Domain structure of proteoheparan sulphate from confluent cultures of human embryonic skin fibroblasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Fransson LA
PROVIDER: S-EPMC1152803 | biostudies-other | 1985 Nov
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Fibroblasts in culture were incubated with [3H]leucine and [35S]sulphate for 1-24 h. A large glucuronic acid-rich and a small iduronic acid-rich dermatan sulphate proteoglycan were isolated with the use of isopycnic density-gradient centrifugation, ion-exchange and gel chromatography. After 3 h the accumulation in the cell layer of the small proteoglycan reached a steady state, whereas the large one continued to increase, albeit more slowly. In the medium both proteoglycans accumulated 'linearly', although the large one appeared somewhat later than the small one. The composition of the polysaccharide chains and the size of the protein cores did not vary during the experiment. The two proteoglycans were synthesized at approximately similar rates, but were distributed differently in the culture. The small proteoglycan was mainly confined to the medium, whereas the large one was found in the medium as well as in a cell-associated pool. There was an intracellular accumulation of iduronic acid-rich dermatan sulphate as free polysaccharides.
| S-EPMC1153662 | biostudies-other
Project description:35SO42- - and [3H]-leucine-labelled proteoglycans were isolated from the medium of a fibroblast culture, from an EDTA extract of the monolayer, and from consecutive dithiothreitol and guanidine hydrochloride extracts of the cells. Proteoglycans of different sizes were isolated from the extracts by gel chromatography on Sepharose 4B. In the medium and the EDTA extract the largest proteoglycans contained only 35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparan [35S-labelled galactosaminoglycan, whereas all other fractions contained in addition heparin [35S]sulphate. The galactosaminoglycan-containing proteoglycans of the various extracts were separated into a larger component, containing chondroitin sulphate-like side chains, and a smaller component, containing dermatan sulphate. The larger proteoglycan of the medium showed reversible association-dissociation behaviour when chromatographed on Sepharose CL2B in phosphate-buffered saline and 4M-guanidine hydrochloride respectively. This property remained after removal of extraneous proteins by CsCl-density-gradient centrifugation in guanidine hydrochloride. The association was markedly increased by the addition of high-molecular-weight hyaluronic acid.
| S-EPMC1161649 | biostudies-other
Project description:Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.
| S-EPMC1162744 | biostudies-other
Project description:The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.
| S-EPMC1133692 | biostudies-other
Project description:Scar formation following skin injury can be a major psychosocial and physiological problem. However, the mechanisms of scar formation are still not completely understood. Previous studies have shown that wound healing in oral mucosa is faster, associates with a reduced inflammatory response and results to significantly reduced scar formation compared with skin wounds. In the present study, we hypothesized that oral mucosal fibroblasts from human gingiva are inherently distinct from fibroblasts from breast and abdominal skin, two areas prone to excessive scar formation, which may contribute to the preferential wound healing outcome in gingiva. To this end, we compared the phenotype of human gingival and skin fibroblasts cultured in in vivo-like three-dimensional (3D) cultures that mimic the cells' natural extracellular matrix (ECM) niche. To establish 3D cultures, five parallel fibroblast lines from human gingiva (GFBLs) and breast skin (SFBLs) were seeded in high density, and cultured for up to 21 days in serum and ascorbic acid containing medium to induce expression of wound-healing transcriptome and ECM deposition. Cell proliferation, morphology, phenotype and expression of wound healing and scar related genes were analyzed by real-time RT-PCR, Western blotting and immunocytochemical methods. The expression of a set of genes was also studied in three parallel lines of human abdominal SFBLs. Findings showed that GFBLs displayed morphologically distinct organization of the 3D cultures and proliferated faster than SFBLs. GFBLs expressed elevated levels of molecules involved in regulation of inflammation and ECM remodeling (MMPs) while SFBLs showed significantly higher expression of TGF-? signaling, ECM and myofibroblast and cell contractility-related genes. Thus, GFBLs display an inherent phenotype conducive for fast resolution of inflammation and ECM remodeling, characteristic for scar-free wound healing, while SFBLs have a profibrotic, scar-prone phenotype.
| S-EPMC3946595 | biostudies-literature
Project description:Interferon gamma (IFN gamma) is often considered to be an antifibrotic cytokine because it inhibits collagen synthesis in fibroblasts. Here we report the effects of recombinant human IFN gamma on sulphated glycosaminoglycan chains produced by normal skin fibroblasts from adult donors. IFN gamma (250 i.u./ml) induced an increase in incorporation of D-[1-3H]glucosamine into glycosaminoglycans, either secreted into the culture medium or associated with the cell layer. The structures of these molecules were analysed by using various cleavage agents (heparinases I and II, heparitinase/chondroitinases ABC and AC/periodate oxidation) followed by size-exclusion and anion-exchange HPLC. No modification was detected in the structure of the heparan sulphate chains. In contrast, the cytokine induced changes in the microcomposition of chondroitin/dermatan sulphate chains. More precisely, we found a decrease in the iduronic acid content, associated with down-regulation of the 4-O-sulphation on the GalNAc residues. In contrast, the 6-O-sulphation on these GalNAc residues was potentiated by the cytokine. These results indicate that IFN gamma is able to modulate not only collagen but also the structure of galactosaminoglycans synthesized by human skin fibroblasts.
