The presence of a novel extracellular hyaluronidase in squid cranial cartilage.

A new type of hyaluronidase was isolated from squid cranial cartilage. The enzyme seems to be localised extracellularly, since it is extracted from the tissue by 0.5 M sodium acetate, pH 7.0, in the presence of proteinase inhibitors. Degradation studies suggest that the enzyme belongs to the family of endoglycosidases generating oligosaccharides of rather large size. The best activity of the enzyme was observed at pH 7.0 and 37 degrees C and the optimum buffer for digestion was 0.15 M Tris acetate. It is inactive in sodium phosphate, morpholine acetate and HEPES buffers. The enzyme degrades aggrecan, hyaluronan, chondroitin sulphate and oversulphated chondroitin sulphate.

Diagnostic and classification value of metalloproteinases in squamous human laryngeal carcinoma.

Metalloproteinases (MMPs) are a class of enzymes largely involved in tumour progression and metastasis. At least twenty different enzymes are recognized that are also present under normal state of tissues. Their activity is regulated by their presence as proenzymes and by the concomitant presence of the respective tissue inhibitors (TIMPs). The present study describes the alterations of MMPs observed in human laryngeal carcinoma with respect to tumour classification and compares their activity in normal and cancerous tissues and biopsy specimens. Samples from five patients who underwent laryngectomy, from five biopsies and three from autopsies were used. The MMPs of normal and malignant human laryngeal cartilage and of biopsy specimens were identified immunochemically and by zymography using gelatin or casein as substrates. Healthy cartilage from autopsies was found to contain almost exclusively MMP-1, proMMP-2 and proMMP-9. Normal parts from laryngectomies contained, in addition, significant amounts of active MMP-2. The respective malignant parts contained both MMP-2 and -9 in increased amounts in their latent and active forms. Similar profile of MMPs was also identified in tissues surrounding affected cartilage. These alterations were found to be in good accordance with tumour stage and were also observed in biopsy samples. Thus, analysis of MMPs in biopsies can be used together with the clinicopathological parameters for the classification or early diagnosis of laryngeal tumours.

Advances in analysis of glycosaminoglycans: its application for the assessment of physiological and pathological states of connective tissues.

Glycosaminoglycans are a class of biological macromolecules found mainly in connective tissues as constituents of proteoglycans, covalently linked to their core protein. Hyaluronan is the only glycosaminoglycan present under its single form and possesses the ability to aggregate with the class of proteoglycans termed hyalectans. Proteoglycans are localised both at the extracellular and cellular (cell-surface and intracellular) levels and, via either their glycosaminoglycan chains or their core proteins participate in and regulate several cellular events and (patho)physiological processes. Advances in analytical separational techniques, including high-performance liquid chromatography, capillary electrophoresis and fluorophore assisted carbohydrate electrophoresis, make possible to examine alterations of glycosaminoglycans with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this review we present the chromatographic and electromigration procedures developed to analyse and characterise glycosaminoglycans. Moreover, a critical evaluation of the biological relevance of the results obtained by the developed methodology is discussed.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Keratan sulphate in cerebrum, cerebellum and brainstem of sheep brain.

Keratan sulphate was identified in sheep brain. We describe here the isolation and partial characterization of keratan sulphate from cerebrum, cerebellum and brainstem of young sheep brains. The galactosaminoglycan was isolated by using ion-exchange chromatography and gel filtration after exhaustive digestion with papain of the delipidated tissues, followed by alkaline borohydride degradation and chondroitinase ABC and heparinases I, II and III treatment. The material isolated by ion-exchange chromatography from each tissue was eluted as single but polydispersed peak from Sephadex G-75, with average molecular masses 8.4, 7.9 and 8.8 kDa for cerebrum, cerebellum and brainstem, respectively. Keratanase I and II totally degraded keratan sulphate from cerebrum and brainstem, but only partially that from cerebellum. The content of keratan sulphate was found to be about 215, 173 and 144 microg/g dry delipidated tissue for cerebrum, brainstem and cerebellum, respectively.

The interactions of cartilage proteoglycans with collagens are determined by their structures.

