ABSTRACT
Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.
Subject(s)
Cartilage, Articular/chemistry , Extracellular Matrix Proteins , Glycoside Hydrolases , Keratan Sulfate/chemistry , Proteoglycans/chemistry , Aggrecans , Antibodies, Monoclonal , Carbohydrate Sequence , Humans , Immunochemistry , Keratan Sulfate/immunology , Keratan Sulfate/isolation & purification , Lectins, C-Type , Molecular Sequence Data , Molecular Structure , Proteoglycans/immunology , Proteoglycans/isolation & purification , beta-GalactosidaseABSTRACT
Contact of various cells with extracellular matrix molecules modulates their cellular functions and phenotype. Most investigations have employed dishes coated with purified matrix constituents or plain collagen I lattices omitting the effects of other important matrix components such as proteoglycans. In this study we analyze the effect of purified glycosaminoglycans (GAGs) on human fibroblasts and human umbilical vein endothelial cells (HUVEC) embedded within collagen I/III lattices. HUVEC contracted collagen I/III gels far less efficiently than fibroblasts and addition of heparan sulfate and heparin almost completely inhibited contraction. In collagen gels HUVEC down-regulated collagenase mRNA while increasing collagen I, IV mRNA expression. Addition of heparin and heparan sulfate reversed the collagen IV mRNA induction whereas hyaluronic acid and chondroitin sulfate enhanced fibronectin and collagenase transcripts. Fibroblasts readily contracted collagen gels, and mRNA levels for fibronectin, collagenase and interleukin-6 were stimulated. Gel contraction was mostly unaffected by the different glycosaminoglycans. Fibroblasts responded to the addition of dermatan sulfate, heparan sulfate and heparin with a decrease in fibronectin, collagenase and interleukin-6 mRNA. Binding studies revealed saturable binding sites on fibroblasts and HUVEC for 35S-labelled heparin, demonstrating specificity for heparin and heparan sulfate over other GAGs in competition experiments. This study implies that glycosaminoglycans participate in cell-matrix interactions by effectively modulating the cellular phenotype via high affinity binding sites.
Subject(s)
Collagen/pharmacology , Extracellular Matrix/drug effects , Fibroblasts/drug effects , Glycosaminoglycans/pharmacology , Umbilical Veins/drug effects , Animals , Binding Sites , Cattle , Cells , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Extracellular Matrix/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Heparin/metabolism , Heparitin Sulfate/metabolism , Humans , Phenotype , RNA, Messenger/metabolism , Skin/cytology , Skin/drug effects , Swine , Umbilical Veins/cytology , Umbilical Veins/metabolismABSTRACT
In specimens taken from the posterior lip of the cervix uteri we determined the collagenase activity and the glycosaminoglycan concentration. In biopsies obtained from the lower uterine segment during cesarian section we measured cytokines (IL-8, IL-2, TNF alpha) and matrix metalloproteinases (MMP-8, MMP-9). We found that the release of collagenases is critically involved in the process of cervical dilatation. The glycosaminoglycan concentration increases during pregnancy and shows remarkable changes of the distribution patterns of the different glycosaminoglycans. The parturition is characterized by a dramatic loss of most of the glycosaminoglycans. Furthermore, the IL-8 shows a close correlation to the clinical feature of cervical ripening and is closely associated with the release of MMP-8 and MMP-9. Summarizing the process of cervical maturation and dilatation is a complex enzymatic controlled process with substantial remodelling of the cervical extracellular matrix. The cytokines IL-8 seems to play an essential role in triggering the process of cervical dilatation.
