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Biomedical subjects

M Paulsson

Publications and source records attributed to M Paulsson.

At least 73 records · Page 4Linked to original sources
PubMed1996

Increased degradation and altered tissue distribution of cartilage oligomeric matrix protein in human rheumatoid and osteoarthritic cartilage.

We investigated the degradation and tissue distribution of cartilage oligomeric matrix protein in normal, osteoarthritic, and rheumatoid arthritic articular cartilage of the human knee. Cartilage was subjected to sequential extractions with buffers containing neutral salt, with EDTA, and finally with guanidine/HCl and then was analyzed by Western blotting with a polyclonal antiserum to human cartilage oligomeric matrix protein. Western blots of the nine neutral salt extracts from normal cartilage revealed mostly intact pentameric molecules of cartilage oligomeric matrix protein, in contrast to the 13 osteoarthritic and five rheumatoid arthritic cartilage samples that demonstrated marked degradation of cartilage oligomeric matrix protein as noted by a predominance of reduction-sensitive bands at approximately 150 kDa and nonreduction-sensitive bands in the 67-94 kDa range. The EDTA and guanidine/HCl extracts from all groups were similar and showed mostly intact molecules of cartilage oligomeric matrix protein, with smaller amounts of degraded cartilage oligomeric matrix protein identical to those resolved by the Western blots of the neutral salt extracts. Western blots of matched pairs of synovial fluid and cartilage extracts demonstrated cartilage oligomeric matrix protein fragments of the same molecular mass. Competitive enzyme-linked immunosorbent assay revealed significantly less cartilage oligomeric matrix protein in rheumatoid articular cartilage than in either normal or osteoarthritic cartilage. In contrast to normal cartilage, where cartilage oligomeric matrix protein was predominantly localized to the interterritorial matrix throughout all zones of the matrix, with increased staining in the deeper cartilaginous zones, the most intense staining in osteoarthritic cartilage was in the superficial zones of fibrillated cartilage, with little to no immunostaining in the midzones and relatively poor staining in the deeper cartilaginous zones. This distribution was the inverse of that for proteoglycans, as demonstrated by toluidine blue staining, where proteoglycans were depleted primarily from the superficial fibrillated cartilage. In mild to moderately affected rheumatoid cartilage, the tissue distribution of cartilage oligomeric matrix protein was similar to the distribution of proteoglycans, with relatively uniform staining of the interterritorial and territorial matrics. In more severely affected rheumatoid cartilage, the superficial zones demonstrated punctate immunostaining for cartilage oligomeric matrix protein in the interterritorial and territorial matrics, and staining was restricted to the territorial matrix in the deep cartilaginous zones. It is evident from this study that (a) noncollagenous proteins such as cartilage oligomeric matrix protein are greatly affected in arthritis, (b) degradation fragments released from the matrix into the synovial fluid reflect the processes occurring within the matrix, and (c) different zones of the articular cartilage are susceptible to degradation of cartilage oligomeric matrix protein in the different disease processes.

Adult↗
PubMed1996

Tissue transglutaminase and factor XIII in cartilage and bone remodeling.

While it is well established that factor XIII functions in crosslinking of the fibrin clot during blood coagulation and in wound healing, the physiological role of tissue transglutaminase is still unclear. Recent studies suggest that the expression of tissue transglutaminase correlates with (terminal) differentiation of cells and that the enzyme may play a role in extracellular matrix remodeling. In cartilage, tissue transglutaminase expression is restricted to hypertrophic chondrocytes and the enzyme is externalized at a distinct step in the chondrocyte maturation program. Upon activation by Ca2+, the transglutaminase modifies matrix constituents in a way that might predispose the matrix for the subsequent mineralization. Crosslinks of the structure gamma-glutamyl-epsilon-lysine are also abundant in bone matrix, but the transglutaminase expressed by osteoblasts and presumably involved in crosslinking of newly formed osteoid is likely to be distinct from both tissue transglutaminase and factor XIII. Matrix proteins thought to be crosslinked by transglutaminases in cartilage and bone matrix include glycoproteins such as osteonectin, osteopontin, fibronectin, fibrillin, and collagens II, III, V, and XI. Expression of the A subunit of factor XIII is restricted to megakaryocytes in the bone marrow cavity, and factor XIIIa is abundant in platelets that probably provide the major source for factor XIII in plasma.

