Hyaluronate synthesized by cultured skin fibroblasts derived from patients with Werner's syndrome.

Hyaluronate in cultured skin fibroblasts derived from patients with Werner's syndrome, who excrete large amounts of urinary hyaluronate, was investigated. The amount of hyaluronate secreted into the medium by Werner's fibroblasts was 2-3-times that of normal fibroblasts, whereas no difference in enzyme activities related to the degradation of hyaluronate was found. Werner's fibroblasts were then cultured in the presence of [3H]glucosamine, and the amount of [3H]hyaluronate and its chain lengths in the medium and matrix (trypsinate) fractions were compared with those of normal cells. No significant difference in the chain length of hyaluronate was observed between normal and Werner's fibroblasts. On the other hand, a significant increase of hyaluronate was found in the matrix fraction of Werner's fibroblasts when the cells reached confluency. In addition, a hyaluronate of small chain length was found in the matrix fraction of Werner's fibroblasts, although this was absent from that of normal cells. It was concluded that the constituents of the extracellular matrix of Werner's fibroblasts differed from those of normal cells, characterized by the presence of a large amount of hyaluronate and a relatively small hyaluronate chain.     

Influence of the fatty acid composition of high-density lipoprotein phospholipids on the cholesterol efflux from cultured fibroblasts

The purpose of this work was to determine whether the changes induced by dietary manipulations in the chemical composition of high-density lipoproteins (HDL) (particularly phospholipid fatty acid composition) modified their capacity to promote [3H]cholesterol efflux from cultured fibroblasts. Plasma HDL were obtained from subjects fed for six successive long periods on diets consisting of one predominant fat: peanut oil, corn oil, olive oil, soybean oil, low erucic acid rapeseed oil or milk fats. The [3H]cholesterol efflux from cells in the presence of plasma HDL was studied by means of normal adult human fibroblasts in culture. The [3H]cholesterol efflux from fibroblasts appeared to be independent of the overall composition of HDL and of the degree of saturation of the HDL phospholipid fatty acids, but it was correlated with the phospholipid fatty acid chain length. The [3H]cholesterol efflux from fibroblasts is highly and positively correlated with the sum of the HDL phospholipid C20, C22, C24 fatty acids, and negatively correlated with the sum of the HDL phospholipid C18 fatty acids.     

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.     

In vitro and in vivo effects of protein phosphatase inhibitors, microcystins and nodularin, on mouse skin and fibroblasts

Three microcystins, YR, LR and RR and nodularin, all of which are hepatotoxic compounds, inhibited dose-dependently the activity of protein phosphatase 2A in and the specific [3H]okadaic acid binding to a cytosolic fraction of mouse skin, as strongly as okadaic acid. However, microcytins and nodularin did not induce any effects on mouse skin or primary human fibroblasts. Microinjection of microcystin YR into primary human fibroblasts induced morphological changes which were induced by incubation with okadaic acid. Microcystins and nodularin penetrate into the epithelial cells of mouse skin and human fibroblasts with difficulty, which reflects tissue specificity of the compounds.     

Cation-independent mannose 6-phosphate receptor contains covalently bound fatty acid

The cation-independent mannose 6-phosphate receptor (215,000 daltons) was isolated from embryonic bovine tracheal cells and embryonic human skin fibroblasts labelled with [3H]palmitic acid. The tritium label was detected in the protein upon fluorographic analysis of SDS-polyacrylamide gels of the purified receptor. The label was not sensitive to hydroxylamine, methanolic KOH, or beta-mercaptoethanol, but labelled fatty acid was recovered from the protein by acidic methanolysis. Labelled receptor protein could not be isolated from cells grown in the presence of [3H]myristic acid. The results suggest the presence of amide-linked palmitic acid in the structure of the cation-independent mannose 6-phosphate receptor.     

Altered glycosaminoglycan production in cultured osteogenesis-imperfecta skin fibroblasts.

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.     