Subject(s)
Cervix Uteri/physiology , Labor Stage, First/physiology , Labor, Obstetric/physiology , Pregnancy/physiology , Biopsy , Cesarean Section , Collagenases/metabolism , Collagenases/physiology , Female , Glycosaminoglycans/analysis , Glycosaminoglycans/metabolism , Humans , Interleukin-2/metabolism , Interleukin-8/metabolism , Matrix Metalloproteinase 8 , Matrix Metalloproteinase 9 , Tumor Necrosis Factor-alpha/metabolism , Uterus/metabolismABSTRACT
The central function of the cervix to maintain pregnancy is biochemically characterized by an increased synthesis of collagen, proteins, glycosaminoglycans (GAG) and fibronectin within the extracellular matrix, thus leading to an increase of cervical volume without significant changes of cervical consistency. During the time of cervical ripening we found a marked reduction of collagen concentration, a 2.5-fold increase in GAG content, a significant fall in dermatan sulfate concentrations from 41% to 15% of total GAG content, a 12-fold increase in hyaluronate concentrations, and a marked reduction in fibronectin, demonstrated by immunhistochemical methods. Thus, the loss of collagen and sulfated GAGs may facilitate distensibility in the ripened cervix, while the significant gain in hyaluronate associated with hydratation may explain the soft and swollen consistency. In this connection increased hyaluronate concentrations and degradation of fibronectin may play a trigger role for subsequent cervical dilatation. The dramatic changes of the cervix during parturition occurring within a few hours require the rapid activation and action of catabolic enzyme systems. Our studies showed a significant increase of sialidase-, collagenase- and elastase activities during cervical dilatation. These proteinases originate from polymorphonuclear leucocytes (pml), which accumulate in cervical capillaries at the onset of labor; this is followed by a massive leucocyte infiltration of the cervical stroma at the beginning of cervical dilatation and a degranulation of the pml at further dilatation, thus releasing collagenase and other proteinases. This process is limited by the immediate post partum insudation of the cervix by plasma containing highly potent proteinase inhibitors. The clinical aim of our basic biochemical studies is to develop new concepts in the causal treatment of cervical pathology during pregnancy.
Subject(s)
Cervix Uteri/physiology , Collagen/metabolism , Fibronectins/metabolism , Glycosaminoglycans/metabolism , Labor Onset/physiology , Proteins/metabolism , Uterine Contraction/physiology , Animals , Collagenases/physiology , Endopeptidases/physiology , Extracellular Matrix/physiology , Female , Humans , Immunohistochemistry , PregnancyABSTRACT
Cervical biopsies were taken during the first trimester from primigravidae and plurigravidae at different time points after intracervical application of prostaglandin E2-gel. Collagenase activity was determined by a highly specific technique using native, triple helical collagen as substrate. Elastase-alpha 1-proteinase-inhibitor complex (elastase) was measured by a commercially available assay, and glycosaminoglycan (GAG) analyses were performed as described by Greiling et al. (5, 6). The maximum activity of collagenase was found 2 hours after PGE2 application in plurigravidae and 4 hours after application in primigravidae. Elastase activity rose nearly 7-fold to maximum values 4 hours after PGE2 application. The total GAG concentrations and the dermatan sulfate concentrations increased by approximately 10%, while the hyaluronic acid concentrations were found to be elevated significantly by nearly 50% in the PGE2-primed cervices. We conclude that a time-dependent enzymatic collagen degradation by collagenases and other proteinases and an increase in hyaluronic acid concentrations are the significant biochemical events underlying PG-induced cervical ripening.
PIP: Clinicians applied prostaglandin E2 (PGE2) in viscous gel form to the cervixes of 38 primiparous and multiparous women in Germany and Hungary to ripen the cervix for 1st trimester abortion. They took cervical biopsies at different times after application to determine collagenase and proteinase activity and to learn the role of glycosaminoglycan (GAG) changes in PGE2-induced cervical ripening. PGE2 induced significant cervical ripening in all cases. Collagenase activity peaked 2 hours and 4 hours after PGE2-application in multigravidae and primigravidae, respectively. The increases in collagenase activity at the peak point were much higher than pretreatment levels (p .05). Elastase activity increased from 854 to 3170 ng/100 mg dry weight 4 hours after PGE2 application. Total GAG levels and dermatan sulfate levels rose insignificantly by about 10% but the hyaluronic acid levels increased significantly by almost 50%. These findings suggest that the most important biochemical events responsible for PG-induced cervical ripening include collagenases and other proteinases degrading collagen within a specified period of time and an increase in hyaluronic acid levels.