Animals↗
PubMed1996

Expression and activation of tissue transglutaminase in apoptotic cells of involuting rodent mammary tissue.

Apoptosis is a form of cell death in which cellular integrity is maintained and neither cytoplasmic nor nuclear content is released. The Ca(2+)-dependent tissue transglutaminase (tTG) is an enzyme that forms protein cross-links between specific glutamyl and lysyl side-chains of intra- and extracellular proteins, therefore it may be responsible for the structural stabilization observed during the death process. In this study, the expression of tTG was investigated following the physiological process of forced weaning which results in an almost complete elimination of secretory epithelium by apoptosis and remodelling of the tissue structure. A dramatic induction of tTG was detected by immunoblotting of total mammary gland homogenates prepared from the involuting glands. The concentration of epsilon(gamma-glutamyl)-lysine crosslinks was also elevated in these samples, showing that the enzyme is activated during apoptosis. To determine the distribution of tTG and its relationship to apoptotic cells, paraffin-embedded specimens were studied by RNA in situ hybridization and immunohistochemical methods as well as using in situ labeling for nuclear fragmentation. All of these approaches indicated that mammary secretory epithelium expressed tissue transglutaminase coincident with the onset of apoptosis. The apoptotic and tTG-expressing cells were found to be identical as demonstrated by a histological double-labeling technique.

Animals↗
PubMed1995

Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204 rhabdomyosarcoma cells.

Collagens V and XI are thought to form a core around which the major interstitial collagens, I and II, respectively, are organized during fibrillogenesis. We previously reported the presence of a heterotypic form of collagens V and XI, [alpha 1(XI)]2 alpha 2(V), in cultures of A204 rhabdomyosarcoma cells [Kleman, J.-P., Hartmann, D. J., Ramirez, F., & van der Rest, M. (1992) Eur. J. Biochem. 210, 329-335]. This collagen forms a matrix which remains highly insoluble, even when cells were cultured in the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase and thereby of "classical" collagen cross-linking. When the cells were cultured in the presence of putrescine, a competitive inhibitor of transglutaminase-catalyzed protein cross-linking, a drastic increase in collagen solubility was observed. This result indicates that a transglutaminase contributes to the covalent stabilization of the collagen matrix of these cells. A204 rhabdomyosarcoma cells express tissue transglutaminase as revealed by specific antibodies, and enzyme activity was detected in the cell layer during culture and in cell extracts. Both collagens V and XI are specific glutaminyl substrates for tissue transglutaminase in vitro, as shown by incorporation of [3H]putrescine. The highly homologous alpha 1 chains of collagens V and XI were the major targets for the cross-linking. Trypsin cleaved the [3H] label from the alpha 1 chain of collagen V, demonstrating that the cross-linking occurs in the non triple helical propeptide domains.

Amino Acid Sequence↗
PubMed1995

Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification.

The extracellular matrix protein BM-40 (osteonectin, SPARC) has recently been shown to be a major target for transglutaminase-catalyzed cross-linking in differentiating cartilage. In the present study we demonstrate that recombinant human BM-40 can be modified with [3H]putrescine in a 1:1 molar ratio by transglutaminaseC (tissue transglutaminase). Residues Gln3 and Gln4 were identified as major amine acceptor sites. This was confirmed with several mutant proteins, including deletions in the N-terminal domain I of BM-40, site-directed mutagenesis of the reactive glutamines, and fusion of the seven-amino acid-long N-terminal sequence (APQQEAL) to an unrelated protein. The results showed that the N-terminal target site is sufficient for modification by transglutaminase but at a low level. For high efficiency amine incorporation an intact domain I is required. The conservation of at least one of the transglutaminase target glutamines in the known vertebrate BM-40 sequences and their absence in an invertebrate homologue point to an important, but yet unknown, role of this modification in vertebrates.

Amino Acid Sequence↗
PubMed1995

Tendon extracellular matrix contains pentameric thrombospondin-4 (TSP-4).