Synthesis of glycosaminoglycans in fibroblasts from abortuses with trisomy,triploidy, and from children with Down's syndrome

Summary A comparative study has been made of glycosaminoglycan (GAG) accumulation in human fibroblasts with trisomy 7 and triploidy from spontaneous abortuses, fibroblasts with triploidy from induced abortuses, fibroblasts from patients with Down's syndrome and diploid fibroblasts from age-matched controls. The study demonstrated that the incorporation of [³H]glucosamine into hyaluronic acid by fibroblasts with trisomy 7 and triploidy, established from spontaneous abortuses, and from two out of three induced abortuses with triploidy, was 2.6–5.3 times lower than control incorporation. One strain of fibroblasts from an induced abortus with triploidy (IMG-1062) did not show any differences in GAG production when compared with diploid fibroblasts. However, the strains from children with Down's syndrome revealed normal or even increased levels of hyaluronic acid production. The data support the contention that the decreased hyaluronic acid synthesis in fibroblasts with an abnormal karyotype is related to spontaneous abortion.     

Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations.

Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.     

RU486 inhibits induction of aromatase by dexamethasone via glucocorticoid receptor in cultured human skin fibroblasts

Effects of RU486 on the induction of aromatase by dexamethasone via glucocorticoid receptor were determined using cultured human skin fibroblasts. Competition of [3H]dexamethasone binding to the cytosol receptor was 7 times stronger with RU486 than with dexamethasone. The order of the strength of competition was RU486 greater than dexamethasone greater than betamethasone greater than prednisolone greater than hydrocortisone. RU486 abolished a specific 8.6 S [3H]dexamethasone binding peak in the cytosol, determined using a sucrose density gradient analysis. Dexamethasone markedly induced aromatase and this event was strongly suppressed by RU486, in a dose-dependent manner, in the cultured skin fibroblasts. A linear correlation between the strength of competition and the induction of aromatase of various glucocorticoids was observed. RU486 non-competitively inhibited aromatase induction by dexamethasone determined from a double reciprocal plot of aromatase activity, with respect to [3H]androstenedione concentration in the presence of RU486. These results show that RU486 is a peripheral noncompetitive antiglucocorticoid on aromatase induction by glucocorticoid in human skin fibroblasts and that aromatase induction is a good marker for the biological function of glucocorticoid receptor in human skin fibroblasts.     

Role of peroxisomal oxidation in the conversion of arachidonic acid to eicosatrienoic acid in human skin fibroblasts.

Human skin fibroblasts converted [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]20:4) to eicosatrienoic acid (20:3), but appreciable amounts of radiolabeled 20:3 were not detected in corresponding incubations with [1-(14)C]20:4. This indicates that the main pathway for synthesizing 20:3 from arachidonic acid in the fibroblast involves oxidative removal of the carboxyl group of arachidonic acid. Fibroblasts deficient in long-chain acyl coenzyme A dehydrogenase (LCAD) converted [3H]20:4 to [3H]20:3. However, Zellweger fibroblasts that are deficient in peroxisomal fatty acid oxidation did not, indicating that the oxidative removal of the carboxyl group occurs in the peroxisomes. [3H]Hexadecatrienoic acid (16:3) was the main product that accumulated when [3H]20:4 was incubated with normal, LCAD deficient, and very long-chain acyl coenzyme A dehydrogenase (VLCAD) deficient fibroblasts, but Zellweger fibroblasts did not form this product. Normal fibroblasts converted [3H]16:3 to radiolabeled 20:3 and arachidonic acid. These findings suggest that some of the 16:3 produced from arachidonic acid by peroxisomal beta-oxidation can be recycled and that this recycling process constitutes a novel pathway for the conversion of arachidonic acid to 20:3 in human fibroblasts.     