In the present work, the interaction of aggrecan, decorin and biglycan isolated from pig laryngeal cartilage and of the three squid cartilage proteoglycans with collagen type I and II was studied. The interaction was examined under conditions allowing the formation of collagen fibrils. It was found that biglycan interacted strongly with collagen type II and not with type I and the interaction seemed to proceed exclusively through its core proteins. Decorin interacted with collagen type I but not with type II. Aggrecan interacted very poorly with both collagen types. The two squid proteoglycans of large size, D1D1A and D1D2, interacted only with collagen type I through both glycosaminoglycans and core proteins. The third squid proteoglycan of small size, D1D1B, interacted poorly only with collagen type I. The results suggested that the interactions of cartilage proteoglycans with collagen were mainly due to the primary structure of both molecules, and would contribute to the maintenance of the integrity of the tissue. The biochemical significance of these interactions might be more critical in aged vertebrate cartilage, where loss of aggrecan and increase of the small proteoglycans was observed, a large proportion of which is found in the extracellular matrix free of glycosaminoglycan chains.

Determination and structural characterisation of dermatan sulfate in the presence of other galactosaminoglycans.

Chondroitin sulfate and dermatan sulfate are galactosaminoglycans that have similar size and charge density thus making difficult their separation and accurate determination from tissue preparations. A procedure was developed, which was based on the specific action of chondroitinase B, that allowed the determination of dermatan sulfate content in a mixture of chondroitin sulfate/dermatan sulfate, its molecular mass (Mr), and iduronic acid content and distribution throughout the chain. According to this procedure, the galactosaminoglycan sample was treated with chondroitinase B and its profile, upon gel chromatography on Sepharose CL-6B, was compared to that of the initial sample. The differences in uronic acid content of the fractions of the gel chromatographies were plotted and a secondary profile was constructed, which corresponded to the elution profile of intact dermatan sulfate in the sample. From this profile, the size distribution of dermatan sulfate was obtained and its Mr was calculated. In addition, the accurate content of dermatan sulfate in the sample was determined. The digest contained oligosaccharides of variable size that were separated on BioGel P-10. From the separated oligosaccharides the distribution of iduronic acid throughout the dermatan sulfate chains was determined. The procedure was applied to the determination and partial characterisation of dermatan sulfate from sheep nasal cartilage, in which it is reported for the first time that it contains a significant proportion of dermatan sulfate chains of low iduronic acid content.

A solid phase assay for the determination of heparan sulfate and its application to normal and cancerous human cartilage samples.

A sensitive and accurate quantitative assay for the measurement of minor amounts of chondroitin/dermatan sulfate and heparan sulfate that does not require specific apparatus or reagents is described. The assay involves labeling of chondroitin sulfate A following reaction of carboxyl groups with biotin hydrazide in the presence of carbodiimide. ELISA plate wells were coated with glutaraldehyde and then spermine was coupled to it via a Schiff's base bond. In such activated wells, the biotinylated molecules were readily bound and detected after the interaction with avidin-peroxidase conjugates and the subsequent enzymic assay. Chondroitin/dermatan sulfate and heparan sulfate competed this interaction in a linear manner. Disaccharides derived from chondroitin sulfate A did not act as competitors, while heparan sulfate disaccharides showed significant competition. From the competition, before and after digestion with either chondroitinase ABC or heparitinases, the amounts of chondroitin sulfate and heparan sulfate in a sample could be calculated. The assay was applied for the determination of sulfated glycosaminoglycans in normal and cancerous human laryngeal cartilage samples. By using this procedure, the accurate determination, especially, of heparan sulfate in a mixture of glycosaminoglycans was achieved, which otherwise would require the use of very expensive technology.

Polydispersity and heterogeneity of squid cranial cartilage proteoglycans as assessed by immunochemical methods and electron microscopy.

The three populations of squid cranial cartilage proteoglycans, D1D1A, D1D1B and D1D2 appeared to have a high degree of polydispersity. Gel electrophoresis and immunoblotting analysis showed that polydispersity was mainly due to the variable size of chondroitin sulphate E chains. This was further ascertained after rotary shadowing electron microscopy of proteoglycan core proteins and glycosaminoglycan side chains and statistical analysis of the sizes measured for both components. Enzymic treatment of the proteoglycan core proteins produced different peptides from each population, suggesting that the observed heterogeneity of the proteoglycans is due to their core proteins. Antibodies were raised in rabbits against all proteoglycans and enzyme-linked immunosorbent analysis of proteoglycan core proteins revealed that the proteoglycans, even heterogeneous, shared many common epitopes. Part of the common proteoglycan epitopes were found to be located in chondroitin sulphate E chains. Heterogeneity of squid proteoglycans was also investigated by studying their interactions with collagen and it was found that only the two populations of high molecular mass, D1D1A and D1D2, were able to interact with only collagen type I, the latter stronger than the former.

A solid-phase assay for quantitative analysis of sulfated glycosaminoglycans at the nanogram level. Application to tissue samples.

