Alterations in human gingival glycosaminoglycan pattern in inflammation and in phenytoin induced overgrowth

Glycosaminoglycans were extracted from normal, inflamed and phenytoin induced overgrowth of human gingival tissue by proteolysis and alcohol precipitation. Extracts were run in a Dowex-1 column and the fractions were treated with mucopolysaccharidases. Cellulose acetate electrophoresis was carried out with or without enzyme digestion for identification of individual glycosaminoglycans. Glycosaminoglycans were found to be decreased in inflammation but were observed to increase in the overgrowth. Hyaluronic acid was found to be increased in both the pathological conditions. Dermatan sulphate, chondroitin sulphate and heparan sulphate were observed to be decreased in inflammation. In overgrowth, dermatan sulphate and chondroitin sulphate were found to increase while the presence of heparan sulphate was not significant. The changes in the pattern of individual glycosaminoglycan in the two varied conditions are discussed.Abbreviations GAG glycosaminoglycan - MPS mucopolysaccharide - DS dermatan sulphate - HS heparan sulphate - CS chondroitin sulphate - HA hyaluronic acid - KS keratan sulphate     

Characterization of glycosaminoglycans in urine from patients with nephrotic syndrome and control subjects, and their effects on lipoprotein in lipase

Previously we found that the α₁-acid glycoprotein fraction from urine of patients with the nephrotic syndrome stimulated the lipoprotein lipase reaction in vivo and in vitro. The activator was separated from the α₁-acid glycoprotein and identified as a glycosaminoglycan. The studies reported here were undertaken to characterize and quantify the glycosaminoglycans contained in urine of patients with the nephrotic syndrome and to compare these to the glycosaminoglycans in urine of control subjects. We found that free low molecular weight glycosaminoglycans, heparan sulfate and chondroitin 4-sulfate, are excreted in both patients with the nephrotic syndrome and controls, however, patients with the nephrotic syndrome excreted much less of both glycosaminoglycans. The free form of heparan sulfate was found to be the activator which stimulated the lipoprotein lipase reaction in vitro in the presence of apolipoprotein C_II. In addition, the urine from patients with the nephrotic syndrome contained a protein-glycosaminoglycan complex which was absent in control urine. Glycosaminoglycans in the complex could be released by papain digestion or by trichloroacetic acid. Our evidence indicates that this glycosaminoglycan fraction is a low charge form of chondroitin sulfate.     

Evidence for the existence of a multichain proteoglycan of heparan sulphate

1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.     

The characterization of a protein–polysaccharide isolated from Kurloff cells of the guinea pig

Kurloff cells of guinea pigs increase in number and accumulate in the spleen on oestrogen treatment. Because they contain metachromatic inclusions and are considered to be lymphocytes they were examined as a possible model for mucopolysaccharidoses like Hurler's syndrome, where some lymphocytes are also metachromatic. Oestrogen treatment produced a large increase in a glycosaminoglycan resembling chondroitin 4-sulphate in chemical analysis, chromatographic behaviour and i.r. spectrum but with an additional strong band at 805cm(-1). Material isolated without proteolysis behaved on gel chromatography as a multiple-chain protein-polysaccharide whose molecular size was decreased by proteolysis. It contained xylose and galactose in molar proportions with serine, compatible with the presence of the same linkage region as in cartilage chondroitin 4-sulphate proteins and which likewise underwent alkaline beta-elimination. Kurloff glycosaminoglycan chains were significantly longer than chondroitin sulphate chains of cartilage protein-polysaccharides as assessed by gel chromatography and the molar ratios of galactosamine to xylose or to serine. Kurloff cells thus contain intact rather than partially degraded protein-polysaccharide and hence are not analogous to Hurler cells, and their electron micrographs were also different. The purified Kurloff protein-polysaccharide and glycosaminoglycan isolated here has been shown by Marshall, Swettenham, Vernon-Roberts & Revell (1970) to be toxic specifically to macrophages at extremely low concentrations in vitro, unlike chondroitin sulphate of protein-polysaccharides from cartilage. The toxic constituent may account for the i.r.-absorption band at 805cm(-1). Although active incorporation of [(35)S]sulphate occurs at early stages of Kurloff-cell induction (Marshall et al. 1970), the fully developed Kurloff cell studied here showed very low incorporation in vitro and in vivo, suggesting that the inclusions are specialized for the storage of the toxic material.     

