Glycosaminoglycan synthesis by the early embryonic chick heart

Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of ³H-glucosamine-derived labeled UDP-N-acetyl-d-glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. ³H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium

The primary corneal stroma is produced by the overlying epithelium. The endothelium appears between 4 and 5 days, fibroblasts at 6 days, and at 12 days the epithelium stratifies. We investigated the synthesis of glycosaminoglycan (GAG) by the epithelium during this developmentally significant period. The sulfated GAG synthesized by isolated 4–6-day-old corneal epithelia during the first 24 hr in vitro are entirely accountable for as chondroitin sulfates and heparan sulfates. Nearly 50% of the total sulfated GAG synthesized by epithelia on Millipore filters is lost to the medium, but only 30–40% is lost when frozen killed lens capsule or stroma is the substratum. Retention of isotope by the tissue is correlated with visible matrix polymerization. The relative amount of heparan sulfate synthesized by the developing epithelium 24 hr in vitro decreases from about 50% of the total sulfated GAG for 4-day-old epithelium to 12% for 12-day-old epithelium. A similar decrease in heparan sulfate synthesis occurs with time in culture. The relative amount of GAG identified as chondroitin sulfate and heparan sulfate is the same when ³H-glucosamine is used to label GAG as when ³⁵SO₄ is used. We conclude that the corneal epithelium produces only sulfated polysaccharides. Since hyaluronate is synthesized by whole 5-day-old corneas, it must be the product of the endothelium.     

Composition and degree of sulfation of glycosaminoglycans from tissues of different animal species: heterogeneity and tissue specificity of heparan sulfates

The composition and degree of sulfation of glycosaminoglycans (GAG) in proteoglycans from various animal tissues were studied. It was shown that sulfated GAG contain chondroitin sulfates AC and B as well as heparan sulfates. The bulk of GAG in the majority of tissues under study is represented by 2-3 types of heparan sulfate molecules differing in the degree of sulfation. According to the degree of sulfation heparan sulfates from all tissues studied can be classified into three groups. Homologous tissues of various animal species are characterized by a similar composition and the degree of sulfation The data obtained are discussed in terms of the feasible role of proteoheparan sulfates in specific cell-to-cell interactions.     

Glycosaminoglycans vary in accumulation along the neuraxis during spinal neurulation in the mouse embryo

We have utilized the method of whole embryo culture for metabolic labeling of mouse embryos with [3H]glucosamine during closure of neural folds at the posterior neuropore (27- to 29-somite stage). Accumulations of newly synthesized glycopeptides, lactosaminoglycans, hyaluronate, and sulfated glycosaminoglycans (GAG) were assessed by ion-exchange chromatography of glycoconjugates isolated from labeled embryos. Accumulation of hyaluronate and sulfated GAG was greatest in the posterior neuropore and decreased progressively toward the hindbrain where neurulation was already complete. Hyaluronate comprised a progressively smaller proportion of total newly synthesized glycoconjugate from the posterior neuropore toward the cranial region and glycopeptides showed the opposite trend. Sulfated GAG and lactosaminoglycans showed no consistent differences in relative abundance along the neuraxis. Autoradiographic analysis of newly synthesized glycoconjugates revealed especially heavy incorporation into developing basement membranes, beneath the neuroepithelium and around the notochord, in the posterior neuropore and recently closed neural tube regions, but not at more cranial levels of the neuraxis. Predigestion of sections with a specific hyaluronidase showed a significant quantity of this glycoconjugate to be hyaluronate. These results are consistent with a role for neuroepithelial and notochordal basement membrane hyaluronate in spinal neurulation.     

Binding of hyaluronate to the surface of cultured cells

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.     

Glycosaminoglycans in the vitreous body of patients with retinal detachment.

The variation and changes of glycosaminoglycans in human vitreous body from patients with retinal detachment were studied. The isolated glycosaminoglycans from normal vitreous were identified as hyaluronate, which is the main component (92%) and chondroitin sulphate (8%). In contrast, in pathologic samples up to 18% of total glycosaminoglycans were identified as chondroitin.sulphate. In addition, in pathologic vitreous two fractions of glycosaminoglycans about 10% were identified as undersulphated chondroitin and heparan sulphate. The hydrodynamic size of hyaluronate differs between normal and pathologic samples. In samples from the patients with detached retinas the hyaluronic acid was of small hydrodynamic size.     

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.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Change in the synthesis of glycosaminoglycans by fibrotic vitreous induced by erythrocytes

Vitreous fibrosis was induced in rabbit eyes by intravitreal injection of erythrocytes. The fibrotic vitreous removed from experimental animals were then incubated with [³H]glucosamine at 37°C for 24 h. The newly synthesized ³H-labeled glycosaminoglycans were isolated by 4 M guanidium hydrochloride extraction followed by pronase digestion. The ³H-labeled glycosaminoglycans were then characterized by gel filtration column chromatography and by specific enzymatic degradation, i.e., hyaluronidase, chondroitinase AC, and/or chondroitinase ABC. The disaccharides derived from chondroitinase ABC degradation were identified by thin-layer chromatography. We previously demonstrated that 91% of the total glycosaminoglycan synthesized by normal vitreous was hyaluronic acid. Our present results indicate that in the fibrotic vitreous, the synthesis of hyaluronic acid was decreased to 26%, whereas the synthesis of chondroitin sulfate increased to 59% of the total newly synthesized glycosaminoglycans. These results suggest that cells present in fibrotic vitreous resemble fibroblasts with respect to their activities in glycosaminoglycans synthesis.     

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.     

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Incorporation of [³⁵S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N-sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N-sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.     

