the existence of a concentration gradient of the cytokine. Our observations regarding the inhibitory effects of TGF-beta 1 on fibroblast migration into 3D collagen gels stand in marked contrast to various published reports indicating that this cytokine stimulates the migration of human skin fibroblasts through the pores of polycarbonate filters as used in modified Boyden chamber assays; this discrepancy underscores the importance of the substratum in modulating cellular response to cytokines. Our results are discussed in terms of the possible combined contribution of MSF and TGF-beta 1 to wound healing.

scholarly journals The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells

1976 ◽  
Vol 158 (3) ◽  
pp. 567-573 ◽  
Author(s):  
B Y J T Yue ◽  
J L Baum ◽  
J E Silbert
Keyword(s):  
Hyaluronic Acid ◽  
Stromal Cells ◽  
Heparan Sulphate ◽  
Deae Cellulose ◽  
Dermatan Sulphate ◽  
Keratan Sulphate ◽  
Monolayer Cultures ◽  
Sulphated Glycosaminoglycans ◽  
Cellulose Column ◽  
Sulphated Glycosaminoglycan

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Sign in / Sign up

Export Citation Format

Share Document