Subject(s)
Abortion, Induced , Cervix Uteri/metabolism , Connective Tissue/metabolism , Dinoprostone/pharmacology , Cervix Uteri/drug effects , Collagenases/metabolism , Dinoprostone/administration & dosage , Female , Glycosaminoglycans/metabolism , Humans , Kinetics , Pancreatic Elastase/metabolism , Parity , Pregnancy , alpha 1-Antitrypsin/metabolismABSTRACT
OBJECTIVE: To investigate the content and distribution patterns of glycosaminoglycans in the human cervix during pregnancy and parturition. METHODS: We obtained a total of 87 specimens from nonpregnant and pregnant women. Biopsies (weight 50-200 mg) were taken from the posterior lip of the cervix. Hyaluronic acid, dermatan sulfate, chondroitin sulfates, and heparan sulfate were separated on a Dowex 1 x 2 column and identified. RESULTS: The total amount of glycosaminoglycans increased during pregnancy from 2800 to 5000 nmol/g dry weight. The highest values were observed at the onset of labor (7100 nmol/g dry weight), followed by a sharp decrease during parturition. The clinical features of cervical ripening and dilatation were also associated with remarkable changes in glycosaminoglycan patterns. CONCLUSION: Besides collagenolysis during pregnancy, the glycosaminoglycans are also important regulators of cervical function. The different clinical features of the human cervix are characterized not only by variation in the total glycosaminoglycan content but also by changes in the proportions of the different glycosaminoglycans.
Subject(s)
Cervix Uteri/metabolism , Glycosaminoglycans/metabolism , Labor, Obstetric/metabolism , Pregnancy/metabolism , Connective Tissue/metabolism , Female , Humans , Menopause/metabolism , Postpartum Period/metabolism , Pregnancy Trimester, First , Pregnancy Trimester, ThirdABSTRACT
Proteoglycans of the human B lymphoblastoid cell line LICR-LON-HMy2 were metabolically labeled with [35S]sulfate. High-density fractions of 35S-labeled material separated by CsCl gradient ultracentrifugation were further purified by anion exchange chromatography and gel filtration. Two proteoglycans, isolated from cell lysates and culture supernatants, were characterized by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with enzymatic degradation. Treatment with chondroitinase AC completely degraded the glycosaminoglycan moiety of the proteoglycans. Three to 4 chondroitin sulfate chains (average molecular mass = 26 kDa) were estimated for each of the two proteoglycans. Differences between the proteochondroitin sulfates (CSPG) were observed in the content of N-linked oligosaccharides. After chondroitinase AC treatment the resulting band in SDS-PAGE of the secreted CSPG was sensitive to treatment with endoglycosidase F (Endo F) which further reduced the molecular mass from 30 to 21.5 kDa, whereas the band of the cellular CSPG after chondroitinase AC treatment (molecular mass = 30 kDa) remained resistant to Endo F treatment. The composition of amino acids was different in the protein cores, suggesting differences in the primary structure. Both CSPG contained a high percentage of glycine and serine. For both CSPG a molecular mass of approximately 135 kDa was deduced from the hydrodynamic sizes of the glycosaminoglycan chains obtained after alkaline/borohydride treatment and the migration of the protein/oligosaccharide complexes in SDS-PAGE. 75% of all [35S]sulfate-labeled molecules were found in the culture supernatant and 25% in the cellular fraction. 35S-Labeled material in the culture supernatant consisted exclusively of intact CSPG, whereas 35S-Labeled molecules in the cellular preparation consisted largely of free chondroitin sulfate chains. Only 8.3% of the cellular material, isolated from the microsomal fraction, was intact CSPG. In pulse-chase experiments maximal secretion of CSPG was found after 4 h, comprising approximately 40% of totally synthesized CSPG. From these experiments we tentatively conclude that a small proportion of CSPG synthesized by LICR-LON-HMy2 cells is membrane-associated, a larger portion is secreted, and another portion is intracellularly degraded.
