In situ interaction of cartilage proteoglycans with matrix proteins.

The interaction of proteoglycans with other matrix proteins via thiol-disulphide interchange was explored. Chick sternal cartilage was extracted with 4 M guanidine hydrochloride in the presence and absence of N-ethylmaleimide and the proteoglycans from the centrifugation A2 fractions were isolated. Those from extracts without N-ethylmaleimide were linked with reducible bonds with 10-15 proteins-glycoproteins including the link proteins, the 148 kDa and 36 kDa proteins. The same was observed with extracts of pig laryngeal and sheep nasal cartilage. The linked proteoglycans from sheep amounted to 2-3% of the extractable uronic acid and belonged to two populations. The major fraction was included by Sepharose 6B (Mr 110 000) had twice as long chondroitin sulphate chains, higher 4-sulphated residues and a high content of aspartic acid and leucine-rich protein. The larger proteoglycans had a size and composition similar to those of aggregating proteoglycans.

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.

Cartilage keratan sulphate: changes in chain length with ageing.

Keratan sulphate from sheep nasal cartilage of five different ages was isolated by a combination of methods. The mean length of the chains progressively increased with ageing, as assessed by the molar ratio of glucosamine to galactosamine or galactosaminitol. The mean length ranges from eight monosaccharides for the younger to seventeen monosaccharides for the older animals. The results suggest that the increase in keratan sulphate content of cartilage may be due to the increase in the length and not in the number of chains.

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.

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.

Analysis of the acid polysaccharides from squid cranial cartilage and examination of a novel polysaccharide.

The polysaccharides of cranical cartilage were isolated by ethanol precipitation after papain digestion and beta-elimination procedures and were fractionated chromatographically on CPC-cellulose. In addition to the previously described, heavily oversulphated chondroitin sulphate, the tissue contained small amounts of hyaluronic acid, which, however, co-eluted with the chondroitin sulphate from the CPC-cellulose. Approx. 20% of the isolated polysaccharides consisted of an acidic polysaccharide which to our knowledge is not previously described. This polysaccharide consists mainly of glucuronic acid, galactose and mannose in a molar ratio of 1:2:1. Gel chromatography of the preparation indicated a polydisperse molecule with an apparent average molecular weight of 39 200 on weight basis (Mw) and 31 400 on number basis (Mn).

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.

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.

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.

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.

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.

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.

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.

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.

Age-related changes of proteoglycan subunits from sheep nasal cartilage.

Proteoglycan subunits of sheep nasal cartilage from animals of five different ages were studied. There is a continuous reduction in the size and chondroitin sulphate content of the aggregable and non-aggregable subunits with ageing. For each age group, the non-aggregable are poorer in protein and keratan sulphate than the corresponding aggregable molecules. Irrespective of age, the amount of proteoglycan protein extracted from each gramme wet cartilage is the same. The amino acid composition and the proportion of the aggregable proteoglycans are also the same.

Studies on protein-polysaccharides from pig laryngeal cartilage. Heterogeneity, fractionation and characterization.

1. Protein-polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline beta-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate-protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.