Cell surface glycosaminoglycans: Identification and organization in cultured human embryo fibroblasts

A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have invetigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with ³H-glucosamine and Na₂³⁵SO₄ and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 μg/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.     

Glycosaminoglycan synthesis in the chick gastrula

Explanted definitive primitive streak to four somite chick embryos were labeled with [H³]glucosamine or S³⁵O₄ and the glycosaminoglycans were isolated and characterized. On the basis of susceptibility to Streptomyces hyaluronidase, which specifically degrades hyaluronic acid, hyaluronic acid is the major glycosaminoglycan produced by these embryos (at least 84%). On the basis of electrophoretic mobility, about 10% of the [H³]glucosamine-labeled glycoaminoglycan is sulfated. At least 55% of the sulfate-labeled glycosaminoglycan is sensitive to testicular hyaluronidase, and 36–39% is resistant to testicular hyaluronidase, but sensitive to nitrous acid treatment. About 94% of the labeled glycosaminoglycans can be accounted for in ratios of 22:1:5:1 as hyaluronic acid:chondroitin sulfate:heparan sulfate. No stage-related changes were observed. It is suggested that hyaluronic acid synthesis at this time might be related to the appearance of extensive cell-free spaces.     

A spectrophotometric microassay for sulfated glycosaminoglycans using a laser densitometer

The absorption spectrum of the dye 1,9-dimethylmethylene blue shifts if complexed with sulfated glycosaminoglycans. The present method uses the decrease in A633 rather than the increase in A535, described in a recent method, to measure the sulfated glycosaminoglycan content of biological samples. A conventional spectrophotometer was used to estimate the levels of sulfated glycosaminoglycan in papain extracts from intestinal wall tissue, by measuring both the A535 and the A633 and comparing them with a chondroitin sulfate standard: a highly significant correlation (r = 0.974, n = 17) was obtained. Also, interference by substances like RNA, DNA, and hyaluronic acid was similar for both methods. These results allowed us to employ a laser densitometer with a helium/neon laser emitting at 633 nm to improve the sensitivity and the capacity of the assay. The combination of a small reaction volume and a high-intensity light source allows the detection of less than 0.1 microgram chondroitin sulfate, a 40-fold improvement in sensitivity as compared with the original method. A very significant correlation (r = 0.885, n = 17) existed between results obtained with the macroassay, using a spectrophotometer, and those found by employing the microassay, using the laser densitometer. The use of microtiter plates and the screening potential of the densitometer yields an assay which is fast, very sensitive, and suitable for processing large numbers of samples.     

Micropattern immobilization of polysaccharide

Two types of polysaccharides, sulfated hyaluronic acid and heparin, were pattern-immobilized on a poly(ethylene terephthalate) and polystyrene film, respectively, in a specific pattern by photolithography. Sulfated hyaluronic acid was prepared from hylaronic acid by the treatment of sulfur trioxide/pyridine complex. Heparin was purchased and used without further treatment. The polysaccharides were coupled with azidoaniline. The derivatized polysaccharides were cast on a film from aqueous solution. After drying, the film was photo-irradiated in the presence or absence of a photomask. The micropatterning was confirmed by staining with a cationic dye. Platelet adhesion was reduced on the sulfated hyaluronic acid-immobilized areas. The immobilized sulfated hyaluronic acid significantly reduced thrombus formation. On the other hand, cells were cultured on the heparin-immobilized film. In the presence of fibroblast growth factor (FGF), the growth of mouse fibroblast STO cells was enhanced only on the heparin-immobilized regions. This result indicated that micropattern-immobilized heparin activated FGF for cell growth activity.     

Glycosaminoglycan polysulfate-induced stimulation of hyaluronic acid synthesis in rabbit knee synovial membrane: involvement of binding protein and calcium ion

