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J Mollenhauer

Publications and source records attributed to J Mollenhauer.

At least 55 records · Page 3Linked to original sources
PubMed1997

Annexin V interactions with collagen.

Annexin V was originally identified as a collagen-binding protein called anchorin CII and was isolated from chondrocyte membranes by affinity chromatography on native type II collagen. The binding of annexin V to native collagen type II is stable at physiological ionic strength when annexin V is reconstituted in liposomes. The binding to native collagen types II and X, and to some extent to type I as well, was confirmed using recombinant annexin V. A physiological role for annexin V interactions with extracellular collagen is consistent with the localization of annexin V on the outer cell surface of chondrocytes, microvilli of hypertrophic chondrocytes, fibroblasts and osteoblasts. A breakthrough in our understanding of the function of annexin V was made with the discovery of its calcium channel activity. At least one of several putative functions of annexin V became obvious from studies on matrix vesicles derived from calcifying cartilage. It was found that calcium uptake by matrix vesicles depend on collagen type II and type X binding to annexin V in the vesicles and was lost when collagens were digested with collagenase: calcium influx was reconstituted after adding back native collagen II or V. These findings indicate that annexin V plays a major role in matrix vesicle-initiated cartilage calcification as a collagen-regulated calcium channel.

Animals↗
PubMed1997

DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours.

Loss of sequences from human chromosome 10q has been associated with the progression of human cancer. Medulloblastoma and glioblastoma multiforme are the most common malignant brain tumours in children and adults, respectively. In glioblastoma multiforme, the most aggressive form, 80% of the tumours show loss of 10q. We have used representational difference analysis to identify a homozygous deletion at 10q25.3-26.1 in a medulloblastoma cell line and have cloned a novel gene, DMBT1, spanning this deletion. DMBT1 shows homology to the scavenger receptor cysteine-rich (SRCR) superfamily. Intragenic homozygous deletions has been detected in 2/20 medulloblastomas and in 9/39 glioblastomas multiformes. Lack of DMBT1 expression has been demonstrated in 4/5 brain-tumour cell lines. We suggest that DMBT1 is a putative tumour-suppressor gene implicated in the carcinogenesis of medulloblastoma and glibolastoma multiforme.

Adult↗
PubMed1997

Analysis of 3H-proline-labeled protein by rapid filtration in multiwell plates for the study of collagen metabolism.

Quantitative and qualitative analysis of cell cultures labeled with 3H-proline for monitoring collagen synthesis is time-consuming and occasionally generates large quantities of radioactive waste. This present work describes the application of a microwell filtration system for the analysis of collagen metabolism in chondrocytes. It is based on trichloroacetic acid (TCA) precipitation of 3H-proline-labeled proteins onto polyvinylidene difluoride membranes fitted in a 96-well plate and subsequent analysis of precipitated proteins by liquid scintillation counting, amino acid analysis and sodium dodecyl sulfate (SDS) gel electrophoresis. This method allows for the initial processing of 96 samples within 2 h, has high sensitivity and accuracy (linearity > or = 200 cpm/sample) for quantitative measurements and a capacity of up to 10 micrograms collagen/microwell. The ratio of the radioactive protein collected by this filtration assay compared to that collected by molecular sieve chromatography on Sephadex G-25 was linear over a broad range, indicating full compatibility of data and a high reproducibility for both assay systems. The quality of protein separation by SDS-polyacrylamide gel electrophoresis (PAGE) from samples obtained by the filtration assay and by dialysis was virtually identical. These features make the assay particularly suited for pulse-chase experiments and for monitoring protein degradation.

Animals↗
PubMed1996

Collagen and proteoglycan production by bovine fetal and adult chondrocytes under low levels of calcium and zinc ions.

The experiments described herein tested the effects of CaCl2 and ZnCl2, added at various concentrations in the culture medium, upon the synthesis of collagen and proteoglycan by adult and fetal (articular, epiphyseal and hypertrophic) bovine chondrocytes maintained in high density multilayer cultures. CaCl2 concentrations below 0.5 mM or the addition of 1-50 microM ZnCl2 to the medium selectively promoted the production of collagen by all four populations of chondrocytes but had no effect on fibroblasts. Further, these changes had no statistically significant effect on the incorporation of 35S-sulfate into macromolecules or on the synthesis of gelatinase A, measured by gelatin zymography. The addition of CaCl2 and ZnCl2 at these concentrations did not result in a change in the relative proportion of non-crosslinked 3H-collagen molecules (synthesized in the presence of beta-aminopropionitrile) partitioning in the cell layer and medium compartments, and did not appreciably alter the pattern of collagens synthesized by any of the cell populations. The hypertrophic cells synthesized high levels of collagen type X in the presence as well as absence of exogenously added cations. However, CaCl2 at 10 mM caused a marked upregulation of collagen type X synthesis by a preparation of chondrocytes derived from the entire growth plate, consistent with the view that calcium at that concentration stimulated the differentiation of some of the cells into hypertrophic chondrocytes.