| S-EPMC1217697 | biostudies-other
Project description:Foetal human lung fibroblasts, grown in monolayer, were allowed to incorporate (35)SO(4) (2-) for various periods of time. (35)S-labelled macromolecular anionic products were isolated from the medium, a trypsin digest of the cells in monolayer and the cell residue. The various radioactive polysaccharides were identified as heparan sulphate and a galactosaminoglycan population (chondroitin sulphate and dermatan sulphate) by ion-exchange chromatography and by differential degradations with HNO(2) and chondroitinase ABC. Most of the heparan sulphate was found in the trypsin digest, whereas the galactosaminoglycan components were largely confined to the medium. Electrophoretic studies on the various (35)S-labelled galactosaminoglycans suggested the presence of a separate chondroitin sulphate component (i.e. a glucuronic acid-rich galactosaminoglycan). The (35)S-labelled galactosaminoglycans were subjected to periodate oxidation of l-iduronic acid residues followed by scission in alkali. A periodate-resistant polymer fraction was obtained, which could be degraded to disaccharides by chondroitinase AC. However, most of the (35)S-labelled galactosaminoglycans were extensively degraded by periodate oxidation-alkaline elimination. The oligosaccharides obtained were essentially resistant to chondroitinase AC, indicating that the iduronic acid-rich galactosaminoglycans (i.e. dermatan sulphate) were composed largely of repeating units containing sulphated or non-sulphated l-iduronic acid residues. The l-iduronic acid residues present in dermatan sulphate derived from the medium and the trypsin digest contained twice as much ester sulphate as did material associated with the cells. The content of d-glucuronic acid was low and similar in all three fractions. The relative distribution of glycosaminoglycans among the various fractions obtained from cultured lung fibroblasts was distinctly different from that of skin fibroblasts [Malmström, Carlstedt, Aberg & Fransson (1975) Biochem. J.151, 477-489]. Moreover, subtle differences in co-polymeric structure of dermatan sulphate isolated from the two cell types could be detected.
| S-EPMC1183669 | biostudies-other
Project description:Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.
| S-EPMC2956221 | biostudies-literature
Project description:The domain structure of heparan sulphate chains from an endothelial low-density proteoglycan was examined using specific degradations of the chains while attached to the intact proteoglycan. 'Inner' chain fragments, remaining on the protein core, were separated from 'outer' fragments by gel chromatography, and were subsequently released from the protein core by alkaline cleavage. The structure of 'inner' and 'outer' chain fragments was then examined and compared. Using deaminative cleavage we obtained evidence that the first N-sulphated glucosamine residue is variably positioned some 10-17 disaccharides from the xylose-serine linkage of the proteoglycan. Digestion with heparinase yielded 'inner' and 'outer' fragments covering a broad range of different sizes, indicating a scarce and variable distribution of sulphated iduronic acid in the native chains. N-sulphated glucosamine occurred more frequently in the 'outer' fragments. We also studied the affinity of the endothelial heparan sulphate chains towards two presumptive biological ligands, namely antithrombin III and lipoprotein lipase. A major part of the endothelial heparan sulphate chains showed a weak affinity for antithrombin III and the affinity was essentially lost on heparinase digestion. On lipoprotein lipase-agarose the endothelial heparan sulphate chains were eluted at the same salt concentration as heparin, and the binding persisted, although with decreased strength, after digestion with heparinase.
| S-EPMC1151520 | biostudies-other
Project description:Spatiotemporal wave activities in excitable heart tissues have long been the subject of numerous studies because they underlie different forms of cardiac arrhythmias. In particular, understanding the dynamics and the instabilities of spiral waves have become very important because they can cause reentrant tachycardia and their subsequent transitions to fibrillation. Although many aspects of cardiac spiral waves have been investigated through experiments and model simulations, their complex properties are far from well understood. Here, we show that intriguing complex-periodic (such as period-2, period-3, period-4, or aperiodic) spiral wave states can arise in monolayer tissues of cardiac cell culture in vitro, and demonstrate that these different dynamic states can coexist with abrupt and spontaneous transitions among them without any change in system parameters; in other words, the medium supports multistability. Based on extensive image data analysis, we have confirmed that these spiral waves are driven by their tips tracing complex orbits whose unusual, meandering shapes are formed by delicate interplay between localized conduction blocks and nonlinear properties of the culture medium.
| S-EPMC1177368 | biostudies-literature