Subject(s)
Chondroitin Sulfate Proteoglycans/biosynthesis , Amino Acids/analysis , B-Lymphocytes , Cell Line , Cell Membrane/metabolism , Centrifugation, Density Gradient , Chondroitin Sulfate Proteoglycans/isolation & purification , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Humans , Kinetics , Molecular Weight , Proteoglycans/chemistry , Proteoglycans/isolation & purification , Sulfates/metabolism , Sulfur RadioisotopesABSTRACT
An in vitro model of frustrated phagocytosis was developed in which granulocytes interact with well-defined slices of hyaline cartilage. The composition of the purified proteoglycan fragments released from the cartilage slices by N-formyl-methionyl-leucyl-phenylalanine-stimulated granulocytes was studied after 30, 60 and 90 min incubation time. It was shown that the proteoglycan fragments do not change their composition during incubation. The only change observed during incubation was an increase in the quantity of the fragments. The protein content of the proteoglycan fragments is 7.0-8.6%, corresponding to a peptide chain of 24-28 amino acids, and the relative molecular mass of the total fragment is Mr = 37,600-39,200. On average, each proteoglycan fragment contains two chondroitin sulphate chains (Mr = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (Mr = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide, whereas no N-glycosidic oligosaccharide could be detected. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin 4-sulphate, 12.9-19.4% chondroitin 6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the proteoglycan fragments resemble those of native proteoglycans, the conclusion may be drawn that the degeneration of the proteoglycans occurs by proteases that attack preferably the chondroitin sulphate-rich region of the core protein. This is the first inflammation model of joint destruction, which demonstrates the elution of soluble specific proteoglycan degradation products of defined size.
Subject(s)
Cartilage , Chondroitin Sulfates/chemistry , Granulocytes/physiology , Nasal Septum , Neutrophils/physiology , Peptide Fragments/chemistry , Phagocytosis , Proteoglycans/metabolism , Amino Acids/analysis , Chondroitin Sulfates/isolation & purification , Chromatography, Gel , Chromatography, High Pressure Liquid , Disaccharides/isolation & purification , Hexosamines/analysis , Humans , In Vitro Techniques , Models, Biological , Molecular Weight , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Peptide Fragments/isolation & purification , Phagocytosis/drug effectsABSTRACT
Maturation of the cervix during pregnancy is an essential pre-requisite for an uncomplicated delivery at term. Physiological cervical ripening is characterised by a diffuse loosening of the collagenous connective tissue with widely scattered collagen fibrils and an increased amount of extracellular ground substance. These morphological changes are similar to those after prostaglandin (PG)-pre-treatment of the cervix. The local application of PG leads to a marked mulifocal loosening of the collagen fibre bundles with "activated" fibroblasts, characterised by a fine granular loosening of the cytoplasm, vacuolised enlarged mitochondria and an increased number of cytoplasmatic vesicles close to the cell surface. In the course of pregnancy the volume of the cervix increases by a significant rise in synthesis of collagen, protein, glycosaminoglycan and fibronectin. The change in consistency during late pregnancy corresponds to a significant decrease in dermatan sulphate coninciding with a marked increase in hyaluronic acid concentration associated with increased water uptake. Contrary to the already published literature, enzymatic collagen degradation does not play an important role in physiological cervical maturation. The action of catabolic enzymes (collagenases, glycosidases), liberated from polymorphonuclear leukocytes invading the extracellular matrix, is responsible for the rapid dilatation of the cervix at parturition. This process is limited by the immediate postpartum insudation of the cervix by plasma containing highly potent proteinase inhibitors (e.g. alpha 2-macroglobulin). PG-induced cervical ripening is associated with a time-limited enzymatic collagen degradation, an increased synthesis of non-collagenous proteins and a significant increase in hyaluronic acid concentration. Our basic biochemical findings in cervical ripening and dilatation during parturition may greatly contribute to the development of new concepts in the causal treatment of cervical pathology during pregnancy.
