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Biomedical subjects

M Wendel

Publications and source records attributed to M Wendel.

At least 19 recordsLinked to original sources
PubMed2002

Ultrastructural distribution of osteoadherin in rat bone shows a pattern similar to that of bone sialoprotein.

Osteoadherin (OSAD) is a keratan sulfate proteoglycan recently isolated from bovine and rat bone. Based on results obtained from in vitro experiments, the protein was shown to bind osteoblasts via the integrin receptor alpha v beta 3. Due to OSAD's capacity to bind hydroxyapatite crystals, a role for the protein in the mineralization process has also been suggested. To test these hypotheses in an in vivo model, the ultrastructural localization of OSAD in bone, tibial (metaphyses and diaphyses), and calvarial samples from normal 10 to 12-day-old rats were examined by immunohistochemical techniques at the ultrastructural level. In addition to the qualitative studies, quantitative measurements of OSAD marker density were performed in relevant compartments. Immunolabeling for OSAD was located to the mineralized bone matrix, with highest concentration of marker at the border between bone and cartilage remnants in the metaphyseal trabeculi. Intracellular labeling was low and no systemic accumulation of OSAD markers was observed at the cell-matrix interface. The observed distribution pattern of OSAD is strikingly similar to that of bone sialoprotein (BSP), confirmed by double labeling. The results of the current study support a role for OSAD in the mineralization process. In this process BSP is assumed to be a nucleator of hydroxyapatite crystals, and OSAD could work in concert with BSP to regulate nucleation. However, the mechanisms involved remain to be elucidated.

Animals↗
PubMed2001

Recurrent peritoneal mesothelioma with long-delayed recurrence.

Peritoneal mesothelioma is a rare malignancy with a poor prognosis. Etiology is unclear and presentation of this disease is often atypical. There are very few cases of long-term survival in patients with peritoneal mesothelioma. We report the case of a patient with peritoneal mesothelioma of 19 years' duration. To the best of our knowledge, there have been no reports of recurrent peritoneal mesothelioma with such long-delayed recurrence and survival. Recurrent peritoneal mesothelioma in the current patient was confirmed by comparing the biopsies of omentum taken in 1979 with that taken in 1997. An extensive literature search was undertaken to look for previous reports of recurrent peritoneal mesothelioma. Aggressive and intensive chemotherapy appeared to provide a longer survival time. Our patient had paraneoplastic manifestations that included paraneoplastic hepatopathy and a wasting syndrome. Peritoneal mesothelioma is a rare malignancy with one to two cases per million in the United States. It is known to exhibit unusual paraneoplastic manifestations, including paraneoplastic hepatopathy. Treatment is often unsuccessful, with a median survival time of approximately 18 months from diagnosis. A better understanding and aggressive management of this disease appear to help prolong survival.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

Osteoadherin, a cell-binding keratan sulfate proteoglycan in bone, belongs to the family of leucine-rich repeat proteins of the extracellular matrix.

Osteoadherin is a recently described bone proteoglycan containing keratan sulfate. It promotes integrin (alphav beta3)-mediated cell binding (Wendel, M., Sommarin, Y., and Heinegârd, D. (1998) J. Cell Biol. 141, 839-847). The primary structure of bovine osteoadherin has now been determined by nucleotide sequencing of a cDNA clone from a primary bovine osteoblast expression library. The entire translated primary sequence corresponds to a 49,116-Da protein with a calculated isoelectric point for the mature protein of 5.2. The dominating feature is a central region consisting of 11 B-type, leucine-rich repeats ranging in length from 20 to 30 residues. The full, primary sequence contains four putative sites for tyrosine sulfation, three of which are at the N-terminal end of the molecule. There are six potential sites for N-linked glycosylation present. Osteoadherin shows highest sequence identity, 42%, to bovine keratocan and 37-38% identity to bovine fibromodulin, lumican, and human PRELP. Unique to osteoadherin is the presence of a large and very acidic C-terminal domain. The distribution of cysteine residues resembles that of other leucine-rich repeat proteins except for two centrally located cysteines. Northern blot analysis of RNA samples from various bovine tissues showed a 4.5-kilobase pair message for osteoadherin to be expressed in bone only. Osteoadherin mRNA was detected by in situ hybridization in mature osteoblasts located superficially on trabecular bone.