Characterization of glycosaminoglycans from normal and fluoride treated rabbit iliac crest

Glycosaminoglycans from rabbit iliac crest was isolated and characterised. Glycosaminoglycans isolated from iliac crest reveals the presence of chondroitin sulphate A, chondroitin sulphate C and hyaluronic acid. However, glycosaminoglycans isolated from fluoride treated rabbit iliac crest shows the presence of dermatan sulphate (or chondroitin sulphate B) in addition to the above mentioned components. Significance of the appearance of dermatan sulphate in response to fluoride treatment is discussed.     

Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.     

Proteoglycan Distribution Pattern During Aging in the Nematode Caenorhabditis elegans: An Ultrastructural Histochemical Study

Glycosaminoglycans are important constituents of the extracellular matrix of vertebrates, where distinct changes in their distribution pattern occur during aging. However, little is known about their changes in the nematode Caenorhabditis elegans, which ages extremely rapidly compared to mammals.The presence of glycosaminoglycans was analysed in cross-sections of all organs of the nematode, in three different age groups (60, 144, 228h), using the electron-dense dye Cuprolinic Blue in conjunction with the critical electrolyte concentration method and specific glycosaminoglycan degrading enzymes. The nematodes (strain DH 26) were grown at 25.5°C.The results indicate the presence of an organ-specific distribution pattern.Chondroitin-4-sulphate and/or chondroitin-6-sulphate are present in the epicuticula. Chondroitin-4-sulphate and/or chondroitin-6-sulphate and dermatan sulphate are detected in the mesocuticula. If stained by conventional methods the mesocuticula shows an empty fissure, which is filled by chondroitin sulphates and dermatan sulphate as shown by Cuprolinic Blue staining and enzymes. Heparan sulphate is found in the terminal web of intestinal cells while dermatan sulphate is revealed in the central cores of microvilli. An unknown polyanion staining at high electrolyte concentrations is observed in the gonads. Age-related changes do not impair the composition of the glycosaminoglycan fraction.In conclusion an unexpected highly differentiated pattern of glycosaminoglycans with high stability during aging exists.     

Fine structural and ultracytochemical studies on the lymphocytes in three types of genetic mucopolysaccharidoses

Lymphocytes from 6 patients with 3 types of genetic mucopolysaccharidoses (Hurler's syndrome, Hunter's syndrome and Morquio's syndrome) contained numberous vacuoles in their cytoplasm. The size of the vacuoles ranged from approximately 300 nm to 750 nm. The percentage of the lymphocytes with vacuoles varied from 10% to 38%. The vacuoles showed acid phosphatase activity, which indicated their lysosommal nature. Staining with dialyzed iron solution usually localized acid mucosubstance in the peripheral region of these vacuoles after glutaraldehyde fixation. Ferritin and horseradish peroxidase were observed in the vacuoles after incubation of the patient's lymphocytes with these tracers. This finding indicates the participation of endocytosis in the formation of these vacuoles.     

The nature of the non-ultrafilterable glycosaminoglycans of normal human urine

1. The non-ultrafilterable acidic glycosaminoglycans from pooled urine of normal men, aged about 20, were isolated and characterized. The isolation procedure included digestion with sialidase and pronase, and fractionation by stepwise elution from an ECTEOLA-cellulose column. The glycosaminoglycans in each fraction were separated from each other by preparative electrophoresis in sodium barbital buffer and in barium acetate. 2. Approximate relative amounts of the different glycosaminoglycans were: chondroitin sulphate 60%, chondroitin 2%, hyaluronic acid 4%, dermatan sulphate 1%, heparan sulphate 15% and keratan sulphate 18%. Chondroitin sulphate-dermatan sulphate hybrids seemed to occur in trace amounts. 3. Chondroitin sulphate, heparan sulphate and keratan sulphate were heterogeneous with respect to degree of sulphation. Two distinct groups of chondroitin sulphate fractions were found, with sulphate/hexosamine molar ratios of about 0.5 and 1 respectively. The sulphate/hexosamine molar ratios in the heparan sulphate fractions varied from 0.5 to 0.9; the N-sulphate/hexosamine ratio was about 0.5 in all fractions. The sulphate/hexosamine molar ratios in the keratan sulphate fractions varied from 0.2 to 0.7.     