We have previously reported that naturally occurring sulfated glycosaminoglycans having a chondroitin-type structure and glycosaminoglycan polysulfate (GAGPS, a persulfated derivative of chondroitin sulfate) caused a specific stimulation of hyaluronic acid synthesis in rabbit knee synovial membranes [H. Nishikawa et al. (1985) Arch. Biochem. Biophys. 240, 146-153]. In the present study, the interaction of [3H]GAGPS and the surface of the rabbit knee synovial membranes and the relationship between this interaction and the stimulatory effect of GAGPS on the hyaluronic acid synthesis were examined in order to define the stimulatory mechanism of hyaluronic acid synthesis by GAGPS. A part of the [3H]GAGPS taken up by the synovial membranes was released from the membranes by trypsin treatment. The interaction of [3H]GAGPS and the surface of the isolated synovial membranes was diminished by pretreatment of the membranes with proteases or chelating reagents. Pretreatment of synovial membranes with trypsin or ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid had little effect on the basal hyaluronic acid synthesis but caused the loss of GAGPS-induced stimulation of hyaluronic acid synthesis accompanied by significant decrease (20% P less than 0.05-P less than 0.01) in the interaction between GAGPS and the surface of the synovial membranes. Dermatan sulfate having a chondroitin-type structure also stimulated hyaluronic acid synthesis but this effect was not additive to the stimulation of hyaluronic acid synthesis by GAGPS. Heparin had no effect on either the basal hyaluronic acid synthesis or the GAGPS-induced stimulation of hyaluronic acid synthesis. These results indicate that binding of GAGPS to certain distinct protein components on the surface of synovial membranes is involved in the stimulatory mechanism of hyaluronic acid synthesis by GAGPS, and that the binding may be mediated by Ca2+ ion. The binding was also found to be specific for sulfated glycosaminoglycans having a chondroitin-type structure.     

Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells

The synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.     

Dermatan sulfate is the major metachromatic glycosaminoglycan in the integument of the anuran Bufo ictericus

Glycosaminoglycans from the ventral and dorsal integuments of the anuran Bufo ictericus were characterized based on biochemical and histochemical methods. Dermatan sulfate is the major metachromatic glycosaminoglycan found in these tissues, but small amount of heparan sulfate was also detected. The average molecular mass of the dermatan sulfate is approximately 20 kDa, similar to the glycosaminoglycan isolated from mammalian skin. In addition, the amphibian integument contains high amounts of hyaluronic acid, especially in the ventral area. We also observed that the glycosaminoglycans occur in the anuran integument as irregular deposits through the spongious dermis and in the mast cells, as revealed by histochemical analysis using Alcian blue, dimethylmethylene blue and toluidine blue stains. The concentration and composition of glycosaminoglycans found in the amphibian integument resemble those from mammalian skin except for the higher concentration of hyaluronic acid in the amphibian tissue. Possibly, this observation indicates that the function of the sulfated glycosaminoglycan in these tissues has been preserved during evolution, although the amphibian integument and the human skin have their own particular physiology.     

Analysis of collagen cyanogen bromide peptides using electrophoresis in continuous concave gradient polyacrylamide gels.

A rapid and simple spectrophotometric method is described for the estimation of microgram quantities of glycosaminoglycans following the formation of soluble complexes with alcian blue dye. The method is based on the different absorption spectra of the dye and dye-glycosaminoglycan complexes. No heating, centrifugation, lengthy equilibration, or sophisticated instrumentation which hamper other methods are required. Samples are mixed with freshly prepared dye solution and absorbance readings at 480 nm are compared to an appropriate standard curve. Albumin and individual monosaccharides do not interfere with the assay but high concentrations of chloride ion do. The method is suitable for the estimation of total glycosaminoglycan levels in biological fluids such as urine and blood.     

Simultaneous Preparation and Quantitation of Proteoglycans by Precipitation with Alcian Blue

Conditions for specific interaction between Alcian blue and proteoglycans were optimized by comparing the differential spectra of Alcian blue obtained with purified chondroitin sulfate dissolved in water with the spectra obtained with nasal cartilage proteoglycans dissolved in synovial fluid. A method was then designed that provides specific precipitation of proteoglycans or glycosaminoglycans in 4 M guanidine-HCl in the presence of protein, hyaluronic acid, or nucleic acids. The specificity is achieved by using a low pH in combination with detergent and high salt concentration. Stepwise addition of reagents is necessary to avoid binding of Alcian blue to proteins and nucleic acids. All polyanions, except polysulfates, are first neutralized by lowering the pH to 1.5. By including detergent in this step, the hydrophobic protein regions are blocked and not accessible for binding with the dye. These regions could otherwise bind Alcian blue by hydrophobic interaction. When the Alcian blue reagent is added after, only the polysulfated molecules will remain charged and free to interact with Alcian blue. At least 0.4 M guanidine-HCl is required to abolish the negative interference by proteins. All sulfated glycosaminoglycans are precipitated at 0.4 M guanidine-HCl. With increasing guanidine-HCl concentrations, the different glycosaminoglycans are precipitated in accordance with the critical electrolyte concentration of the respective glycosaminoglycan. The Alcian blue precipitation can be performed at different concentrations of guanidine-HCl in order to separate different classes of proteoglycans. Excess dye and contaminating proteins are removed by a wash in a DMSO-MgCl₂ solution and the precipitate is dissolved in a mixture of guanidine-HCl and propanol. For quantitation, the absorbance is recorded in a microplate reader with the 600-nm filter, the assay being linear between 0.5 and 20 μg proteoglycan. Since no digestion of samples with protease is needed, the proteoglycans are recovered in native form. The proteoglycan-Alcian blue complexes dissociate in the guanidine-HCl/propanol mixture and the proteoglycans can be selectivelyprecipitated with propanol. The dye is used for quantitation and the proteoglycans can be utilized for further analysis.     