Amino Acid Sequence↗
PubMed1998

Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone.

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride-containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mof the proteoglycan was 85, 000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin alphavbeta3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts.

Amino Acid Sequence↗
PubMed1998

Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex.

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.

Adenosine Triphosphatases↗
PubMed1998

Mouse models for extracellular matrix diseases.

Mutations of a number of genes encoding for extracellular matrix (ECM) proteins in mice have provided new insights regarding their role during development and disease. Many mouse strains have helped to verify the link between mutation and disease in humans, and others have produced unexpected phenotypes and identified new functions for ECM proteins. Finally, some null mutations in ECM genes provide no phenotypic alterations in mice, confronting the scientific community with a new challenge to search for their functions. This review lists all mouse strains with spontaneous and experimentally induced mutations in ECM genes. The phenotypes of these mice are discussed in comparison with the human diseases.

Animals↗
PubMed1997

Cartilage matrix macromolecules in lavage fluid of temporomandibular joints before and 6 months after diskectomy.

The purpose was to investigate if antigenic fragments of aggrecan and cartilage oligomeric matrix protein (COMP) are detectable by enzyme-linked immunosorbent assay in lavage fluids from the temporomandibular joint (TMJ) and to examine if the relative content of these cartilage markers changes during development of osteoarthrosis (OA) after diskectomy. Lavage fluid was obtained at surgery and 6 months postoperatively in 13 patients. Computed tomography or magnetic resonance imaging was without evidence of hard-tissue changes prior to surgery in all patients. In 9 of the patients, sufficient material for analysis was obtained at both examinations. Aggrecan and COMP were detectable in all but 2 fluids, in which the COMP levels were below detection limit. The aggrecan/COMP ratio increased in all 9 patients during the 6-month period, indicating increased release of aggrecan relative to COMP fragments. The changed aggrecan/COMP ratio possibly reflects increased cartilage turnover during development of OA. Changes compatible with OA were present on computed tomography in all cases at the 6-month follow-up. This study shows that the lavage procedure is feasible for obtaining synovial fluid from the TMJ for immunochemical analyses of tissue-derived macromolecules.

Adolescent↗
PubMed1996

Enhancement of hepatitis C virus NS3 proteinase activity by association with NS4A-specific synthetic peptides: identification of sequence and critical residues of NS4A for the cofactor activity.

The NS3 proteinase of hepatitis C virus utilizes NS4A as a cofactor for cleavages at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region of the viral polyprotein. To characterize NS4A for its role in modulating the NS3 proteinase activity at various cleavage sites, synthetic peptides spanning various parts of NS4A were synthesized and tested in a cell-free trans-cleavage reaction using purified NS3 proteinase domain and polyprotein substrates. The NS3 proteinase domain was expressed in Escherichia coli, purified, denatured, and refolded to an enzymatically active form. We found that a 12-amino-acid peptide containing amino acid residues 22 to 33 in NS4A (CVVIVGRIVLSG) was sufficient for cofactor activity in NS3-mediated proteolysis. The peptide enhanced the cleavage at the NS5A/5B site and was necessary for NS3-mediated cleavage at NS4A/4B and NS4B/5A. Sequential amino acid substitution within the designated peptide identified residues I29 and I25 as critical for potential cofactor activity. We provide evidence that the NS4A peptide and the NS3 catalytic domain form an enzymatically active complex. These data suggest that the central 12-amino-acid peptide (aa 22-33) of NS4A is primarily important for the cofactor activity through complex formation with NS3, and the interaction may represent a new target for antiviral drug development.

Amino Acid Sequence↗
PubMed1995

Isolation, characterization, and primary structure of a calcium-binding 63-kDa bone protein.