High-Affinity [3H]Choline Accumulation in Cultured Human Skin Fibroblasts

[3H]Choline can be transported across cell membranes by high-affinity (KT less than 5 microM) and low-affinity (KT much greater than 5 microM) systems. High-affinity choline accumulation (HACA) has been demonstrated in synaptosomes made from cholinergic brain regions such as the hippocampus and caudate-putamen. In cell culture, HACA has been demonstrated in glia and avian telencephalon, dissociated spinal cord, and muscle fibroblasts. We examined [3H]choline accumulation in a single normal human fibroblast line cultured from skin biopsy. [3H]Choline accumulation was temperature-dependent and linear with incubation time up to 6 min at 0.125 microM-choline. The apparent KT for [3H]choline was 5 microM, which is similar to that observed in avian fibroblasts. Isoosmotic replacement of Na+ with either Li+ (144 mM) or sucrose (288 mM) severely reduced [3H]choline accumulation (by 70-90%). Pre-incubation with ouabain (100 microM), sodium orthovanadate (100 microM), or 2,4-dinitrophenol (100 microM), or replacement of Ca2+ by Mg2+ had little or no effect on subsequent [3H]choline accumulation. [3H]Choline accumulation was inhibited by hemicholinium-3 (HC-3); after pre-incubation in HC-3 at 37 degrees C for 10 min, the IC50 (at 0.125 microM-choline) was 5.6 microM. The HC-3 sensitivity, Na+ dependence, and low KT suggest that human skin fibroblasts have a high-affinity transport system for choline.     

Effect of sphingosine and other amphiphilic amines on the biosynthesis of phosphatidylethanolamine and other glycerolipids in isolated rat hepatocytes.

The importance of ethanolamine and sphingosine as precursors of phosphoethanolamine was investigated by incubating them with [3H]glycerol and isolated rat hepatocytes. Sphingosine (0.1--0.5 mM) stimulated the synthesis of phosphatidylethanolamine from [3H]glycerol, but the stimulation by ethanolamine was more pronounced. Furthermore, more phosphoethanolamine accumulated in the heptatocytes after incubation with ethanolamine than after incubation with sphingosine. It is concluded that ethanolamine is the most important phosphoethanolamine precursor in rat liver. Higher concentrations of sphingosine caused accumulation of [3H]phosphatidate and inhibition of total glycerolipid synthesis in isolated hepatocytes, when incubated in the presence of [3H]glycerol. These effects were very similar to those of fenfluramine and norfenfluramine described previously. Simpler cationic amphiphilic amines, like oleoylamine and octadecyltrimethylammonium bromide, also caused these effects. Variation of alkyl chain length and amphiphile charge showed that both a positive charge and a certain alkyl chain length were necessary for interference with phosphatidate metabolism. A much wider range of compounds inhibited total glycerolipid synthesis from [3H]glycerol.     

Biosynthesis of proteodermatan sulfate in cultured human fibroblasts

Biosynthesis and secretion of proteodermatan sulfate produced by cultured human skin fibroblasts were investigated employing immunological procedures. During an incubation period of 10 min in the presence of [3H]leucine, two core protein forms of Mr = 46,000 and 44,000, respectively, were synthesized. They were converted to mature proteodermatan sulfate with a half-time of approximately 12 min. Fifty per cent of total mature proteodermatan sulfate were found in the culture medium after a 35-min chase. Six to eight per cent remained associated with the cell layer after a chase of 6 h. In the presence of tunicamycin, fibroblasts synthesized a single core protein of Mr = 38,000 that was converted to mature proteodermatan sulfate and secreted with similar kinetics as the N-glycosylated species. Subtle differences in the molecular size of core proteins were noted when cell-associated and secreted proteodermatan sulfate were degraded with chondroitin ABC lyase, but core proteins free of N-linked oligosaccharides were identical. Labeling with [3H]mannose revealed that secreted proteodermatan sulfate contains two or three complex-type or two complex-type and one high-mannose-type N-linked oligosaccharide chains. The N-glycans are bound to a 21-kDa fragment of the core protein. After incubation in the presence of [3H]glucosamine, the [3H]galactosamine/[3H]glucosamine ratio was 3.76 and 3.30 for secreted and cell-associated proteodermatan sulfate, respectively. Evidence for the presence of O-linked oligosaccharides could not be obtained. Small amounts of core protein free of dermatan sulfate chains were secreted when the cultures were treated with p-nitrophenyl-beta-D-xyloside.     

