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.     

Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

Biosynthesis of glycosaminoglycans by cultured mastocytoma cells

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [³⁵S]sulphate (and in some cases [6-³H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [³H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.     

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

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, Na(2) (35)SO(4) and d-[2-(3)H]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, [(35)S]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 [(3)H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased (35)S/(3)H ratio (nmol of [(35)S]sulphate incorporated/nmol of [(3)H]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 [(3)H]glucose. 3. After 34h exposure to a ;corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [(35)S]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.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

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.     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.     

Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1. When pig ear skin slices were cultured for 18h in the presence of 1μg of tunicamycin/ml the incorporation of d-[³H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5μg of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-¹⁴C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[³H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[³H]glucosamine but not of (U-¹⁴C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[³H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[³H]glucosamine and ³⁵SO₄²⁻ into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.     

Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor

The effect of mouse epidermal growth factor (mEGF) on the synthesis of glycosaminoglycans and glycoproteins by human fibroblasts has been studied. The addition of physiological concentrations (10^?9 M) of mEGF to quiescent cultures preincubated in the absence of serum was found to elicit an increased incorporation of ³H-glucosamine into the glycosaminoglycans and glycoproteins of both the cellular and extracellular fractions. Although the growth response to the factor, as measured by DNA replication, was minimal under these conditions as compared with the effect of serum, the mEGF-induced incorporation of glucosamine into these cellular constituents and into the extracellular glycoproteins was comparable to that elicited by serum shift-up. Serum, however, caused a significantly larger incorporation of glucoasmine into extracellular, acid-soluble glycosaminoglycans, which were shown to contain hyaluronic acid as the major component. As previously demonstrated, the growth response to mEGF can be enhanced several fold by an mEGF-binding arginine esterase, which is normally associated with the factor in vivo, and by ascorbate. The esterase was found to increase markedly the mEGF-induced incorporation of glucosamine into extracellular hyaluronic acid, while the addition of ascorbic acid did not significantly alter glucosamine incorporation.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

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.     

Glucose deficiency inhibits glycosaminoglycans synthesis in fibroblast cultures

We decided to study the effect of glucose deprivation on glycosaminoglycan (GAG) synthesis and degradation in fibroblast cultures, vitality of these cells and a correlation of these processes with the expression of oxygen/glucose-regulated proteins (ORP150/GRP170). The incorporation of [³H]-glucosamine into both newly synthesised hyaluronic acid and sulphated GAGs and [³⁵S]-sulphate into GAGs was used as an index of glycosaminoglycan synthesis. Quantitative evaluation of newly synthesised GAGs degradation was determined by pulse-chase experiments. We demonstrated that fibroblasts incubated in high glucose medium synthesised significant amounts of GAGs. Most of them were secreted into the culture medium. The shortage of glucose resulted in about 40% reduction in synthesis of GAGs, both those secreted into culture medium and remaining in the cell layer. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised glycosaminoglycans was protected against intracellular degradation. Proportionally less GAGs were degraded in cultures incubated in low glucose than in high glucose media. These phenomena were accompanied by an increase in the expression of chaperon – ORP150 in cultures growing in low glucose medium. We suggest that the increased expression of ORP150 is a factor which prolongs the cell vitality and protects glycosaminoglycans against intracellular degradation induced by glucose deprivation.     

Characterization of an alpha-glucan from chick embryo sternum whose synthesis is stimulated by L-3,5,3'-triiodothyronine and human serum.