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.     

Studies on the in vitro incubation procedure for [35S]sulphate labelling of articular cartilage glycosaminoglycans

Articular cartilage from cow and calf femoral condyles was incubated in Tyrodes solution containing [35S]sulphate for different periods up to 80 min. Glycosaminoglycans from the cartilage tissue and incubation medium were fractionated on Cetylpyridinium chloride and ECTEOLA cellulose microcolumns. The incorporation of [35S]sulphate into all individual fractions of chondroitin sulphate and keratan sulphate was found to be linear from 20 to 80 min incubation time. As a rule the total specific activities of keratan sulphate and chondroitin sulphate were similar for both calves and cows. The proteoglycan material recovered from the medium amounted to about 1% of the tissue dry weight and was found to have a higher chondroitin sulphate: keratan sulphate ratio than the corresponding cartilage tissue for both calf and cow. The solubility profiles for the newly synthesised glycosaminoglycans, obtained from determination of the radioactivity in the individual fractions, were compared with those of glycosaminoglycans already present. These curves indicated that newly synthesised chondroitin sulphate had a higher average molecular size than that present in the tissue whereas the newly synthesised keratan sulphate had a smaller average molecular size. These newly synthesised components were also detected in the proteoglycans recovered from the incubation medium.     

Studies on the in vitro incubation procedure for [35]sulphate labelling of articular cartilage glycosaminoglycans

Articular cartilage from cow and calf femoral condyles was incubated in Tyrodes solution containing [³⁵S]sulphate for different periods up to 80 min. Glycosaminoglycans from the cartilage tissue and incubation medium were fractionated on Cetylpyridinium chloride and ECTEOLA cellulose microcolumns.The incorporation of [³⁵S]sulphate into all individual fractions of chondroitin sulphate and keratan sulphate was found to be linear from 20 to 80 min incubation time. As a rule the total specific activities of keratan sulphate and chondroitin sulphate were similar for both calves and cows.The proteoglycan material recovered from the medium amounted to about 1% of the tissue dry weight and was found to have a higher chondroitin sulphate: keratan sulphate ratio than the corresponding cartilage tissue for both calf and cow.The solubility profiles for the newly synthesised glycosaminoglycans, obtained from determination of the radioactivity in the individual fractions, were compared with those of glycosaminoglycans already present. These curves indicated that newly synthesised chondroitin sulphate had a higher average molecular size than that present in the tissue whereas the newly synthesised keratan sulphate had a smaller average molecular size. These newly synthesised components were also detected in the proteoglycans recovered from the incubation medium.     

Sequential distribution of keratan sulphate and chondroitin sulphate epitopes during ameloblast differentiation

Proteoglycans are complex macromolecules containing one or more glycosaminoglycan chains and exhibiting a variety of biological functions in connective tissues. The aim of the present study was to immunolocalize the distribution of keratan sulphate and chondroitin sulphate epitopes during initial enamel formation in order to study temporo-spatial expression patterns of these macromolecules. Third molars of four-months-old pigs were used for immunolocalization of keratan sulphate and chondroitin sulphate epitopes in the developing enamel layer. Tooth organs were prepared for paraffin sections in order to perform indirect immunohistochemistry. The results demonstrated a mutually exclusive positioning between these two epitopes. Keratan sulphate epitopes were observed in pre-secretory pre-ameloblasts and adjacent stratum intermedium while chondroitin sulphate epitopes were demonstrated in secretory ameloblasts and adjacent stratum intermedium. Our findings suggest that proteoglycans containing glycosaminoglycan chains may play a regulatory role during enamel mineralization.     