Subject(s)
Cervix Uteri/drug effects , Labor, Induced/methods , Progesterone/administration & dosage , Cervix Uteri/pathology , Collagen/metabolism , Female , Gestational Age , Glycosaminoglycans/metabolism , Humans , PregnancyABSTRACT
Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.
Subject(s)
Cornea/chemistry , Dermatan Sulfate/chemistry , Keratan Sulfate/chemistry , Proteoglycans/ultrastructure , Alginates/chemistry , Animals , Cattle , Chondroitin Sulfates/chemistry , Heparin/chemistry , Heparitin Sulfate/chemistry , Humans , Hyaluronic Acid/chemistry , Methylcellulose/chemistry , Microscopy, Electron , Molecular Conformation , RabbitsABSTRACT
The proteolytic degradation products of nasal hyaline cartilage proteoglycans produced by polymorphonuclear leukocyte lysosomal enzymes were investigated. The protein content of the degradation products is 7.0-8.6% corresponding to a peptide chain of 24-28 amino acids and the relative molecular mass of the total fragment is M(r) = 37,600-39,200. On an average, each proteoglycan fragment contains two chondroitin-sulphate chains (M(r) = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (M(r) = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin-4-sulphate, 12.9-19.4% chondroitin-6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the degradation products resemble those from native proteoglycans, it is suggested that the degradation of the proteoglycans occurs by proteinases that attack preferably the chondroitin sulphate region of the core protein.
Subject(s)
Cartilage/chemistry , Hyalin/chemistry , Proteoglycans/analysis , Amino Acids/analysis , Chondroitin Lyases , Chondroitin Sulfates/analysis , Chromatography, High Pressure Liquid , Glycosaminoglycans/analysis , Hexosamines/analysis , Humans , Keratan Sulfate/analysis , Molecular Weight , Peptide HydrolasesABSTRACT
Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.
Subject(s)
Cornea/analysis , Disaccharides/analysis , Glycosaminoglycans , Glycoside Hydrolases , Keratan Sulfate , Animals , Carbohydrate Conformation , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Neuraminic Acids/analysis , Oligosaccharides/analysis , Swine , beta-GalactosidaseABSTRACT
Glycosaminoglycans (GAG) were described by histochemical staining procedures in the normal urothelium of the urinary bladder; they are supposed to be involved in the antibacterial defense mechanism. Our quantitative analysis, however, demonstrated only heparan sulfate in the normal calf urothelium (less than 600 nmol/Gm d.wt.); only trace amounts of other GAG were to be analyzed. High concentrations of GAG were found in the submucosa and muscle layers as to be expected in mesoderm originating tissues. According to these results there were no GAG with the exception of heparan sulfate at the surface of the normal urothelium; therefore, glycoproteins detected in mumol/Gm d.wt. ranges are more likely to be involved in the antibacterial defense mechanism.
Subject(s)
Glycosaminoglycans/analysis , Urinary Bladder/analysis , Animals , Cattle , Epithelium/analysis , Heparitin Sulfate/analysis , HistocytochemistryABSTRACT
Light scattering due to the formation of insoluble complexes between the long-chain quaternary ammonium salt N-cetylpyridinium chloride and glycosaminoglycans was utilized for a relative simple, sensitive and precise determination of total and specific types of glycosaminoglycans by laser nephelometry. The addition of the ammonium salt to solutions of various glycosaminoglycans in 0.03 mol/l NaCl produces a time-dependent increase in light scattering, which reaches a maximum between 14 and 18 h of complex formation, irrespective of the type of glycosaminoglycan studied. Only keratan sulphate does not generate light scattering, and is therefore not detectable by the procedure. The scattering of laser light by certain types of sulphated glycosaminoglycans (e.g. heparan sulphate, heparin) depends more on the degree of sulphation (charge density) than on chain length within a certain range. Optimum light scattering was found at 28 mmol/l N-cetylpyridinium chloride and at a ionic strength around 0.03 mol/l NaCl. The detection limits and linear ranges of the individual glycosaminoglycans were evaluated. For the determination of chondroitin sulphate, laser nephelometry is at least 8 times more sensitive and much more simple than the modified carbazole method (glucuronic acid). The intra-assay and inter-assay coefficients of variation are about 4% and 7%, respectively. Laser nephelometry is much more sensitive than turbidimetry. Complex synthetic mixtures of glycosaminoglycans and biological fluids were accurately differentiated by successive chemical and enzymatic degradation of the respective glycosaminoglycans followed by the measurement of the resulting reduction of laser light scattering. In synovial fluids from non-inflammatory joint diseases, light scattering (units/ml) was about 4.5 times higher than in synovial fluids from inflammatory articular lesions. In both pathologic conditions nearly all of the light scattering can be attributed to hyaluronic acid.
