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N Hauser

Publications and source records attributed to N Hauser.

At least 19 recordsLinked to original sources
PubMed1999

Matrilin-2, a large, oligomeric matrix protein, is expressed by a great variety of cells and forms fibrillar networks.

Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.

Animals↗
PubMed1999

[Cartilage oligomeric matrix protein (COMP): the role of a non-collagen cartilage matrix protein as a marker of disease activity and joint destruction in patients with rheumatoid arthritis and osteoarthritis].

UNLABELLED: Today, we can assess criteria to predict the tissue destruction and progression of Rheumatoid Arthritis (RA) and Osteoarthritis (OA) only in a late stage of the disease. It would be an advantage to have biochemical markers of disease activity and joint destruction to optimize therapy. PATIENTS AND METHODS: In this cross-sectional study with 37 RA and 20 OA patients (disease duration 119 +/- 130 months for RA and 41 +/- 73 months for OA), ESR, CRP, disease activity score (DAS), the functional status of RA (American College of Rheumatology), and the radiological scoring systems of Larsen and Kellgren/Lawrence, respectively, were used as parameters for disease activity and joint destruction. Cartilage oligomeric matrix protein (COMP) was measured with an enzyme-linked immunosorbent assay (ELISA) in serum and synovial fluid, COMP fragments with immunoblot in the synovial fluid. RESULTS: The mean COMP value in synovial fluid was 38 ug/ml (RA) and 46 ug/ml (OA); 6.5 ug/ml (RA) and 3.4 ug/ml (OA) in serum. RA patients had a higher amount of small COMP fragments in synovial fluid than OA patients. In RA patients, there was a significant positive correlation between disease activity (DAS) and COMP in synovial fluid and serum, a negative correlation between functional status of RA and serum COMP and between radiologic joint destruction of the knee and serum COMP. In OA patients, there was a significant correlation of joint space width and synovial fluid COMP. DISCUSSION: A high clinical disease activity (DAS) correlated with high COMP values in serum and synovial fluid and with increasing proteolytic activity (higher amount of small COMP fragments especially in RA). An increased turnover of cartilage matrix in joint inflammation might explain this correlation. The correlation of decreased COMP with decreased functional status in RA and increased joint destruction is compatible with a loss of cartilage and less turnover. The correlation between joint space width and increased COMP in OA patients with short disease duration might be explained with a higher turnover of the cartilage matrix in the early stage of the disease.

Adult↗
PubMed1999

Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

Animals↗
PubMed1998

[From chondrocyte culture to joint cartilage replacement. Development of de novo cartilage in vitro].

Local repair of acute or chronic cartilage lesions has not been successful so far. An attempt has been made to use synthetic materials to improve the quality of the repair tissue, but no method has achieved reliable regrowth of normal hyaline cartilage with adequate biomechanical properties and bonding to surrounding tissue. After publication of the first short-term results of chondrocyte transplantation in patients with localized cartilage lesions of the knee joints by a Swedish group in 1994 [1], the situation seems to have changed. Even though the advantages of this method of chondrocyte transplantation is a matter of controversy, the interest in the so-called "Carticel" approach has grown steadily. Indeed, the technique was recently approved by the FDA, on condition of a randomized, "placebo"-controlled trial. In view of this rapid development, we feel that independent experimental studies are urgently needed. In this article we present our own results in synthesizing de novo cartilage from cultured and phenotypically stable chondrocytes in a truly three-dimensional cartilage-like polyanionic matrix. With the experience gained in animals, we expect to set the stage for future experimental therapy in young human patients with early cartilage lesions.

Animals↗
PubMed1998

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids.

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.

Arthritis, Rheumatoid↗
PubMed1997

Inhibition of the electrostatic interaction between beta-amyloid peptide and membranes prevents beta-amyloid-induced toxicity.

The accumulation of beta-amyloid peptides (Abeta) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Abeta is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Abeta toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Abeta1-42 and the toxic fragment Abeta25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) reduction. Formation of the toxic beta-sheet conformation by Abeta peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Abeta1-40 and Abeta25-35 to negatively charged lipid vesicles and Abeta induced cell toxicity. These results suggest that Abeta toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.

Amyloid beta-Peptides↗
PubMed1997

Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays.

Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.

Carbohydrate Sequence↗
PubMed1997

Ultrastructure and cytochemical detection of alkaline phosphatase in long-term cultures of osteoblast-like cells from rat calvaria.

Two methods of collecting osteoblast-like cells from newborn rat calvaria were tested, either placing individual glass fragments or tipping dense glass beads onto the endocranial surface of periosteum-free bone. Inoculated at high density, cells collected by using these two methods form large mineralized plates after three weeks of culture. The main purpose of our investigation was to analyze the progressive formation of this mineralized structure and to localize alkaline phosphatase activity. At the beginning of the culture, flattened cells gathered into multilayers and synthesized collagen fibers. Cells in the upper layer became rapidly cuboidal in shape and continued to secrete collagen at their basal pole, whereas other cells became progressively embedded in the extracellular matrix. The upper cells featured ultrastructural characters of osteoblasts, whereas the embedded cells resembled osteocytes. After two weeks, the matrix began to mineralize: crystals appeared on collagen fibers, on matrix vesicles, and on cell debris. During the first days of the culture, the alkaline phosphatase activity was localized on the plasma membranes and on the collagen fibers. Thereafter, only the upper cells and collagen fibers that were juxtaposed to these cells showed alkaline phosphatase activity. In addition, the presence of mineralized matrix prevented the reaction product from being visualized on collagen fibers. The ultrastructural analysis reveals large mineralized plates with a structure resembling that of bone in vivo. This culture appears to be an appropriate model to study bone formation and regulation.