In preparations of cartilage oligomeric matrix protein (COMP) from bovine tendon two contaminating polypeptides of 120 and 135 kDa were detected. N-terminal protein sequencing of these polypeptides showed homology to the N-terminus and to an internal sequence in TSP-4, respectively. TSP-4 was further enriched by heparin affinity chromatography. Electron microscopy of this sample shows primarily five armed particles with globular domains at the periphery connected to a central assembly domain in which smaller N-terminal globular domains can be resolved tightly packed at the center of the particle. We can thereby confirm the pentameric model for TSP-4 proposed by Lawler et al. [(1995) J. Biol. Chem. 270, 2809-2814], on the basis of recombinantly expressed protein. We further show that TSP-4 is abundant in tendon.

Amino Acid Sequence↗
PubMed1995

Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates.

Aggregates of proteoglycans from the Swarm rat chondrosarcoma reassembled in vitro have been studied by rotary-shadowing electron microscopy, and shown to be similar to native structures that have never been dissociated [Mörgelin, Engel, Heinegård and Paulsson (1992) J. Biol. Chem. 267, 14275-14284]. A hyaluronate with defined chain length (HAshort) has now been prepared by autoclaving high-Mr hyaluronate and fractionation to a narrow size distribution by gel filtration. Proteoglycan monomers, core protein, hyaluronate-binding region and link protein were combined with HAshort. Free chains of HAshort and reconstituted complexes with proteoglycan, link protein and aggrecan fragments were examined by electron microscopy after rotary shadowing. Length measurements showed that the hyaluronate was condensed to about half of its original length on binding intact aggrecan monomers, any aggrecan fragment or link protein alone. This strongly implies that hyaluronate adopts a defined spatial arrangement within the central filament of the aggregate, probably different from its secondary structure in solution. No differences in length were observed between link-free and link-stabilized aggregates.

Animals↗
PubMed1995

Cartilage oligomeric matrix protein: isolation and characterization from human articular cartilage.

Cartilage oligomeric matrix protein was purified in a native form from normal adult human articular cartilage. The key steps in the purification scheme were selective extraction with buffer containing EDTA, wheat germ agglutinin affinity chromatography, and removal of the related protein thrombospondin by heparin affinity chromatography. Particles of cartilage oligomeric matrix protein viewed by electron microscopy after rotary shadowing revealed structures similar to the prototype molecule purified from Swarm rat chondrosarcoma. The protein demonstrated a bouquet-like five-armed structure, with peripheral globular domains connected by thin flexible strands to a central assembly domain. Immunohistochemistry revealed age-dependent differences in the protein's distribution in cartilage. In normal human adult articular cartilage, there was a relatively uniform distribution throughout the interterritorial extracellular matrix, whereas in fetal articular cartilage, immunostaining was localized to the extracellular matrix directly adjacent to the chondrocytes. The isolation and characterization of human cartilage oligomeric matrix protein will facilitate its study in pathological conditions of human cartilage.

Adult↗
PubMed1995

Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate.

The expression of tissue transglutaminase in skeletal tissues is strictly regulated and correlates with chondrocyte differentiation and cartilage calcification in endochondral bone formation and in maturation of tracheal cartilage (Aeschlimann, D., A. Wetterwald, H. Fleisch, and M. Paulsson. 1993. J. Cell Biol. 120:1461-1470). We now demonstrate the transglutaminase reaction product, the gamma-glutamyl-epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cross-link specific antibody. Incorporation of the synthetic transglutaminase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypertrophic chondrocytes in the central, calcifying areas of the horseshoe-shaped cartilages. One predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix is osteonectin as revealed by incorporation of the dansyl label in culture. Indeed, nonreducible osteonectin-containing complexes of approximately 65, 90, and 175 kD can be extracted from mature tracheal cartilage. In vitro cross-linking of osteonectin by tissue transglutaminase gives similar products of approximately 90 and 175 kD, indicating that the complexes in cartilage represent osteonectin oligomers. The demonstration of extracellular transglutaminase activity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.

Antibody Specificity↗
PubMed1994

Cartilage oligomeric matrix protein (COMP) is an abundant component of tendon.

An abundant matrix protein was purified under native conditions from adult bovine tendon and identified as cartilage oligomeric matrix protein (COMP) by immunochemical crossreaction, amino acid sequence identity of tryptic peptides derived from both N- and C-terminal regions, and structure revealed by electron microscopy. Immunohistochemistry showed age-dependent differences in distribution of COMP in tendon.

Aging↗
PubMed1994

Native cartilage matrix protein (CMP). A compact trimer of subunits assembled via a coiled-coil alpha-helix.