The heterogeneity of dermatan sulfate and heparan sulfate in rat liver and a shift in the glycosaminoglycan contents in carbon tetrachloride-damaged liver

The glycosaminoglycan of rat liver can be separated into five distinct fractions; a hyaluronic acid franction, a heparan sulfate fraction with a molar ratio of sulfate to hexosamine (S/HexN) around 0.7, a heparan sulfate fraction with a S/HexN ratio around 1.4, a dermatan sulfate fraction with a S/HexN ratio near unity, and a dermatan sulfate fraction with a S/HexN ratio around 1.3.Enzymatic analysis of the two dermatan sulfate fractions indicates that they differ significantly in that the high sulfated fraction contains relatively more N-acetylgalactosamine 4,6-bissulfate units (about 26% of the total hexosamine). In experimental injury produced by carbon tetrachloride, the low sulfated fraction increases as much as 9-fold on a dry weight basis, bearing no linear relationship to the amount of the high sulfated fraction which increases only 2-fold. A significant shift is also observed in the levels of the two heparan sulfate fractions. In this case, however, the high sulfated fraction shows a much more pronounced increase than does the low sulfated fraction. On the basis of these observations, it is suggested that for each of the dermatan sulfate and heparan sulfate classes are at least two pools, distinguished by sulfation degree and perhaps by turnover rate and physiological function.     

The heterogeneity of dermatan sulfate and heparan sulfate in rat liver and a shift in the glycosaminoglycan contents in carbon tetrachloride-damaged liver.

The glycosaminoglycan of rat liver can be separated into five distinct fractions; a hyaluronic acid fraction, a heparan sulfate fraction with a molar ratio of sulfate to hexosamine (S/HexN) around 0.7, a heparan sulfate fraction with a S/HexN ratio around 1.4, a dermatan sulfate fraction with a S/HexN ratio near unity, and a dermatan sulfate fraction with a S/HexN ratio around 1.3. Enzymatic analysis of the two dermatan sulfate fractions indicates that they differ significantly in that the high sulfated fraction contains relatively more N-acetylgalactosamine 4,6-bissulfate units (about 26% of the total hexosamine). In experimental injury produced by carbon tetrachloride, the low sulfated fraction increases as much as 9-fold on a dry weight basis, bearing no linear relationship to the amount of the high sulfated fraction which increases only 2-fold. A significant shift is also observed in the levels of the two heparan sulfate fractions. In this case, however, the high sulfated fraction shows a much more pronounced increase than does the low sulfated fraction. On the basis of these observations, it is suggested that for each of the dermatan sulfate and heparan sulfate classes there are at least two pools, distinguished by sulfation degree and perhaps by turnover rate and physiological function.     

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.     

Sulfated hyaluronic acid and dexamethasone possess a synergistic potential in the differentiation of osteoblasts from human bone marrow stromal cells

The development of novel bioactive biomaterials is urgently needed to meet the needs of an aging population. Both sulfated hyaluronic acid and dexamethasone are candidates for the functionalization of bone grafts, as they have been shown to enhance the differentiation of osteoblasts from bone marrow stromal cells in vitro and in vivo. However, the underlying mechanisms are not fully understood. Furthermore, studies combining different approaches to assess synergistic potentials are rare. In this study, we aim to gain insights into the mode of action of both sulfated hyaluronic acid and dexamethasone by a comprehensive analysis of the cellular fraction, released matrix vesicles, and the extracellular matrix, combining classical biochemical assays with mass spectrometry–based proteomics, supported by novel bioinformatical computations. We found elevated differentiation levels for both treatments, which were further enhanced by a combination of sulfated hyaluronic acid and dexamethasone. Single treatments revealed specific effects on osteogenic differentiation. Dexamethasone activates signalling pathways involved in the differentiation of osteoblasts, for example, CXC-motif chemokine receptor type 4 and mitogen-activated protein kinases. The effects of sulfated hyaluronic acid were predominantly linked to an alteration in the composition of the extracellular matrix, affecting the synthesis, secretion, and/or activity of fibrillary (fibronectin and thrombospondin-2) and nonfibrillary (transglutaminase-2, periostin, and lysyloxidase) extracellular matrix components, including proteases and their inhibitors (matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-3). The effects were treatment specific, and less additive or contrary effects were found. Thus, we anticipate that the synergistic action of the treatment-specific effects is the key driver in elevated osteogenesis.     