Alkaline Phosphatase↗
PubMed1997

Small fragments of cartilage oligomeric matrix protein in synovial fluid and serum as markers for cartilage degradation.

We determined the tissue distribution of cartilage oligomeric matrix protein (COMP) in man and evaluated COMP in synovial fluid (SF) and serum. COMP was purified from human articular cartilage. Polyclonal antibodies were used to detect COMP in tissue cryosections and protein extracts. COMP was determined quantitatively and qualitatively in SF and serum by competitive enzyme-linked immunosorbent assay and immunoblotting. Knee joint SF was taken from nine cadaveric and six living controls, 52 patients with osteoarthritis (OA), 85 patients with rheumatoid arthritis (RA) and 60 patients with other forms of inflammatory arthritis. The degradative potential of SF on native COMP was tested in vitro. The highest concentrations of COMP were measured in articular cartilage and meniscus, the lowest in rib and trachea. Compared with controls, the concentrations of COMP in SF and serum were elevated in 36 and 50% of the patients. A total of 84% of patients with RA and 60% of patients with other forms of inflammatory arthritis showed significant amounts of low-molecular-weight COMP fragments (50-70 kDa) in SF. In contrast, SF fragments were present in only 21% of the OA patients. Furthermore, 13% of SF taken from patients with RA or other forms of inflammatory arthritis were able to degrade COMP in vitro. Using inhibitors, the involvement of serine proteinases could be demonstrated in only 8% of the cases. Based on these results, the absolute levels of COMP in SF and serum, and its fragmentation pattern in SF, seem to be promising as markers of joint tissue metabolism.

Adult↗
PubMed1996

Interaction of cartilage matrix protein with aggrecan. Increased covalent cross-linking with tissue maturation.

Cartilage matrix protein (CMP) is a trimeric protein present in many types of cartilage extracellular matrix. It has recently been purified under native conditions that allowed the proposal of a structural model (Hauser, N., and Paulsson, M. (1994) J. Biol. Chem. 269, 25747-25753). To examine the functional properties of CMP we studied its interaction with aggrecan within cartilage extracellular matrix. Aggrecan-enriched fractions were purified from bovine tracheal cartilage of different ages under nondenaturing and denaturing conditions, respectively, and characterized by a combination of biochemical methods and electron microscopy. The fractions contained a pool of CMP noncovalently associated with aggrecan as well as a pool of CMP that appears covalently cross-linked to the aggrecan core protein. Only about two thirds of the CMP subunits could be released even upon reduction under denaturing conditions. It appears that CMP is attached by a nonreducible covalent interaction of one of its subunits with the protein core. The amount of CMP strongly bound to aggrecan increases with age. Electron microscopy revealed interaction sites for CMP in the extended chondroitin-sulfate attachment domain E2. In old tissue five distinct binding sites for CMP were found while in young cartilage only three of these were occupied. The extent of decoration of E2 with CMP increases with age.

Aggrecans↗
PubMed1996

N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast.

The endoplasmic reticulum (ER) of eukaryotic cells contains a quality control system, that is required for the proteolytic removal of aberrantly folded proteins that accumulate in this organelle. We used genetic and biochemical methods to analyse the involvement of N-glycosylation in the degradation of a mutant derivative of carboxypeptidase yscY in the ER of the yeast Saccharomyces cerevisiae. Our results demonstrate that N-glycosylation of this protein is required for its degradation since an unglycosylated species is retained stably in the ER. Cells that were devoid of the ER-processing alpha 1,2-mannosidase showed reduced degradation of the glycosylated substrate protein. Disruption of CNE1, a gene encoding a putative yeast homologue for calnexin, did not exhibit any effects on the degradation of this substrate protein in vivo. Also, the alpha 1,2-mannosidase-dependent reduction in the degradation rate did not show any correlation with the function of the CNE1 gene product. Our results suggest that the ER of yeast contains a glycosylation-dependent quality control system, as has been shown for higher eukaryotic cells.

Calcium-Binding Proteins↗
PubMed1996

Cloning, sequencing and expression analysis of mouse cartilage matrix protein cDNA.

A cDNA encoding the mouse cartilage matrix protein (CMP) was cloned following the reverse-transcription polymerase chain reaction and rapid amplification of cDNA ends procedures using mRNA isolated from trachea. The open reading frame encodes a product of 500 amino acids. Large parts of the protein have been completely conserved when compared to chicken and human sequences, including all 12 cysteine residues of the mature CMP. In situ hybridization reveals an even distribution of the CMP mRNA in the developing skeleton, which is followed by a zonal distribution paralleling hypertrophy and calcification. From early cartilage differentiation and onwards, CMP transcript is absent in the forming articular surfaces and intervertebral discs. Extraskeletal expression of CMP mRNA was detected in the adult eye.

Amino Acid Sequence↗
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

Beta-amyloid-induced cell toxicity: enhancement of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dependent cell death.

In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of beta-amyloid (Abeta) peptides have been widely studied. At high micromolar concentrations Abeta peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Abeta peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Abeta peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Abeta render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.

Amyloid beta-Peptides↗
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↗
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↗