Incorporation of [3H]thymidine by isolated fetal myoblasts and fibroblasts in response to human placental lactogen (HPL): possible mediation of HPL action by release of immunoreactive SM-C

We investigated the actions of human placental lactogen (HPL) and human growth hormone (HGH) on [3H]thymidine incorporation and the release of immunoassayable somatomedin-C (SM-C) by isolated myoblasts, dermal fibroblasts, and costal cartilage explants taken from human fetuses at 11-21 weeks of gestation. The incorporation of [3H]thymidine by myoblasts and fibroblasts was significantly increased after incubation for 20 hr or 44 hr, and cell number after incubation for 7 days, in the presence of 50-250 ng/ml HPL. Incubation with HPL did not increase [3H]thymidine incorporation into cartilage explants, whereas incubation with HGH failed to enhance the uptake of this isotope by any of the tissues. Following extraction with acid-ethanol, culture medium conditioned by exposure to myoblasts or fibroblasts for 44 hr, and to cartilage explants for 7 days, contained radioimmunoassayable SM-C. Myoblast-conditioned medium contained significantly more SM-C [1,609 +/- 953 mU/mg cell protein (mean +/- SD); n = 10] than did that conditioned by fibroblasts (637 +/- 323; n = 5; P less than 0.02). In 1 week of culture, cartilage explants released 4.1 +/- 1.1 mU/mg wet weight (n = 7). The release of immunoassayable SM-C from cultured cells was significantly increased in the presence of 250 ng/ml HPL in five of eight experiments with myoblasts and two of four experiments with fibroblasts. Neither fibroblasts or myoblasts showed increased SM-C release following exposure to HGH. The results suggest that HPL, but not HGH, is growth-promoting for some human fetal tissues in vitro and that this action is mediated, at least in part, by an increased release of somatomedins.     

A novel 4-methylumbelliferyl-beta-D-xyloside derivative, sulfate-O-3- xylosylbeta1-(4-methylumbelliferone), isolated from culture medium of human skin fibroblasts, and its role in methylumbelliferone-initiated glycosaminoglycan biosynthesis

Human skin fibroblasts were incubated in the presence of 4- methylumbelliferyl-beta-D-xyloside (Xyl-MU). The culture medium was recovered and Xyl-MU derivatives which were initiated by the Xyl-MU acting as a primer were purified. As a result, a novel Xyl-MU derivative was isolated, in addition to previously reported Xyl-MU derivatives such as glycosaminoglycan-MU, Gal-Gal-Xyl-MU, Gal-Xyl-MU, SA-Gal-Xyl-MU, Xyl-Xyl-MU, GlcA-Xyl-MU, and sulfate-GlcA-Xyl-MU. This Xyl-MU derivative was subjected to carbohydrate composition analysis, enzyme digestion, ion-spray mass spectrometric analysis, and Smith degradation. The results indicated that it was sulfate- O -3-Xyl-MU. When Xyl-MU was incubated with [35S]PAPS using a homogenate prepared from the same cultured skin fibroblasts, [35S]sulfate- O -3-Xyl-MU was produced. Moreover, when Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was transferred to Xyl-MU, but when sulfate- O -3-Xyl-MU was incubated with UDP-[3H]Gal, [3H]galactose was not transferred. These results indicate that chain elongation from Xyl-MU is inhibited by sulfation of Xyl-MU, and that Xyl-MU sulfation is involved in the control of Xyl-MU-initiated glycosaminoglycan biosynthesis.      

Time and concentration dependence of the action of cortisol on fibroblasts in vitro

The effect of cortisol on cultured fibroblasts from human skin were studied. After 0–84-h preincubations in the presence of cortisol the cells were labeled for 12 h with [³H]thymidine, [³H]proline or [³H]glucosamine and the radioactivity incorporated into DNA, collagen, total proteins, hyaluronic acid and sulphated glycosaminoglycans was determined.Cortisol (1 · 10^?5 M) caused a rapid, progressive decrease in the synthesis of hyaluronic acid when compared to the controls. Similarly, it decreased the synthesis of sulphated glycosaminoglycans and DNA, but this was seen first after 12- and 24-h preincubations, respectively. The synthesis of collagen and other proteins was significantly increased when the preincubation time was 0–24 h. This stimulation, however, turned to inhibition when an 84-h preincubation was used. It was found that 1 · 10^?7 M cortisol was the lowest concentration which caused the early inhibition in hyaluronate synthesis, while even 1 · 10^?8 M was sufficient after an 84-h preincubation. The syntheses of sulphated glucosaminoglycans and DNA were significantly inhibited by 1 · 10^?8 and 1 · 10^?7 M cortisol, after an 54-h preincubation, respectively. Thus, the studies of cortisol effects on fibroblast functions may result in quite variable conclusions unless the time sequence and the steroid concentration effects are taken into account.     