Subject(s)
Glycosaminoglycans/analysis , Lasers , Cetylpyridinium , Chondroitin Lyases , Humans , Hyaluronoglucosaminidase , Nephelometry and Turbidimetry/methods , Nitrous Acid , Scattering, Radiation , Sodium Chloride , Synovial Fluid/analysisABSTRACT
The catalytic activities of 4 glycosidases (hyaluronate-4-glycanohydrolase (EC 3.2.1.35), beta-N-acetyl-D-glucosaminidase (EC 3.2.1.30), beta-glucuronidase (EC 3.2.1.31), alpha-L-iduronidase (EC 3.2.1.76)), of the arylsulphatases A and B (EC 3.1.6.1) and of the protease cathepsin D (EC 3.4.23.5) were measured in extracts from hepatocytes and non-parenchymal cells and in serum during the development of thioacetamide-induced rat liver fibrosis (22 weeks). In non-parenchymal liver cells the catalytic activities of beta-N-acetyl-D-glucosaminidase, beta-glucuronidase, alpha-L-iduronidase and cathepsin D were increased significantly during chronic liver damage, but that of hyaluronate-4-glycanohydrolase was reduced by 40 to 65% during the period of application of thioacetamide. The catalytic activities of the arylsulphatases were lowered by 65% compared to control values in the 12th week but with advancing liver damage the catalytic activities returned to nearly normal values. Parenchymal cells of rats, which had been liver-damaged for 6 months, contained strongly elevated activities of beta-glucuronidase, beta-N-acetyl-D-glucosaminidase, arylsulphatases A and B, and cathepsin D but only slightly increased activities of hyaluronate-4-glycanohydrolase and alpha-L-iduronidase, respectively. In the serum of liver-damaged rats the activity of alpha-L-iduronidase was strongly elevated, while that of N-acetyl-beta-D-glucosaminidase was only slightly increased. The activities of beta-glucuronidase and of arylsulphatases A and B were decreased during the whole period of treatment. The catalytic functions of hyaluronate-4-glycanohydrolase and of cathepsin D, respectively, were decreased initially, but both enzyme activities were elevated during the more advanced stages of long term thioacetamide treatment.
Subject(s)
Arylsulfatases/metabolism , Cathepsins/metabolism , Glycoside Hydrolases/metabolism , Liver Cirrhosis, Experimental/enzymology , Liver/enzymology , Lysosomes/enzymology , Proteoglycans/metabolism , Sulfatases/metabolism , Animals , Cathepsin D , Liver/pathology , Liver Cirrhosis, Experimental/pathology , Male , Rats , Rats, Inbred Strains , Thioacetamide/toxicityABSTRACT
Cornea cells were isolated from bovine corneae after collagenase treatment. Subcellular fragments were fractionated by density gradient centrifugation. The density gradient run was monitored by determination of the marker enzyme activities for mitochondria, plasma membranes, lysosomes and endoplasmatic reticulum, of the enzyme activities involved in keratan sulfate synthesis and of the protein content. The fractions were further investigated by electron microscopy. Two membrane fractions with keratan sulfate-synthesizing activity (UDP-N-acetylglucosamine:keratan-N-acetylglucosaminyl-transferase, UDPgalactose:keratan galactosyltransferase and keratan sulfotransferase) were detected: a heavy fraction separated from the other organells investigated and a light fraction exhibiting the same density as plasma membranes. The activities of the three enzymes were found in the same density gradient fractions with a similar distribution pattern between the fractions, which suggests a joint localization of these 3 enzymes at the same intracellular sites.