A sensitive and accurate solid-phase methodology for the quantitative analysis of glycosaminoglycans is described. Chondroitin-4-sulfate (CSA) was labelled with biotin hydrazide after the reaction of its carboxyl groups with it in the presence of carbodiimide. Polystyrene plates modified with sequential reaction with glutaraldehyde (GH) and spermine to possess amino groups were used to immobilize electrostatically the biotin labelled CSA. Exogenously added sulfated glycosaminoglycans (GAGS) [variously sulfated chondroitin sulfates and heparan sulfate (HS)] were found to compete to this immobilization in a concentration dependent mode, within a concentration range from 10 up to 300 ng/ml. Glycosaminoglycan-derived oligosaccharides competed to a degree similar to that of intact molecules. Hyaluronan (HA) and keratan sulfate (KS) did not compete the immobilization. The procedure was applied for the rapid and reproducible determination of the sulfated glycosaminoglycans in proteinase digests of small tissue samples or cell cultures with high sensitivity and accuracy.

Aggrecan immobilization onto polystyrene plates through electrostatic interactions with spermine.

A new procedure for the immobilization of proteoglycans and the core protein thereof via their carbohydrate chains onto enzyme-linked immunosorbent assay (ELISA) plate wells is presented. The aggrecan was immobilized via electrostatic interactions with spermine coupled to glutaraldehyde via Schiff's base, the latter being directly anchored onto ELISA wells. The amounts of aggrecan bound by this procedure measured immunochemically were 10-fold greater than those adsorbed by direct coating. The interaction of aggrecan and spermine may be inhibited by very small amounts of sulfated glycosaminoglycans or proteoglycans in a competitive manner, and therefore the system may be used for their quantitation. Bound aggrecan could react with link protein and therefore the system may be used for studying interactions of cartilage macromolecules. The method may also be used for direct quantitation of proteoglycans since the amounts adsorbed, in a given range of concentrations, are directly proportional to the amounts in solution.

Effect of proteoglycans on hydroxyapatite growth in vitro: the role of hyaluronan.

The effect of cartilage proteoglycans on HA seed crystal growth was studied using a system providing constant supersaturation with respect to HA. The monomers were much less effective than the aggregates in reducing the rate of HA growth, which correlates with their affinity for the HA crystals. Hyaluronan, which is a normal constituent of the proteoglycan aggregates, behaved as a strong inhibitor of HA seed crystal growth and had an affinity constant similar to that of proteoglycan aggregates. The results indicate that inhibition of HA seed crystal growth is mediated through the interaction of hyaluronan with HA crystal surface and that the proteoglycans add to the volume of the adsorbate causing steric hindrance.

Self-aggregation of squid cranial cartilage proteoglycans.

Squid cranial cartilage has been found to contain three different proteoglycan populations, two of which form aggregates (Vynios, D.H. and Tsiganos, C. P., Biochim. Biophys. Acta 1033: 139-147, 1990). The aggregation involves interaction of their protein cores as assessed by electron microscopy and biochemical data. Aggregating oligopeptides were isolated after mild trypsin digestion which inhibited self-aggregation of proteoglycans. The aggregation does not involve interaction of the side chains of polar amino acids and evidence is provided that it is mediated through hydrophobic interaction. It is enhanced upon concentration or incubation of the samples at 37 degrees C.

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern.

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.

Effect of low calcium on high-energy phosphates and sarcolemmal Na+/K(+)-ATPase in the infarcted-reperfused heart.

This study was undertaken to compare the effect of low to normal serum calcium on biochemical parameters in the myocardium of dogs subjected to 90 min of coronary artery ligation followed by 30 min reperfusion. The accumulation of calcium, the decrease of adenosine triphosphate (ATP) and creatine phosphate (CP) and the inhibition of sarcolemmal ouabain-sensitive Na+/K(+)-ATPase which are prominent findings in the ischemic-reperfused myocardium, were studied under normal and low serum Ca produced by normal and modified hemodialysis (HD). The results showed a lower accumulation of Ca (P less than 0.002) in the ligated-reperfused myocardium of dogs subjected to low-calcium HD. In the same group of animals ATP was protected to some extent while CP was completely preserved. This may indicate that during reperfusion with low Ca, restored ATP is further utilized for CP regeneration. The activity of Na+/K(+)-ATPase was within normal values in the ligated-reperfused myocardium of the low-calcium group. The significantly (P less than 0.001) negative correlation between tissue calcium concentration and Na+/K(+)-ATPase activity under various conditions examined, provided additional evidence that low calcium is a protective factor of the enzyme activity during ischemia and reperfusion.