The glycosaminoglycans of pig colonic wall connective tissue

Colon wall from pig, stripped of most of the mucosal layer to leave material largely composed of muscle, basement membrane, and extracellular matrix, was subjected to procedures for isolation of glycosaminoglycans. A total ethanol precipitate from a papain digest was fractionated by selective ethanol precipitation in the presence of Ca²⁺. Glycosaminoglycan fractions, freed proteolytically from a high molecular weight glycoprotein component, were further purified by Sepharose CL-6B gel-filtration or DE-52 anion-exchange chromatography. Glycosaminoglycans were identified by chemical composition, ¹³C-NMR spectroscopy and response to chondroitinase and nitrous acid degradations. The content of glycosaminoglycan in the tissue is low (0.05% dry weight) being comprised of dermatan sulphate (38%), heparin (34%), heparan sulphate (18%) and chondroitin sulphates (10%) as a percentage of total glycosaminoglycan content. Hyaluronic acid and keratan sulphate have not been detected. The composition is generally typical of a high muscle content tissue.     

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.     

Distribution of proteoglycans and hyaluronic acid in transverse sections of bovine thoracic aorta

Summary The distribution of hyaluronic acid and proteoglycans in bovine thoracic aorta was studied by Alcian Blue staining of frozen tissue sections under controlled electrolyte conditions with and without prior enzymic digestion. Some sections were digested with chondroitinase ABC, testicular hyaluronidase or bacterial collagenase and subsequent staining permitted conclusions to be drawn about the distribution of specific glycosaminoglycans within the tissue. The total glycosaminoglycan content was maximal in the intima and decreased across the arterial wall to the outermost adventitial layer. The content of proteoglycan containing chondroitin sulphate and/or dermatan sulphate chains paralleled this distribution. However, other glycosaminoglycans also contributed significantly to staining, although there was no evidence for any appreciable concentration of heparin or highly sulphated heparan sulphate.Several experiments indicated that proteoglycan containing chondroitin sulphate and/or dermatan sulphate was associated with elastic laminae which were often seen stained along their periphery. Hyaluronic acid was present at significant concentrations in all locations of the aorta and there was evidence for a similar distribution of heparan sulphate which was possibly also present at a high concentration in the endothelium. Staining of sections after treatment with 4m guanidinium chloride confirmed that this extractant removed most of the proteoglycan from the tissue section.     

Glycosaminoglycans in the bronchial mucus of patients with chronic bronchitis and mucoid impaction of the bronchus

Glycosaminoglycans were isolated from mucus of patients with chronic bronchitis and mucoid impaction of the bronchus, whose contents were approximately 56 mumoles and 80 mumoles of hexosamine per g of dry weight of mucus respectively. Electrophoretic and chemical characterization and enzymatic susceptibility demonstrated that the glycosaminoglycans in mucus from both groups of the patients contained hyaluronic acid as the main constituent, with undersulphated chondroitin as a minor component. In addition, in mucus from the patient with mucoid impaction of the bronchus chondroitin sulphate and heparan sulphate or heparan sulphate-like substance were identified.     

The biosynthesis in vitro of keratan sulphate in bovine cornea

1. Bovine corneas were incubated in vitro with [U-(14)C]glucose. 2. The glycosaminoglycans of corneal stroma were isolated and fractionated on cetylpyridinium chloride-cellulose columns. The major components were keratan sulphate (71%), chondroitin 4-sulphate (17%) and chondroitin 6-sulphate (4%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan biosynthesis of corneal stroma were separated on Dowex 1 (formate form) and the tissue content and cellular concentrations were determined. 4. The rates of synthesis of the intermediates of glycosaminoglycan biosynthesis in corneal stroma were determined. 5. The incorporation of radioactivity from [U-(14)C]glucose into the uronic acid and hexosamine components of the glycosaminoglycans present in corneal stroma were measured and the turnover rates of these polymers were calculated. It was found that keratan sulphate was turning over in about 723h and chondroitin 6-sulphate in 251h.     

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.     

The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-(14)C]glucose and [(35)S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride-cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-(14)C]glucose were found to be 0.79+/-0.16 and 3.2+/-0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-(14)C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [(35)S]sulphate incorporation and were found to be in good agreement with those obtained from [U-(14)C]glucose labelling.