Animals↗
PubMed1994

Structural and functional comparison of anchorin CII (cartilage annexin V) and muscle annexin V.

Annexin V has been isolated from chicken muscle and cartilage either by EDTA extraction or by plasma membrane purification and solubilization with detergent to obtain the hydrophilic and hydrophobic variants. The hydrophobic variant of the cartilage annexin V associated with phosphatidylserine-containing liposomes in a Ca(2+)-independent manner, whereas the EDTA-extracted molecule required Ca2+ for association with the liposomes. The collagen-binding assay used is based on the principle of a cell attachment assay using mildly pepsinized collagen type II or intact collagen type I as the solid-phase substrate. Soluble intact collagen type I or II was added as competitive inhibitor. The lipophilic and the EDTA-extracted anchorins CII from cartilage were inhibited to the same extent by collagen type II on pepsinized collagen type II as the solid-phase substrate. The EDTA-extracted muscle annexin V exhibited a fivefold lower affinity to collagen type II than its counterpart from cartilage. Peptide mapping studies and amino acid sequencing of selected peptides from the hydrophobic cartilage annexin V and the hydrophilic cartilage and muscle annexin V revealed 100% identity to the established chicken annexin V protein sequence in the corresponding amino acids 7-29 and 118-126. These results indicate that annexin V may occur in multiple pools within one cell type and/or tissue and that its biological function may depend on the subcellular distribution as well as the microenvironment in the tissue.

Amino Acid Sequence↗
PubMed1994

A constitutive 65 kDa chondrocyte protein as a target antigen in adjuvant arthritis in Lewis rats.

The autoantigen in adjuvant arthritis in Lewis rats is still unknown despite the knowledge that the 65 kDa mycobacterial heat-shock protein (hsp) is involved in the disease process. T cells and antibodies obtained from rats with adjuvant arthritis respond to chondrocyte membrane antigen(s). In Western blots a 65 kDa chondrocyte membrane protein (CH65) is stained by sera from arthritic rats. In addition, spleen cells from rats with adjuvant arthritis proliferate in vitro to chondrocyte membranes and CH65 as antigens. Furthermore, pretreatment of rats with CH65 or mycobacterial hsp65 but not human hsp60, induces a significant retardation of the onset of adjuvant arthritis in Lewis rats. The data suggest that CH65 is a potential autoantigen involved in the pathogenesis of adjuvant arthritis in Lewis rats.

Animals↗
PubMed1994

Isolation and characterization of a cartilage-specific membrane antigen (CH65): comparison with cytokeratins and heat-shock proteins.

We report the isolation and characterization of a 65,000 MW chondrocyte autoantigen (CH65) which may be involved in rheumatoid arthritis. This chondrocyte-specific antigen reacted with sera from patients with rheumatoid arthritis (RA). CH65 did not cross-react with a polyclonal antibody raised against microbial heat-shock protein (hsp) 65, anti-human hsp 65 monoclonal antibodies (mAb) (LK1 and LK2), anti-microbial hsp 65 mAb (IA10, IIC8 and WTB-78H1) and anti-cytokeratin 8, 18, 19 mAb (NCL5D3MAb). CH65 could be purified from chicken chondrocyte membranes by ammonium sulphate precipitation and a novel electro-gel-filtration method. The amino acid analysis yielded an unusually high degree of glycine, serine and asparagine residues. The internal amino acid sequence obtained by tryptic digestion revealed homologies with the cytokeratin family. Despite these homologies, CH65 lacked immunological cross-reactivity with commercial anti-cytokeratin antibodies. Mice mAb generated against the purified CH65 (C6) were used to identify the protein as a tissue-specific constitutive protein membrane from chondrocytes. Sera from patients with RA cross-reacted with purified CH65. The stress or heat-shock protein (hsp 65), implicated in the development of experimental and clinical arthritis, showed no immunological cross-reactivity with CH65 in Western blots. These findings suggest that CH65 may represent an interesting cartilage-specific new antigen in RA. The availability of this antigen in purified form and specific mAb may offer useful tools in arthritis research.