Cartilage matrix protein (CMP), a major component of many types of cartilage, is a noncollagenous glycoprotein with a molecular mass of 148 kDa consisting of three identical subunits. With the aim of performing a more comprehensive characterization, we purified CMP in a native conformation from fetal bovine rib cartilage avoiding the denaturing solvents previously used. CMP could be selectively extracted with EDTA-containing buffer which indicates a divalent cation-dependent anchorage in the cartilage matrix. Determination of the amino-terminal sequence of the bovine protein confirmed its identity when compared with published cDNA sequences of chicken and human CMP. Electron microscopy revealed the presence of three ellipsoid subunits which are connected at one end. Sequence analysis indicated the presence of a coiled-coil alpha-helical assembly domain formed by the COOH-terminal end of the subunits. The trimeric structure was retained after complete reduction under native conditions which shows that the coiled-coil domain is stable also in the absence of interchain disulfide bonds.

Amino Acid Sequence↗
PubMed1994

Cartilage oligomeric matrix protein and thrombospondin 1. Purification from articular cartilage, electron microscopic structure, and chondrocyte binding.

Cartilage oligomeric matrix protein (COMP) and thrombospondin 1 (TSP1) were purified in a native form from normal bovine articular cartilage. The key step in the purification scheme was selective extraction with EDTA-containing buffer. Final separation of these two molecules was achieved by heparin affinity chromatography. Particles viewed by electron microscopy after rotary shadowing and negative staining revealed structures similar to their prototype molecules; from the Swarm rat chondrosarcoma for COMP, or from platelets for TSP1. Attachment of primary bovine chondrocytes to purified matrix proteins was investigated. Cells attached to COMP but not to the structurally related TSP1 indicating separate functions for these proteins in cartilage.

Amino Acid Sequence↗
PubMed1994

Characterization of native laminin from bovine kidney and comparison with other laminin variants.

A comprehensive characterization of laminin isoforms requires access to native preparations of laminins of a defined subunit composition. For this purpose an optimized isolation procedure was developed and shown to be broadly applicable to normal mammalian tissues. The protocol does in addition yield side fractions highly enriched in collagens XII and XIV. The major laminin purified from bovine kidney is indistinguishable from mouse Engelbreth-Holm-Swarm (EHS) tumor laminin in electron microscopy, but contains an A chain that migrates in a position intermediate to the Ae and the Am chains on SDS/PAGE. Antisera raised against mouse EHS-tumor laminin crossreact with B chains, but not with the A chain, of kidney laminin. Further, this A chain is not recognized by antisera raised against the Am chain. Laminins from heart and kidney both contain a significant subpopulation with a 190-kDa polypeptide identified as the B1s chain. The Am-containing laminins from heart and placenta differ morphologically from the Ae-containing EHS laminin in having one short arm that does not have the characteristic globule-rod-globule appearance. Further, the Am-containing laminins show a significantly higher thermal stability of the coiled-coil alpha-helical region in the long arm than does Ae-containing EHS laminin, indicating that certain combinations of laminin chains interact more strongly than others.

Animals↗
PubMed1994

Adherence of coagulase-negative staphylococci to heparin and other glycosaminoglycans immobilized on polymer surfaces.

The adherence of clinical isolates of staphylococci to surfaces immobilized with various glycosaminoglycans (GAGs) was studied. In general, cells of strains of coagulase-negative (CNS) staphylococci showed a greater adherence to polyethylene surfaces than did cells of Staphylococcus aureus, as studied by bioluminescence. When the surface was heparinized, the adherence of staphylococcal cells decreased, but CNS cells still adhered in greater numbers than did cells of S. aureus. The adherence of CNS to serum-coated heparinized surfaces was of the same magnitude, or increased compared with nonheparinized surfaces. When the surfaces were preadsorbed with different proteins with known heparin-binding domains, i.e., vitronectin, fibronectin, laminin, or collagen, the S. epidermidis cells showed higher binding to heparinized surfaces than to nonheparinized ones, and also in greater numbers than did other staphylococcal cells. Different CNS strains showed a greater ability to agglutinate polystyrene beads immobilized with heparin than did S. aureus. The adherence of S. epidermidis strain 3380 to polyethylene coated with various GAGs such as heparin and chondroitin, dextran, dermatan, and heparan sulfate was shown to be pH-dependent, with the highest adherence at pH 7.2. This may indicate that CNS have the ability to bind to other domains of host proteins when they are adsorbed to heparinized surfaces, versus to nonheparinized ones.