The disorder of hyaluronic acid metabolism in cultured skin fibroblasts derived from a patient with the Hurler syndrome

The metabolism of hyaluronic acid in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was examined. 1. An increased net incorporation of [(3)H]glucose into the hyaluronic acid fraction of the Hurler-syndrome cells occurred when compared with normal cells. 2. During a ;chase' period, approx. 35% of the radioactivity derived from glucose was lost from the hyaluronic acid fraction of the Hurler-syndrome cells, whereas the normal cells retained all their radioactivity. 3. Although the Hurler-syndrome cells contained a ninefold greater amount of hyaluronic acid than normal cells, simultaneous determination of the specific radioactivity derived from the label revealed a value for the Hurler-syndrome cells one-half that of normal cells. These results are taken to indicate that the Hurler cells synthesize hyaluronic acid de novo at a higher rate than do normal cells. 4. Exposure of Hurler-syndrome cultured fibroblasts to a crude urine corrective-factor preparation (Neufeld & Cantz, 1971), now known to contain alpha-l-iduronidase, the specific Hurler-syndrome corrective factor (Bach et al., 1972), decreased the hyaluronic acid content to near-normal values before any effect was observed on [(3)H]glucose incorporation into the hyaluronic acid fraction. 5. In addition, the hyaluronic acid content of the normal cells decreased after exposure to the corrective factor of urine. 6. The mobilization of hyaluronic acid in Hurler-syndrome and normal cells exposed to the crude corrective-factor preparation of urine caused a decrease in specific radioactivity in the ;corrected' Hurler-syndrome cells and an increase in specific radioactivity in the ;corrected' normal cells.     

Hyaluronic acid: separation and biological implications

Hyaluronic acid (hyaluronan) is a ubiquitous extracellular matrix component, and present at high concentrations in skin, joints and cornea. In the skin, it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes. Hyaluronic acid usually exists as a high molecular mass (600,000-1,000,000) and non-sulfated glycosaminoglycan composed of a disaccharide unit of [bond]3GlcNAc beta 1[bond]4GlcA beta 1[bond]. Hyaluronic acid has been widely used not only for osteoarthritis and ophthalmology but also for cosmetics for skin care. To examine the biological activities of hyaluronic acid, we have to accurately determine the quantity and molecular masses in biological samples. We review recent development in the analysis of hyaluronic acid having various molecular sizes using electrophoretic and chromatographic techniques. Recently, interactions between hyaluronic acid oligomers and hyaluronic acid-binding proteins have attracted the interest for understanding the biological functions. We show some interesting reports on biological interactions of hyaluronic acid and its oligomers with some proteins.     

Effects of wheat germ agglutinin and concanavalin A on the accumulation of glycosaminoglycans in pericellular matrix of human dermal fibroblasts. A comparison with insulin.

The effect of insulin, wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (ConA) on [3H]glucosamine incorporation into pericellular glycosaminoglycans (GAGs) was investigated in two lines of cultured human dermal fibroblasts. Insulin and WGA stimulated [3H]glucosamine incorporation into hyaluronic acid (HA) and heparan sulphate (HS) without any alteration of chondroitin sulphate (CS) and dermatan sulphate (DS) contents. ConA increased [3H]glucosamine incorporation into HS, CS and DS, but had no effect on [3Hglucosamine incorporation into HA. PNA affected neither the content, nor the composition of GAGs. In contrast to PNA, ConA and WGA stimulated glycolysis and demonstrated an evident antiproliferative effect on dermal fibroblasts. Thus, both the insulin-like action of WGA and ConA on cultured dermal fibroblasts and the differences between the effects of lectins on modulation of GAGs synthesis appear to be determined by their chemical structure.     

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

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.