Incorporation of [³H]glucose into macromolecular components of 12-day chick embryo sternum incubated in vitro was stimulated by both human serum and l-3,5,3′-triiodothyronine. Under all conditions, 65–70% of the radioactivity was incorporated into glycosaminoglycans. About 10% of the radioactivity was incorporated into a fraction separable by ion-exchange chromatography which was stimulated two- to sixfold by addition of 2–10 nm triiodothyronine and 5–20% ($\text{v}\text{v}$) human serum. Further characterization of this fraction by paper electrophoresis at pH 3.5 showed the presence of two components, one apparently anionic and one neutral. All of the increase in incorporation of [³H]glucose was into the former species. Acid hydrolysis of this material showed that it contained only glucose. Treatment with α-amylase released 78% of the label as maltotriose and maltose; digestion with crystalline β-amylase released 75% as maltose; and treatment with glucoamylase and α-amylase released 93% as glucose. There was no incorporation of any amino acid into this fraction, nor could any incorporation of [³²P]phosphate, [³⁵S]sulfate, [³H]uridine, or [³H]acetate be demonstrated. Mild acid hydrolysis (0.1 N HC1, 100 °C, 10–20 min) converted the material to a neutral species with a much lower molecular weight. The results indicate that chick embryo sternum contains a species of glycogen whose synthesis is stimulated by thyroid hormones and other serum factors.     

Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.     

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.     

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.     

伴刀豆蛋白A及其衍生物对培养软骨细胞蛋白多糖代谢的影响

观察了ＣｏｎＡ对培养软骨细胞ＰＧ合成代谢的影响。证实ＣｏｎＡ能够使培养的软骨细胞高分子硫酸化ＰＧ的合成增加３～４倍,其分子量、硫酸化部位和硫酸化程度与对照组相比无明显差异,是具有正常结构的软骨型ＰＧ。ＣｏｎＡ对低分子型ＰＧ的合成未见明显的影响。ＣｏｎＡ促进ＰＧ合成的作用可由ＭｅＭａｎ完全解除,比具有同样效应的激素、生长因子都强,并有明显的凝集素特异性。推测ＣｏｎＡ的作用可能与软骨细胞膜或细胞内的分化诱导因子的受体或软骨中存在的ＣｏｎＡ软骨细胞分化因子有关。     

Incorporation of [14C]glucosamine into synaptosomes in vitro

Abstract— Synaptosomes isolated from rat cerebral cortex by zonal centrifugation in-corporated radioactive glucosamine into macromolecules in vitro as glucosamine, galactosamine, N-acetylneuraminic acid, and glucuronic acid. The largest percentage of incorporated radioactivity was recovered in the particulate fraction in which radioactive carbohydrates were bound in covalent linkage requiring acid hydrolysis or enzymatic digestion for release. Less than 20 per cent of the particulate radioactivity represented incorporation into gangliosides. Some 20 per cent of the radioactivity was incorporated into proteins as glucosamine, identified in hydrolysates by paper chromatography and by the amino acid analyser. After incubation, radioactivity was demonstrable in the proteins as sialic acid by paper chromatography and specific enzymic digestion; and as glucuronic acid by chromatography, electrophoresis, and digestion with hyaluronidase. Incorporation of carbohydrate was stimulated by sodium and potassium at concentrations demonstrated to enhance incorporation of amino acids, and involved the macro-molecules of all subsynaptosomal fractions. Significant incorporation of radioactivity was found in the synaptic plasma membrane. The synthesis of glycoproteins was suggested by simultaneous incorporation of [¹⁴C]glucosamine and [³H]leucine into glycopeptides subsequently hydrolysed and subjected to polyacrylamide gel electrophoresis and two-dimensional paper chromatography and electrophoresis. Such studies demonstrated that amino acids and carbohydrates may be incorporated into glycoproteins of the synaptic membrane and suggest the possibility of local synthesis as well as modification of material brought to the nerve ending by axoplasmic flow.     

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.     

The effect of solubilized bone matrix fractions from different mammalian species on glycosaminoglycan synthesis by cultured fibroblasts

Human and bovine bone matrices were extracted with salt solutions of different composition and the extracts tested for stimulation of incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the hyaluronic acid and chondroitin sulfate of the cell pellet, the cell surface and the medium fractions of human synovial cells in culture. Stimulatory activity was extracted with a solution of 0.3 M EDTA in 2.5 M NaCl from bovine but not human bone. Subsequent extraction of the residues with 4 M guanidinium hydrochloride yielded activity from both matrices. A major stimulation of incorporation of radioactivity was observed in the cell surface fractions. Human synovial cells constitute a more sensitive assay system for the stimulatory activity than rabbit synovial cells.