Amino Acids↗
PubMed1993

Triplet repeat sequences in human DNA can be detected by hybridization to a synthetic (5'-CGG-3')17 oligodeoxyribonucleotide.

The seemingly autonomous amplification of naturally occurring triplet repeat sequences in the human genome has been implicated in the causation of human genetic disease, such as the fragile X (Martin-Bell) syndrome, myotonic dystrophy (Curshmann-Steinert), spinal and bulbar muscular atrophy (Kennedy's disease) and Huntington's disease. The molecular mechanisms underlying these triplet amplifications are still unknown. We demonstrate here that a synthetic (CGG)17 oligodeoxyribonucleotide can be utilized as hybridization probe to visualize some of the triplet repeats in the human genome. This technique may help in studies aimed at the elucidation of the amplification mechanism.

Autoradiography↗
PubMed1993

Characterization of macrophage proteins bearing the functional leukotriene D4-binding site of an anti-cysteinylleukotriene monoclonal antibody.

Protein conformations of the putative cysteinylleukotriene (LT) receptor of macrophages were characterized using anti-idiotypic IgG (AIAb) against an anti-LT monoclonal Ab (LTmAb). The AIAb nature of two rabbit antisera were demonstrated with titers of up to 1:1000 against F(ab')2 from the LTmAb (in an enzyme-linked immunoassay) which also inhibit LTD4 binding to the LTmAb (in a radioimmunoassay), whereas non-immunized rabbit serum was not reactive. The specific reactivity of Fc-purified AIAb towards LTmAb was measured by two fractions obtained after passage over columns of Sepharose either coupled with LTmAb (fraction A, representing immunoglobulins not absorbed to LTmAb) or coupled with homologous immunoglobins (fraction B, representing immunoglobulins not absorbed to homologous IgG). The difference in immunoreactivity between both fractions showed that fraction B contains AIAb against a LT-recognizing domain of the LTmAb (in enzyme-linked immunoassays coated with LTmAb and homologous IgG) and AIAb against the functional LT-binding site of LTmAb (in radioimmunoassay). Using the antisera, Western-blot analysis with peritoneal cell proteins detected signals at 236, 198, 118, 99, 75, 25 and 18 kDa. Dithiothreitol-reduced proteins were detected at 25 kDa and 18 kDa. In general, this suggested recognition of a 236-kDa oligomeric protein composed of subunits with molecular masses of 25 kDa and 18 kDa, including intramolecular disulfide bridges all bearing an epitope similar to the LTmAb. From these conformations, an overlay assay with [3H]LTD4 favoured a 75-kDa protein. Immunohistochemical analysis demonstrated that the recognized proteins may be located at cell membranes, because (a) in an ELISA, enriched plasma membrane preparations from peritoneal cells showed a threefold increase in reactivity to the AIAb, compared to the original cell homogenate; (b) after Western-blot analysis, the membrane-enriched protein fraction exhibited stronger protein signals than the microsomal fraction and the original cell homogenate; (c) regions of AIAb binding on the surface of cultured mouse peritoneal macrophages were detected by indirect immunofluorescence. Taken together, this study demonstrated AIAb binding to macrophage membrane-associated proteins bearing the LTD4-binding site of LTmAb, which may include identification of the putative LT receptor.

Animals↗
PubMed1993

Preferential cellular and humoral immune reactivities to native and denatured collagen types IX and XI in a patient with fatal relapsing polychondritis.

We describe a patient with histologically confirmed relapsing polychondritis, an episodic systemic disorder. Although the etiology is unknown and its pathogenesis is incompletely understood, there is evidence strongly suggesting immunologically mediated mechanisms. Enzyme linked immunosorbent assays, immunoblotting and cellular immune responses using lymphocyte proliferation assays showed strong parallel humoral and cellular immune reactivities against collagens type IX and XI. There was also a considerable response to collagen type II which, however, was less pronounced compared to collagen type IX and was directed to native epitopes. Our findings demonstrate a highly distinct immune response to minor matrix collagens in a destructive cartilage disease and thus strongly argue against nonspecific anticollagen immune reactions simply representing epiphenomena resulting from cartilage damage.

Aged↗
PubMed1992

The humoral immune response to heat shock proteins.