Subject(s)
Chondroitin Sulfate Proteoglycans/biosynthesis , Cornea/metabolism , Glycosaminoglycans/biosynthesis , Keratan Sulfate/biosynthesis , N-Acetylglucosaminyltransferases , Proteoglycans/biosynthesis , Subcellular Fractions/metabolism , Sulfotransferases , Animals , Cattle , Cornea/ultrastructure , Fibroblasts/metabolism , Galactosyltransferases/isolation & purification , Glucosyltransferases/isolation & purification , In Vitro Techniques , Lumican , Membranes/metabolism , Sulfurtransferases/isolation & purification , Carbohydrate SulfotransferasesSubject(s)
Chondroitin Sulfate Proteoglycans/biosynthesis , Cornea/metabolism , Glycosaminoglycans/biosynthesis , Keratan Sulfate/biosynthesis , Proteoglycans/biosynthesis , Sulfotransferases , Sulfurtransferases/isolation & purification , Animals , Cattle , Chromatography, DEAE-Cellulose , Hydrogen-Ion Concentration , In Vitro Techniques , Kinetics , Lumican , Microsomes/enzymology , Peptide Chain Termination, Translational , Temperature , Carbohydrate SulfotransferasesABSTRACT
The glycosaminoglycan (GAG) content in human urothelial carcinomas was biochemically determined and compared to that of normal urothelium and bladder wall of the calf. The total GAG content was elevated in urothelial carcinomas, and the distribution pattern differed from that of normal urothelium and bladder wall. Whereas urothelial carcinomas contained heparan sulphate, dermatan sulphate, chondroitin 4-sulphate and chondroitin 6-sulphate, only heparan sulphate could be detected in the normal urothelium. The GAG determination was based on hexosamine analysis and thin layer chromatography after elution on Dowex 1 X 2 columns.
Subject(s)
Carcinoma, Transitional Cell/analysis , Glycosaminoglycans/analysis , Urinary Bladder Neoplasms/analysis , Urinary Bladder/analysis , Aged , Animals , Cattle , Chromatography, Thin Layer , Female , Humans , Male , Middle AgedABSTRACT
Peptidokeratan sulfate from bovine cornea was degraded by a combination of desulfation, exo-enzymic digestion and finally digestion with endo-beta-N-acetylglucosaminidase D. The same procedure was carried out both with [3H]fucose-labelled and [3H]mannose-labelled peptidokeratan sulfate. Data obtained by methylation analysis of peptidokeratan at the different degradation steps, as well as action of endo-beta-N-acetylglucosaminidase D, showed that the binding-region in proteokeratan sulfate from bovine cornea is identical with a structure found in various GlcNAc(beta 1-N)-Asn-linked mannosyl glycoproteins. The existence of a chitobiose unit between asparagine and mannose was proved by action of endo-beta-N-acetylglucosaminidase D. The existence and position of an (alpha 1 leads to 6)-linked fucosyl residue at the Asn-bound GlcNAc was demonstrated by action of alpha-fucosidase, endo-beta-N-acetylglucosaminidase D and by gel chromatography on Bio-Gel P-4. By gas chromatography/mass spectrometry studies, the existence of a 1,4,6-trisubstituted beside a 1,4-disubstituted GlcNAc in the binding-region oligosaccharide was shown. Other results reported here are according to analytical data previously published (Keller, R., Stein, T., Stuhlsatz, H.W., Greiling, H., Ohst, E., Müller, E. & Scharf, H.-D. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, 327-336).