Proteoglycans of alveolar bone of diabetic and non-diabetic mice: a histochemical study

The effect of diabetes mellitus on the interdental alveolar bone has been long debated. The present study reported the distribution of glycosaminoglycans (GAG) in normal and diabetic alveolar bone using histochemical techniques. Animals were rendered diabetic and killed at 2, 4, 6 and 9 weeks after injections. Tissues were stained with Alcian blue 8GX dye (pH 2.5) to demonstrate GAG and the intensity of the staining reactions compared with age-matched controls. During the experiment, weights of control animals did not change significantly; weights of diabetic animals were significantly less than initial weights from 0-6 weeks (p less than 0.001), but became nearly equal by 9 weeks. Staining intensity of diabetic bone demonstrated initial decrease (0-4 weeks) followed by a marked increase (4-9 weeks) suggesting an early decline in bone GAG levels followed by increased bone GAG levels as compared to age-matched control and initial levels. Bone GAG levels were significantly different between diabetics and age-matched controls at 2 (p less than 0.005) 4 (p less than 0.001), 6 (p less than 0.001) and 9 (p less than 0.001) weeks after streptozotocin injections. Digestion with chondroitinase AC, ABC and streptomyces hyaluronidase suggested significant differences between control and diabetic bone matrix in the levels of chondroitin 4 and 6 sulfates (p less than 0.05) and hyaluronic acid (p less than 0.001) but not dermatan sulfate. In control and diabetic bone, chondroitin sulfates were located within the bone matrix, dermatan sulfate within bone matrix and Sharpey fiber bundles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and turnover of glycosaminoglycans in rat kidneys.

The turnover of sulfate label in crude glycosaminoglycan fractions from rat kidney cortex, medulla, and papilla has been determined. Heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronate have been separated electrophoretically and their specific activities determined after injection of labeled sulfate or glucose. The half-lives of the sulfated glycosaminoglycans are within the ranges found for other organs and tissues, but hyaluornate has a somewhat faster turnover in the kidney than elsewhere.     

Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body

Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous body by stepwise fractionation on AG-1X2(Cl^?) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1 : 2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.     

Heparin and heparan sulfate delimit nephron formation in fetal metanephric kidneys

Formation of nephrons from primitive mesenchyme in fetal kidneys is induced by ureteric buds. Nephron induction is closely coordinated with branching morphogenesis of the ureteric bud. Having previously shown that branching of the primitive ureter is associated with de novo synthesis of chondroitin sulfate proteoglycan and release of free heparan sulfate glycosaminoglycan chains, we asked whether glycosaminoglycans influence nephron development. Fetal mouse kidneys were incubated in organ cultures containing heparan sulfate, heparin, chondroitin sulfate, or hyaluronate. After 48 hr the number of nephrons at each developmental stage was enumerated by light microscopic analysis of serial tissue sections. Kidneys incubated in heparin or in heparan sulfate contained up to 10-fold fewer nephrons than did kidneys incubated in control conditions or in chondroitin sulfate or hyaluronic acid. Maturation of nephrons, however, was unaffected. Inhibition of nephron development was associated with binding of labeled heparin to primitive mesenchyme and altered tissue distribution of fibronectin. Branching morphogenesis was impaired in kidneys exposed to heparin but not to heparan sulfate or to de-N-sulfated, N-acetylated heparin. The capacity of glycosaminoglycans to inhibit nephron formation depended on sugar composition and O-sulfation but not GAG chain size or charge density. Thus, heparan sulfate may have the capacity to specifically control formation of nephrons in fetal metanephric kidneys in vitro.     

Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100°C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing ³⁵S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution in necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.     

Comparative characteristics of the composition and degree of sulfation of glycosaminoglycans in resting and actively proliferating tissues

A comparative study of the composition and degree of sulfation of glycosaminoglycans (GAG) in proteoglycans isolated from normal animal tissues and from actively proliferating embryonic, tumour and regenerating tissues was carried out. Significant differences in the ratios of various types of sulfated GAG were revealed. The relative content of chondroitin sulfate AC in actively proliferating tissues was considerably increased. It was found that all types of sulfated GAG in actively proliferating tissues, with the exception of regenerating tissue, are characterized by a lower degree of sulfation as compared to GAG from resting tissues. The experimental results are discussed in terms of the role of proteoglycans in the organization of extracellular matrix and in control of cell proliferation.     

Glycosaminoglycans in Embryonic Mouse Teeth and the Dissociated Dental Constituents

Abstract. The nature, amounts, and distribution of glycos-aminoglycans (GAG) before and during odontoblast terminal differentiation were studied. GAG have been isolated from intact mouse tooth germs and from dissociated dental epithelia and dental papillae after labeling with [³H] glucos-amine or ³⁵SO₄²⁻ as precursor. The kinds and relative amounts of ³H-labeled GAG were analyzed by chromatography on a DEAE-cellulose column and cellulose thin-layer sheets. The amounts of individual GAG relative to total GAG were determined from the elution profiles, whereas their nature was identified by the selective removal of chromatographic peaks after enzymatic or chemical degradation. We found hyaluronate and probably a minute quantity of heparan sulfate in the dental epithelium, while hyaluronate, heparan sulfate, and chondroitin sulfate were the main types of GAG in the dental papilla. The chondroitin sulfate recovered was further fractionated by cellulose thin-layer chromatography into two isomers, namely chondroitin-4-sulfate (the major component) and chondroitin-6-sulfate. Changes in the elution profile from DEAE-cellulose chromatography of tooth GAG extracted from different developmental stages suggest that modifications of GAG occur during odontogenesis. Alcian blue staining localized large amounts of hyaluronate and sulfated GAG along the epithelio-mesenchymal junction. Tissue specificity and changing patterns of GAG were demonstrated during odontogenesis.