Isolation and characterization of two glycoproteins from hyaline cartilage.

Two acidic glycoproteins of molecular mass 92 kDa and 56 kDa were purified from 4 M guanidine hydrochloride extracts of chick sternal cartilage, by density gradient centrifugation, ion-exchange chromatography, gel chromatography and SDS/PAGE. The glycoproteins differed in their amino acid and carbohydrate compositions. They were identified by the immunoblotting technique in extracts of chick articular cartilage from various sites and in extracts of cartilage from other species. The proteins are synthesized by the chondrocytes and show a partial cross-reactivity between their antisera.

Chondroitin proteoglycans from squid skin. Isolation, characterization and immunological studies.

Two populations of proteochondroitins were isolated from 4 M guanidine hydrochloride extracts of squid skin by a combination of ion exchange, gel chromatography and density gradient centrifugation. The proteoglycans, Mr 4.8 x 10(5) and 2.8 x 10(5), contained four and two chondroitin chains respectively and unusual oligosaccharides with uronic acid and sulphate groups, and had different amino acid and neutral sugar composition. The chondroitin chains isolated after alkaline borohydride treatment contained varying amounts of glucose, galactose, mannose, fucose and xylose, most likely as branches. Both proteoglycans were antigenic to the rabbit and showed considerable cross-reactivity as assessed by competition experiments using the ELISA technique. The proteoglycans reacted neither with exogenous hyaluronic acid nor with each other to form aggregates.

Squid proteoglycans: isolation and characterization of three populations from cranial cartilage.

Squid cranial cartilage is poor in proteoglycans. They were extracted by 2% SDS and purified by isopycnic centrifugation in the presence of detergent. According to their buoyant density and hydrodynamic size they were fractionated into three structurally different populations of Mr 1.3.10(6), 0.6.10(6) and 1.0.10(6). The proteoglycans of each population differ in the number of oversulphated chondroitin sulphate chains, ranging from two to five, in the number and size of uronic acid and sulphate containing oligosaccharides and in the size of their core protein. The majority, if not all, of the oligosaccharides are linked to the protein via an O-glycosidic bond involving galactosamine and most likely xylose. The chondroitin sulphate chains are segregated on a small peptide segment of the molecule which also contains a large proportion of the oligosaccharides. The proteoglycans have no tendency to interact with hyaluronate.

Extraction and fractionation of proteoglycans from squid skin.

The extractability of squid skin proteoglycans with solutions of varying concentrations of guanidine-HCl, urea and SDS was studied; 4 M guanidine-HCl, being the best extractant, removed 95% of the tissue proteoglycans (glycosaminoglycan uronic acid). The proteoglycans in the 4 M guanidine-HCl extract were fractionated by repeated ion exchange and gel chromatography on Sepharose CL-4B to give three main populations, all being present in about equal proportions. Two populations (Kd 0.34 and 0.56) contained only chondroitin (proteochondroitin) and the other (Kd 0.50) only oversulphated chondroitin sulphate (oversulphated proteochondroitin sulphate). Two minor populations, one containing chondroitin and chondroitin sulphate and the other chondroitin sulphate and oversulphated chondroitin sulphate, were also identified.

Rooster comb hyaluronate-protein, a non-covalently linked complex.

Hyaluronate from rooster comb was isolated by ion-exchange chromatography on DEAE-cellulose from tissue extracts and papain digests. The preparations were labelled with [14C]acetic anhydride and subjected to CsCl-density-gradient centrifugation in 4 M-guanidinium chloride in the presence and absence of 4% ZwittergentTM 3-12. A radioactive protein fraction was separated from the hyaluronate when the zwitterionic detergent was also present. The protein could also be separated from the glycosaminoglycan by chromatography on Sepharose CL-6B eluted with the same solvent mixture. The protein fraction contained three protein bands of Mr 15,000-17,000 as assessed by polyacrylamide-gel electrophoresis in 0.1% SDS, and seemed to lack lysozyme activity. No evidence of other protein or amino acid(s) covalently linked with the hyaluronate was obtained. The hyaluronate-protein complex may be re-formed upon mixing the components, the extent of its formation depending on the conditions used. The results show that, as in chondrosarcoma [Mason, d'Arville, Kimura & Hascall (1982) Biochem. J. 207, 445-457] and teratocarcinoma cells [Prehm (1983) Biochem. J. 211, 191-198] the rooster comb hyaluronate also is not linked covalently to a core protein.