Humoral immune reactions to heat shock proteins (hsp) from microorganisms are one aspect of microbial infections in humans. The production of antibodies which are specific to epitopes present on procaryotic hsp leads also to the appearance of cross-reactive serum antibodies in the host organism that react with human hsp. This article discusses the consequences of such autoreactive antibodies for the host in context with the development of immune tolerance and autoimmune diseases, especially rheumatoid arthritis (RA), and in experimental animal models for arthritis such as adjuvant arthritis in rats. On the basis of epitope cross-reactivity between hsp and other host proteins, a hypothesis is presented for the development of autoimmune disease following the production of hsp-specific antibodies.

Animals↗
PubMed1992

Immunohistochemical study of extracellular material in the aged human synovial membrane.

Types III, IV, VI collagen and laminin distribution in synovial tissue of seven autopsy knee joints from old human donors (69-94 years of age) were investigated with immunocytochemical and immunohistochemical methods. The synovial intima is separated from the subintimal tissue by an intermediate fibrillar zone rich in staining for type III collagen. In the intima basement membrane-like material associated with synovial lining cells stains for type IV collagen and laminin. Fine fibrils surrounding the lining cells stain for type VI collagen. In two of the cases type VI collagen occurs mainly as long-spacing collagen, the distinct aggregated form of type VI collagen. This staining pattern was qualitatively the same in all different regions and cases investigated. However, considerable quantitative differences were seen.

Aged↗
PubMed1992

Metabolic chiral inversion of 2-arylpropionates in rat H4IIE and human Hep G2 hepatoma cells. Relationship to in vivo metabolism.

The inversion of 2-arylpropionic acids (2-APAs) has been investigated in vitro using rat H4IIE and human Hep G2 hepatoma cells in continuous culture. The effect of substrate concentration (15-150 micrograms/mL), cell density (1.5-12 x 10(6) cells/dish) and serum content of the culture medium (0-20%) on inversion was examined in rat H4IIE hepatoma cells using R-ibuprofen as model compound. Increasing R-ibuprofen concentrations and decreasing serum content of the medium resulted in increased inversion whereas variation of cell density had no effect. Furthermore, rat H4IIE and human Hep G2 hepatoma cells were incubated with the individual enantiomers of ibuprofen, ketoprofen and flurbiprofen under optimized culture conditions (serum-free culture medium). The elimination rate constants (kel) and fractions inverted (Fi) were determined. Although inversion occurred slowly in the tumor cells and thus long incubation periods (120 hr) were required, the hepatoma cells were nevertheless able to mimic qualitatively the species and substance specificity of inversion of 2-APAs as observed in vivo.

Animals↗
PubMed1992

Cytotoxic effects of rheumatoid arthritis sera on chondrocytes.

Human sera from patients with rheumatoid arthritis (RA) and also from healthy donors were found to be toxic to cultured chondrocytes. Immunoglobulins were found to bind to the surface of cultured cells and cells in chicken sternal cartilage, as detected by indirect immunofluorescence. In vitro, cell detachment from the substrate was caused by the incubation of chondrocyte monolayers with 5 per cent and less RA serum. Serum treatment caused cytotoxic degradation of the cells. This could be quantified by a chromium release assay. Heat inactivation of the serum abolished the cytotoxicity. The extent of the cytotoxic reaction was related to the complement content of the serum and also to the intensity of the disease, as determined by the Ritchie-index. Other cell types, as chondrosarcoma cells, normal fibroblasts and corneal epithelium, were not affected by RA sera.

Animals↗
PubMed1991

A monoclonal antibody against the sulfidopeptide leukotrienes LTC4, LTD4 and LTE4.

A monoclonal antibody (1A-LDR1) against sulfidopeptide leukotrienes (LT) is described. The mAb shows a nearly identical detection limit of about 0.04 ng for LTC4, LTD4, LTE4 and NacLTE4 in standard fluid phase RIA. Steric modifications, however, diminish the sensitivity, as determined for the examples 5-epi-LTC4, 6-epi-LTC4, 5,6-epi-LTC4 and 11-trans-LTC4. No crossreactivity could be observed for LTB4. Crossreactions with components of the LT peptide chain such as L-cysteine or glutathione, as well as with arachidonic acid, were not detectable. In assessing the accuracy of the LT-RIA, recovery experiments with supernatants of mouse peritoneal macrophages and incubates of gastric mucosa showed a good correlation of r = 0.993 and 0.990, respectively. Results of an inhibition experiment with mouse peritoneal macrophages, incubated with several concentrations of indomethacin and nordihydroguaiaretic acid (NDGA), support the reliability of RIA and ELISA. The new LT-mAB allows an almost complete detection of peptide leukotrienes in one assay.

Animals↗