A novel noncollagenous protein of the mineralized matrix of bovine bone was isolated by ion exchange and gel permeation chromatography. The apparent M(r) of the protein is 63,000 as determined by SDS-polyacrylamide gel electrophoresis. The protein is a rather minor constituent in bone and could not be detected in other connective tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. The 63-kDa protein was detected in the osteoid and around the osteocytes upon immuno-histochemical staining of bovine compact bone. The sequence of the 63-kDa protein was deduced from cDNA clones isolated from a rat calvaria lambda gt11 expression library. The protein contains two centrally located EF-hand Ca(2+)-binding domains. Seven heptad repeats are present indicating the ability of the protein for coiled-coil interactions. Ability to bind calcium was confirmed by 45Ca2+ binding to protein blotted onto nitrocellulose membrane. The protein was synthesized in calvaria explants as detected by immunoprecipitation of radiolabeled protein from the culture medium. Although the protein can be detected in biochemical amounts in bone only, varying amounts of mRNA for this protein were detected in several rat tissues by RNase protection assay with highest levels in rat calvaria. This extracellular protein corresponds to a mouse protein called nucleobindin.

Amino Acid Sequence↗
PubMed1994

Purification of a bone sialoprotein-binding protein from Staphylococcus aureus.

Bone sialoprotein (BSP) is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone and joint tissues [Rydén C., Maxe, I., Franzén, A., Ljungh, A., Heinegård, D. & Rubin, K. (1987) Lancet II, 515]. We now report on the purification of a cell-wall protein from Staphylococcus aureus, strain O24, that possesses affinity for bone sialoprotein. Staphylococcal cell-wall components with capacity to inhibit binding of 125I-labeled BSP to staphylococcal cells were solubilized with LiCl (1.0 M, pH 5.0). Preparative SDS/PAGE and protein-overlay experiments revealed that inhibitory activity present in LiCl extracts resided in a fraction of polypeptides with M(r) 75,000-110,000. Staphylococcal proteins solubilized with LiCl were chromatographed on a Mono-Q anion-exchange column. Inhibitory activity was eluted at 0.6-0.8 M NaCl and could be further purified by affinity chromatography on BSP-Sepharose. Elution of the affinity matrix with 0.1 M glycine, pH 3.0, specifically eluted inhibitory activity. Analysis by SDS/PAGE revealed a single M(r) 97,000 polypeptide in the eluate. The purified M(r) 97,000 protein bound BSP in protein-overlay experiments. LiCl extracts from S. aureus, strain E514 or Staphylococcus epidermidis, strain 7686, both lacking the capacity to bind BSP did not contain the M9r) 97,000 protein. Our data demonstrate the presence of a S. aureus cell-surface BSP-binding protein. This protein could be involved in bacterial tropism in osteomyelitis.

Bacterial Proteins↗
PubMed1994

Dephosphorylation of osteopontin and bone sialoprotein by osteoclastic tartrate-resistant acid phosphatase. Modulation of osteoclast adhesion in vitro.

The tartrate-resistant acid phosphatase (TRAP) of skeletal osteoclasts was found to partially dephosphorylate the bone matrix phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP). TRAP also partially dephosphorylated metabolically [32P]PO4-labeled OPN as well as BSP, whereas comparable amounts of either alkaline phosphatase or prostatic acid phosphatase, at their respective pH optima, were ineffective, indicating a certain preference of TRAP for these phosphoprotein substrates. It has previously (Flores, M., Norgärd, M., Heinegård, D., Reinholt, F. P., and Andersson, G. (1992) Exp. Cell Res. 201, 526-530) been shown that osteoclasts bind to OPN as well as to BSP coated onto glass. We can now show that the partially dephosphorylated proteins no longer support osteoclast binding. These results indicate that the secretion of TRAP from osteoclasts into the resorption area could exert a regulatory influence on the attachment of the cells to the bone surface. This could imply roles in the development of ruffled borders and/or in the regulation of osteoclast motility on the bone surface.