Biosynthesis of heparan sulfate

Microsomal preparations from Englebreth-Holm-Swarm mouse sarcoma were incubated with UDP-N-acetyl[³H] glucosamine and UDP-[¹⁴C]glucuronic acid to form proteoglycan containing [³H,¹⁴C]glycosaminoglycan with equimolar amounts of [³H]glucosamine and [¹⁴C]glucuronic acid. The labelled glycosaminoglycan was totally resistant to degradation by testicular hyaluronidase, but could be degraded readily by a crudeFlavobacter heparinum enzyme preparation which is capable of degrading heparin and heparan sulfate. Chromatography of the [³H,¹⁴C]glycosaminoglycan on DEAE-cellulose provided a pattern with three peaks: the first appearing before hyaluronic acid, the second and largest appearing at the site of hyaluronic acid, and a third appearing slightly beyond hyaluronic acid but before a standard of chondroitin sulfate. When 3-phosphoadenosine 5-phosphosulfate was also included in the reaction mixture, a change appeared in the [³H,¹⁴C]glycosaminoglycan so that chromatography on DEAE-cellulose presented a pattern with a significant amount of material which cochromatographed in the area where heparan sulfate would be found. There was no material that co-chromatographed with the more highly sulfated substance, heparin. This indicates that the microsomal preparation from the Englebreth-Holm-Swarm sarcoma is capable of producing a heparan sulfate-like molecule and is controlled in its sulfation of precursors so that heparin is not formed.     

Effect of retinoic acid on in vitro proliferation activity and glycosaminoglycan synthesis of mesenchymal cells from palatal shelves of mouse fetuses

To clarify the mechanism by which retinoid causes cleft palate, we investigated the effect of retinoic acid (RA) on proliferation activity and glycosaminoglycan (GAG) synthesis in mouse fetuses palatal mesenchymal (MFPM) cells. MFPM cells were incubated for 1-11 days with various concentrations of RA to examine its effect on growth rate. Also, confluent cultures were incubated with [3H]glucosamine or [35S]sulfate in the presence of various concentrations of RA to investigate the effect of RA on GAG synthesis. RA remarkably inhibited the growth of MFPM cells in a dose-dependent manner. RA also inhibited the synthesis of GAGs, with sulfated GAGs being more severely affected than hyaluronic acid. These data suggest that the inhibition of proliferation activity and GAG synthesis of palatal mesenchymal cells might be involved in the induction of cleft palate by retinoic acid.     

Isolation and characterization of proteoglycans synthesized by cultured mesangial cells

Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.     

Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture.

The depression of proteoglycan synthesis in ten-day-old high density chondrocyte cultures was shown to be dependent on both the concentration and time of exposure of the cells to hyaluronic acid. Hyaluronic acid had no effect on the overall protein synthesis by the cultured cells. Using benzyl-beta-D-xyloside an exogenous acceptor, it was shown that glycosaminoglycan biosynthesis by the chondrocytes was not affected by hyaluronic acid. It was concluded that hyaluronic acid was effecting glycosaminoglycan chain initiation, hence proteoglycan biosynthesis, either by specifically depressing the synthesis of the core protein or by repressing the activity of the xylosyltransferase.     

Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture

The depression of proteoglycan synthesis in ten-day-old high density chondrocyte cultures was shown to be dependent on both the concentration and time of exposure of the cells to hyaluronic acid. Hyaluronic acid had no effect on the overall protein synthesis by the cultured cells. Using benzyl-β-D-xyloside an exogenous acceptor, it was shown that glycosaminoglycan biosynthesis by the chondrocytes was not affected by hyaluronic acid. It was concluded that hyaluronic acid was effecting glycosaminoglycan chain initiation, hence proteoglycan biosynthesis, either by specifically depressing the synthesis of the core protein or by repressing the activity of the xylosyltransferase.     

Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.     

糖胺聚糖降解菌株的筛选及鉴定

以土壤为材料,用透明质酸和硫酸软骨素为唯一碳源富集分离菌株,通过BSA-乙酸平板显色法及比色定糖法进行筛选。从80份土壤中筛选出13株糖胺聚糖降解活性的菌株并对其进行了16S rDNA测序鉴定。结果表明,筛选到13株糖胺聚糖降解菌株均具有透明质酸酶和硫酸软骨素酶活性;获得8株尚未报导过的产糖胺聚糖降解酶活性菌株。本研究为开发新型的糖胺聚糖降解酶提供参考。