Histochemical studies on pseudocysts in adenoid cystic carcinoma of the human salivary gland

Summary Pseudocysts are unique structures found in adenoid cystic carcinomata of human salivary glands. They were studied in 13 such cases by histochemical and immunohistochemical means. The pseudocysts contained an abundance of mucoid materials which reacted strongly with both Alcian Blue and dialysed iron ferrocyanide. The mucoid material was digested with chondroitinase ABC and heparitinase, but was resistant toStreptomyces hyaluronidase. The inner surfaces of the pseudocysts were strongly reactive for laminin, whereas the interface between the tumour cell nests and the outer stromal area was intensely reactive for fibronectin. Numerous fibronectin-reactive fibrils and blood coagulation factor XIII (F-XIII)-positive cells were distributed extensively in the outer stromal area. The F-XIII-positive cells were also found within some pseudocysts. The results obtained in the present study have shown that the pseudocysts represent a peculiar structure consisting of basement membrane components; laminin, fibronectin, heparan sulphate and chondroitin sulphate.     

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.     

Immunocytochemical Localisation of Glycosaminoglycan-Like Activity in the Mucus and Associated Tissues of Terrestrial Slug Species (Gastropoda: Pulmonata)

Indirect immunofluorescence assays were conducted on cryotome sections of four terrestrial slug species from three distinct phylogenetic groups, Arion ater (L.), Arion hortensis (Férussac), Tandonia (Milax) budapestensis (Hazay), and Deroceras reticulatum (Müller) using monoclonal antibodies for two glycosaminoglycans (GAGs), heparan sulphate, and chondroitin sulphate. Specific staining for a heparan sulphate-like component was demonstrated in the foot and tail regions of each species and was particularly intense in A. ater and A. hortensis, notably in the epidermis and associated mucus-like material, and in mucus-like material from the pedal gland region of the latter species. Subsequent studies with A. ater confirmed the presence of heparan-sulphate–like activity in the caudal gland duct region. No evidence of specific staining for chondroitin sulphate-like activity was found in any of the slug species. This work suggests that a specific GAG, or a group of closely related GAGs, is a common component of mucus in a range of slug species and of different types of mucus, including trail (pedal) mucus and the more viscous mucus produced by the caudal gland.     

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     

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.     

Proteoglycans mediate malaria sporozoite targeting to the liver

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.     

The effect of preganglionic stimulation on the incorporation of l-[U-14C]valine into the protein of the superior cervical ganglion of the guinea pig

The acid glycosaminoglycans were extracted from the skins of young rats less than 1 day post partum. The isolated products were fractionated by a cetylpyridinium chloride-cellulose column technique and identified by chemical analysis, electrophoretic mobility and susceptibility to testicular hyaluronidase digestion. Hyaluronic acid (56%) dermatan sulphate (15.6%) and chondroitin 6-sulphate (9.1%) were the major components, but chondroitin 4-sulphate, heparan sulphate and heparin were also present, together with two further fractions tentatively suggested to be a heparan sulphate-like fraction and a dermatan sulphate fraction, both of short chain length or low degree of sulphation.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS FROM BRAIN OF CHILDREN WITH PROTEIN-CALORIE MALNUTRITION

Abstract— The uronic acid containing glycosaminoglycans (GAGs) were isolated from the brains of 1-year-old and 4-year-old kwashiorkor children and characterised by constituent analyses. A marked reduction is the total GAG concentration of brain was noticed in both cases of kwashiorkor. In the 1-year-old kwashiorkor brain, hyaluronic acid is the most predominant GAG (73.5 per cent) whereas heparan sulphate, chondroitin sulphates and low sulphated chondroitin sulphate constituted less than 10 per cent. In the 4-year-old kwashiorkor brain, the proportion of hyaluronic acid was 27.5 per cent, low sulphated chondroitin sulphate 31.2 per cent, chondroitin sulphates 28.3 per cent and heparan sulphate 10 per cent. This marked reduction in the concentration as well as qualitative changes in GAG in protein-calorie malnutrition as compared to the normal is discussed in relation to brain function.     

Structural requirements for heparan sulphate self-association

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

X-ray-diffraction patterns from chondroitin 4-sulphate, dermatan sulphate and heparan sulphate

Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating beta-d-(1-->4) and alpha-d-(1-->4).     

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.     

Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans.

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ''matrix'', and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).     

Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.     