Bacterial Adhesion↗
PubMed1994

Laminin isoforms promote attachment of hepatocytes via different integrins.

Three isoforms of laminin were compared for the ability to promote adhesion of primary rat hepatocytes. In tests of initial attachment to these substrates, kidney laminin (alpha k, beta 1, gamma 1, and alpha k, beta 2, gamma 1) was shown to be a more efficient substrate than Engelbreth Holm Swarm (EHS) (alpha 1, beta 1, gamma 1) or heart laminin (alpha 2, beta 1, gamma 1, and alpha 2, beta 2, gamma 1). Hepatocyte attachment to EHS laminin and heart laminin was completely inhibited by antibodies specific for the integrin subunit beta 1, while a combination of beta 1 integrin antibodies and GRGDS peptide was needed for total inhibition of hepatocyte attachment to kidney laminin. Antiserum directed to the integrin subunit beta 3 could not substitute for the GRGDS peptide in this inhibition. Antibodies against the integrin subunit alpha 1 efficiently blocked adhesion of hepatocytes to collagen type I and to the P1 domain of EHS laminin. However, this antibody had essentially no effect on the attachment to kidney laminin and had only a minor inhibitory effect on attachment to heart laminin, to intact EHS laminin, or to the isolated fragment E8 of EHS laminin. Combining the alpha 1 integrin antibody with GRGDS peptide gave no further inhibition on these substrates. These results show that a recently described isoform of laminin from bovine kidney is an efficient substrate for initial attachment of hepatocytes, interacting with at least one beta 1-containing integrin and an RGD-dependent integrin not containing beta 1 or beta 3 subunits. Native EHS laminin uses integrin alpha 1 beta 1 for hepatocyte binding to the center of the cross (fragment P1) and other beta 1 integrin(s) in addition to alpha 1 beta 1 as receptors for the distal part of the long arm (fragment E8).

Animals↗
PubMed1994

Adhesion of coagulase-negative staphylococci and adsorption of plasma proteins to heparinized polymer surfaces.

Protease treatment of cells of coagulase-negative staphylococci reduced the adhesion of bacteria to heparinized polyethylene preadsorbed with serum. Fibronectin (Fn), fibrinogen (Fg), vitronectin, complement factor C3c, plasminogen, laminin and to a low extent albumin were detected on tridodecylmethylammonium chloride (TDMAC)-heparinized polyvinyl chloride (PVC) catheters extirpated from the circulation of patients. Using a perfusion model we show that during the first hours of perfusion with human plasma, Fn and Vn dominate, whereas after 22 h of perfusion Fg is the dominant protein. Field emission scanning electron microscopy and atomic force microscopy studies on TDMAC-heparinized catheters as well as on end-point attached heparinized PVC catheters indicate that quantitatively more Fg than Fn is exposed on these surfaces after prolonged exposure (> 22 h) to human plasma.

Adsorption↗
PubMed1994

Protein adsorption of poly(ether urethane) surfaces modified by amphiphilic and hydrophilic polymers.

A commercial biomedical poly(ether urethane), Pellethane 2363-80AE, was surface modified through the use of amphiphilic polymeric additives, and through surface grafting with poly(ethylene glycol), PEG. Two different amphiphilic polymers, Polymer C and Pluronic PE9400, were used as additives. Polymer C, a segmented polyurethane, was prepared from PEG1500, 4,4'-diphenylmethane diisocyanate and a C16-C18 monoglyceride chain extender. Pluronic PE9400 is a propylene oxide-ethylene oxide tri-block co-polymer obtained from BASF. Adsorption of human albumin and fibrinogen to the modified surfaces was studied by means of radiolabelled proteins. By contact angle measurements and X-ray photoelectron spectra the amphiphilic polymers were shown to accumulate at the polyurethane surfaces. Adsorption of fibrinogen, in particular, was significantly reduced by the amphiphilic additives to levels similar to those obtained for Pellethane surfaces grafted with PEG 20,000. In vitro clotting times for citrate-buffered blood in contact with the amphiphilic surfaces increased as compared with the unmodified ones.

Adsorption↗