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Publications (31)

  • [Show abstract] [Hide abstract] ABSTRACT: Hydroxyapatite (HAP) crystal growth was found to be inhibited by glycosaminoglycans in vitro. The major effect was produced by hyaluronan and of the sulphated glycosaminoglycans by chondroitin-4-sulphate. Chondroitin-6-sulphate did have a minor effect, while oversulphated chondroitin did not inhibit growth of HAP crystals. Chemically desulphated chondroitin sulphate still retains its inhibitory ability. Fragmented glycosaminoglycans by testicular hyaluronidase behaved in a manner similar to the respective intact molecules, even in the case in which the size of the fragments was in the tetrasaccharide range. Constituents of the monosaccharide of the glycosaminoglycans, were also examined with respect to their effect on HAP crystal growth; N-acetylglucosamine being the most potent inhibitor. The results clearly suggest that the chemical structure of the glycosaminoglycans determines their behavior in inhibiting HAP crystal growth. The presence of N-acetylglucosamine, and of only one sulphate ester group in the axial position, seemed to be the most prominent.
    Chapter · May 2007
  • [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Sep 2004 · International Journal of Oncology
  • [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Sep 2004 · Biochimie
  • [Show abstract] [Hide abstract] ABSTRACT: Recently, we reported the isolation and partial characterization of keratan sulphate (KS) from sheep brain. In this study, a panel of monoclonal antibodies (Mab) recognizing epitopes within KS chains and core proteins of KS-containing proteoglycans were used to detect, by immunoblotting, antigenically related molecules extracted from cerebrum, cerebellum and brainstem, respectively. Although the intensity of labelling varied with each of the antibodies, the brain KSPGs were recognized by all the monoclonals used, confirming the presence of KS side chains, which react with the Mabs: 5-D-4, EFG-11, EFG-4, I22, as also the presence of KSPGs related to phosphacan-KS (3H1 proteoglycan). Extracts of all the three brain areas could bind both anti-KS and anti-core protein Mabs, as also anti-HNK-1 monoclonal antibody. Binding was sensitive to keratanases degradation in the cerebrum and brainstem except cerebellum where the presence of a large molecular size hybrid CS/KSPG bearing KS chains partially resistant to keratanases was identified. This population reacts only with 5-D-4, EFG-11 and EFG-4 antibodies. Furthermore, the presence of HNK-1 epitope in CSPGs was detected in the cerebellum and brainstem. In contrast, in the cerebrum the coexistence of HNK-1 epitope and KS in KSPGs was identified. These data suggest that the KSs of sheep brain are part of proteoglycans containing protein and KS antigenic sites related to those of corneal and cartilage KSPG, as also of the brain proteoglycan phosphacan-KS.
    Article · Jan 2003 · Biochimie
  • D H Vynios · N K Karamanos · C P Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Jan 2003 · Journal of Chromatography B
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    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Full-text Article · Jul 2002 · Experimental Gerontology
  • [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Nov 2001 · Biochimie
  • [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Oct 2001 · Biochimie
  • D.A. Theocharis · N Papageorgacopoulou · D.H. Vynios · [...] · C.P. Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · May 2001 · Journal of chromatography. B, Biomedical sciences and applications
  • [Show abstract] [Hide abstract] ABSTRACT: Ozonetherapy is used for the treatment of immunodeficiency syndromes as well as for the treatment of cardiovascular disease. It is also used for the treatment of low back-pain with promising results although it is not yet well established. The aim of the current study is the presentation of the effects of ozonetherapy injected intradiscally or paravertebrally. We present the histological, immunological and biochemical changes in vertebral discs. Our material consist of human specimens as well as New Zealand rabbits.
    Article · Mar 2001
  • D H Vynios · Papadas ThA · A Faraos · [...] · C P Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Feb 2001 · Journal of Immunoassay and Immunochemistry
  • Demitrios H Vynios · Matthias Mörgelin · Nicoletta Papageorgakopoulou · [...] · Constantine P Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Sep 2000 · Biochimie
  • D H Vynios · A Faraos · G Spyracopoulou · [...] · C P Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Jan 2000 · Journal of Pharmaceutical and Biomedical Analysis
  • D H Vynios · S S Vamvacas · D L Kalpaxis · C P Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Jul 1998 · Analytical Biochemistry
  • N Papageorgakopoulou · E Papaefthymiou · D Topalidou · [...] · C.P Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: Germination of the cereals barley, wheat and maize in tritiated water (H3HO; 1850 kBq ml−1) revealed that various enzyme changes occurred. Whole grain extracts of cereals germinated for 24, 48 and 72 h in H3HO were analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), SDS–PAGE–zymography and native electrophoresis for changes in protein profile, proteolytic and phosphohydrolytic activities, respectively. Protein patterns and acid and alkaline phosphohydrolytic enzymes of all cereals were not affected by the presence of tritium during germination for 72 h. In wheat grains a gelatinolytic activity of ≈70 kDa after 24 h and one in maize of ≈200 kDa after 72 h of germination were induced by tritium. In contrast, tritium inhibited the appearance of a ≈60 kDa proteolytic activity of maize after 24 h and a ≈50 kDa proteolytic activity of barley after 72 h of germination.
    Article · Feb 1998 · Environmental and Experimental Botany
  • P Paschalakis · D.H. Vynios · C.P. Tsiganos · [...] · P.G. Koutsoukos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Nov 1993 · Biochimica et Biophysica Acta
  • Demitrios H. Vynios · Matthias Mörgelin · Constantine P. Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Jan 1993 · Matrix (Stuttgart, Germany)
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    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Full-text Article · Apr 1992 · European Journal of Biochemistry
  • Stavros T. Anagnostides · Alexis J. Aletras · Peggy Lymberi · Constantine P. Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Article · Dec 1990 · European Journal of Biochemistry
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    K K Karamanos · Alexis J. Aletras · Christos A. Antonopoulos · [...] · Constantine P. Tsiganos
    [Show abstract] [Hide abstract] ABSTRACT: 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.
    Full-text Article · Sep 1990 · European Journal of Biochemistry