Acid Phosphatase↗
PubMed1994

Distribution and synthesis of bone sialoprotein in metaphyseal bone of young rats show a distinctly different pattern from that of osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two phosphorylated and highly glycosylated cell-binding proteins in bone. Both proteins bind to hydroxylapatite. The cell binding is mediated via an Arg-Gly-Asp (RGD) sequence and previous work indicates that both proteins can bind to the vitronectin receptor (alpha v beta 3). The present work shows that a prevailing localization of BSP in metaphyseal bone of the young rat is at the interface between calcified cartilage and bone. Thus BSP shows a conspicuous enrichment in the osteoid laid down by the invading osteoblasts immediately next to the calcified cartilage. Furthermore, the most prominent amount of BSP mRNA was detected in cells at the epiphyseal/metaphyseal border. As opposed to OPN, no prominent accumulation of BSP immunoreactivity was observed at bone surfaces that face cells. Also the synthesis OPN was most pronounced at sites very different from those of BSP. Thus, the most prominent amount of OPN mRNA was observed in cells close to the metaphyseal/diaphyseal border, where osteoclastic bone resorption is particularly active. Indeed, message was often found in cells surrounding osteoclasts without any detectable message. The distinctly different patterns of synthesis and expression of the two proteins indicate different roles in bone turnover at this stage of development. Thus, it appears that BSP has a specific role during the initial phases of bone formation at the cartilage/bone interface. On the other hand, the pattern of OPN synthesis and expression support and extend our previous data showing OPN particularly enriched at attachment sites of osteoclasts resorbing bone.

Amino Acid Sequence↗
PubMed1993

A major non-collagenous 62 kDa protein from rat bone mineralized matrix is identical to pp63 a phosphorylated glycoprotein from liver.

A protein present as a M(r) 62 k monomer and as several differently sized disulfide-bonded oligomers has been isolated from rat bone mineralized matrix. Its overall tissue distribution determined by ELISA immunoassays showed the protein present only in bone, tooth and in serum while aorta, cartilage, intestine, kidney, liver, muscle, skin, spleen and tendon were all negative. Despite that the 62 kDa protein was abundant and selectively found in bone, no positive cDNA clone could be identified in several rat bone libraries. Positive clones were, however, identified in a rat liver expression library. A cDNA clone of 1.3 kb hybridized in a Northern blotting assay to a 1.8 kb mRNA in rat liver. No hybridization signal was detected with RNA from bone, brain, lung, muscle, spleen and kidney. Sequence analysis of the isolated cDNA clone revealed a 50-bp untranslated region followed by an open reading frame of 357 amino acids. The open reading frame can be divided into a 17-amino acid signal peptide followed by the mature protein of 340 amino acids with alanine as its N-terminal amino acid. A short N-terminal amino acid sequence from the isolated 62-kDa bone protein verified the molecular identity of the cDNA clone. The primary structure of the 62-kDa liver protein was identical to a that of a 63-kDa phosphorylated glycoprotein (pp63) from liver.

Animals↗
PubMed1992

Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage.

An Mr = 524,000 oligomeric protein was isolated from bovine cartilage and designated COMP (Cartilage Oligomeric Matrix Protein). The protein is composed of disulfide-bonded subunits with an apparent Mr of 100,000 each. It is markedly anionic, probably due to its high contents of aspartic acid and glutamic acid, as well as to its substitution with negatively charged carbohydrates. COMP was found in all cartilages analyzed, but could not be detected in other tissues by enzyme-linked immunosorbent assay of guanidine HCl extracts. Within a given cartilage, COMP shows a preferential localization to the territorial matrix surrounding the chondrocytes.

Amino Acids↗
PubMed1992

Regenerative response to membranous and enchondral lyophilized allogeneic bone in rabbit skull defects.

The regenerative responses of inlays of lyophilized allogeneic bone of membranous (skull) and enchondral (tibia) origin were studied in an experimental cranioplasty model in rabbits. The lyophilized bone particles were also bioassayed for inductive bone production in an orthotopic critical size defect rat model. Three trephined calvarial defects were evaluated in each of 14 adult rabbits. The experimental materials were implanted into two of the defects and the third was left empty for control purposes. The implants disclosed no major structural divergences as assessed by scanning electron microscopy. Healing was evaluated by light microscopy and contact radiography after periods of four and 15 weeks. The lyophilized bone allografts of both embryonic origins displayed a similar fashion of bone regeneration, bone marrow reappearance, and volumetric density of trabecular bone substance and displayed no obvious differences between experimental groups or intervals. The two materials exhibited low osteoinductive potential.

Animals↗