A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

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.

Glycosaminoglycans modulate cell-matrix interactions of human fibroblasts and endothelial cells in vitro.

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.

Biochemical events in cervical ripening dilatation during pregnancy and parturition.

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.

[Biochemical principles of cervix ripening and dilatation].

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.

Biochemical changes in human cervical connective tissue after intracervical application of prostaglandin E2.

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.

Glycosaminoglycans in cervical connective tissue during pregnancy and parturition.

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.

Secreted and cellular proteochondroitin sulfates of a human B lymphoblastoid cell line contain different protein cores.

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.

Composition of proteoglycan fragments from hyaline cartilage produced by granulocytes in a model of frustrated phagocytosis.

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.

[Taurine supplementation in cystic fibrosis (CF): effect on vitamin E absorption kinetics].

Oral vitamin E (Vit.E) bioavailability is reduced in CF patients especially in case of malnourishment. Both exocrine pancreatic insufficiency and an altered bile acid composition showing an elevated glycine taurine ratio of conjugated bile acids which is due to excessive loss of bile acids in the stools may contribute to this observation.

[Principles of physiologic and drug-induced cervix ripening--recent morphologic and biochemical findings].

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.

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy.

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.

Analysis of the proteolytic degradation products of hyaline cartilage proteoglycans.

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.

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

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.

Quantitative analysis of glycosaminoglycans in urothelium and bladder wall of calf.

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.

Laser nephelometric determination of glycosaminoglycans--method and application.

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.

Changes in the catalytic activities of proteoglycan-degrading lysosomal enzymes in parenchymal and non-parenchymal liver cells and in serum during the development of experimental liver fibrosis.

The catalytic activities of 4 glycosidases (hyaluronate-4-glycanohydrolase (EC 3.2.1.

Biosynthesis of proteokeratan sulfate in the bovine cornea. 2) Isolation of subcellular membrane fragments from bovine cornea cells with keratan sulfate synthesizing activity.

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.

Glycosaminoglycans in urothelial carcinomas.

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.

Structure of the linkage-region between polysaccharide chain and core protein in bovine corneal proteokeratan sulfate.

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.

Studies on the polydispersity and heterogeneity of proteokeratan sulfate from calf and porcine cornea.

After proteolysis of the calf and porcine corneae with papain 66 (calf) and 56 (hog) polysaccharide-containing fractions were obtained by chromatography on Dowex 1X2 and fractionating precipitation with ethanol (calf) and by chromatography on Dowex 1X2 and CPC-cellulose (hog). The sulfatation degrees (mol sulfate/mol hexosamine) and molecular weights (Mw) of 13 peptidokeratan sulfate fractions from calf cornea and of 9 such fractions from porcine cornea were 0.41-1.

Studies on the characterization of the linkage-region between polysaccharide chain and core protein in bovine corneal proteokeratan sulfate.

1) A new method of enrichment of the linkage-region in corneal proteokeratan sulfate is described, which consists of desulfation of peptidokeratan sulfate, followed by chromatography on Con A-Sepharose 4B and enzymatic degradation with beta-D galactosidase and beta-N-acetyl-D-glucosaminidase. 2) After permethylation, hydrolysis, reduction with sodium borohydrid and acetylation gas chromatography/mass spectrometry analyses were performed. The followings products could be detected as their peracetates: 2,3,4-tri-O-methylfucitol; 2,3,4,6-tetra-O-methylmannitol; 3,4,6-tri-O-methylmannitol; 2,4-di-O-methylmannitol; 2,3,4,6-tetra-O-methylgalactitol; 2,4,6-tri-O-methylgalactitol; 2,4-di-O-methylgalactitol.