Synthesis of glycosaminoglycans by cloned bovine endothelial cells cultured on collagen gels

Sulphated glycosaminoglycans have been analysed in cloned bovine aortic endothelial cells cultured on collagen gels after incubation with [3H]glucosamine and Na2(35)SO4. Radioactive products were analysed in the culture medium, in sequential collagenase and trypsin extracts of the cell monolayer and the associated extracellular matrix, and in the remaining viable cells. Heparan sulphate and chondroitin sulphate were found in each compartment: the heparan sulphate had a low degree of sulphation (approximately 0.4 N-sulphate and 0.2 O-sulphate groups per disaccharide unit on average). In the nitrous acid scission products of heparan sulphate, O-sulphated substituents were confined to disaccharide and tetrasaccharide fragments, indicating that local regions of the chain (which might be susceptible to excission by the platelet endoglycosidase) are highly sulphated. Only minor structural differences in heparan sulphate were observed between the various compartments. In contrast the chondroitin sulphate found in the collagenase extract had a higher iduronic acid content than corresponding material in the trypsin extract and the culture medium, indicating that collagenase and trypsin may extract glycosaminoglycans from different regions of the extracellular and pericellular matrix.     

Inhibition by heparin-modulated antithrombin III of amidolysis catalysed by m beta-acrosin.

Purified m beta-acrosin catalysed amidolysis of several p-nitroanilides with C-terminal arginine residues. Antithrombin III inhibited amidolysis catalysed by the enzyme. This effect of antithrombin III was potentiated by heparin, and to a modest extent by heparan sulphate, cellulose sulphate, dextran sulphate and xylan sulphate. De-N-sulphated heparin, de-N-sulphated N-acetylated heparin, heparin of low relative molecular mass, chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid were ineffective.     

Ultrastructural localization and internalization of proteoglycan epitopes in a human non-Hodgkin (B) lymphoma

Summary In a human non-Hodgkin (B) lymphoma xenograft (HT-117) heparan sulphate (HS) proved to be the main cell surface glycosaminoglycan, in contrast to the chondroitin sulphate dominance in normal lymphoid cells. Using anti-proteoglycan (PG) antibodies and immunoelectronmicroscopy, two heparan sulphate proteoglycans (transferrin receptor (TfR) and fibroblast membrane type) and one chondroitin sulphate proteoglycan (articular cartilage type) molecule were co-localized as random clusters on the surface of these lymphoma cells. Double labelling revealed that during internalization, which occurred via endosomes avoiding the lysosomal system, the different proteoglycan (PG) antigens became separated. The TfR and fibroblast membrane type HSPG epitopes reappeared on plasmalemmal vesicles derived most probably from the multivesicular endosomes, representing a unique form of exocytosis. It is suggested that different cell membrane PGs are integrated into subunits of yet unknown function in these human non-Hodgkin (B) lymphoma cells.     

Macromolecular composition of the sarcolemma and endomysium in the rat

The macromolecular composition of sarcolemma and endomysium was studied by classical staining methods for glycosaminoglycans and using immunological techniques for proteins. Both proteoglycans and glycosaminoglycans (heparan sulphate, dermatan sulphate, chondroitin sulphate) could be detected in the sarcolemma. Type IV and type V collagen and laminin were found exclusively in the sarcolemma and endomysium. Type I and type III collagen as well as fibronectin were detected both in the endomysium and perimysium.     

D(+)catechin enhances heparan sulphate content in rat liver

Administration of (D+) catechin (100 mg/kg body wt) to rats resulted in an increase in the amount of total sulphated glycosaminoglycans (GAG) in liver. The increase was more pronounced in the case of heparan sulphate than chondroitin sulphate and dermatan sulphate. The liver slices prepared from catechin-treated rats showed a significant increase in the rate of incorporation of 35S-sulphate into GAG. Similarly there was a concentration-dependent increase in the rate of 35S-sulphate incorporation into GAG by normal liver slices in presence of catechin in vitro. Susceptibility to nitrous acid degradation and chondroitinase ABC digestion showed that more than 80% of the GAG labelled in vivo with 35S-sulphate, was heparan sulphate and about 10% chondroitin sulphate and dermatan sulphate. Gel filtration of the 35S-labelled material isolated from livers of normal and catechin-treated animals over sephacryl S-300 did not show any difference probably excluding the possibility of free GAG chains initiated on catechin or any of its metabolites in vivo. These results indicate that catechin stimulates the synthesis of sulphated GAG, particularly heparan sulphate in liver.