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E Bartnik

Publications and source records attributed to E Bartnik.

At least 19 recordsLinked to original sources
PubMed2006

IL-1beta, but not BMP-7 leads to a dramatic change in the gene expression pattern of human adult articular chondrocytes--portraying the gene expression pattern in two donors.

Anabolic and catabolic cytokines and growth factors such as BMP-7 and IL-1beta play a central role in controlling the balance between degradation and repair of normal and (osteo)arthritic articular cartilage matrix. In this report, we investigated the response of articular chondrocytes to these factors IL-1beta and BMP-7 in terms of changes in gene expression levels. Large scale analysis was performed on primary human adult articular chondrocytes isolated from two human, independent donors cultured in alginate beads (non-stimulated and stimulated with IL-1beta and BMP-7 for 48 h) using Affymetrix gene chips (oligo-arrays). Biostatistical and bioinformatic evaluation of gene expression pattern was performed using the Resolver software (Rosetta). Part of the results were confirmed using real-time PCR. IL-1beta modulated significantly 909 out of 3459 genes detectable, whereas BMP-7 influenced only 36 out of 3440. BMP-7 induced mainly anabolic activation of chondrocytes including classical target genes such as collagen type II and aggrecan, while IL-1beta, both, significantly modulated the gene expression levels of numerous genes; namely, IL-1beta down-regulated the expression of anabolic genes and induced catabolic genes and mediators. Our data indicate that BMP-7 has only a limited effect on differentiated cells, whereas IL-1beta causes a dramatic change in gene expression pattern, i.e. induced or repressed much more genes. This presumably reflects the fact that BMP-7 signaling is effected via one pathway only (i.e. Smad-pathway) whereas IL-1beta is able to signal via a broad variety of intracellular signaling cascades involving the JNK, p38, NFkB and Erk pathways and even influencing BMP signaling.

Aged↗
PubMed2005

Comparison of the chondrosarcoma cell line SW1353 with primary human adult articular chondrocytes with regard to their gene expression profile and reactivity to IL-1beta.

OBJECTIVE: In this study, the human chondrosarcoma cell line SW1353 was investigated by gene expression analysis in order to validate it as an in vitro model for primary human (adult articular) chondrocytes (PHCs). METHODS: PHCs and SW1353 cells were cultured as high density monolayer cultures with and without 1ng/ml interleukin-1beta (IL-1beta). RNA was isolated and assayed using a custom-made oligonucleotide microarray representing 312 chondrocyte-relevant genes. The expression levels of selected genes were confirmed by real-time polymerase chain reaction and the gene expression profiles of the two cell types, both with and without IL-1beta treatment, were compared. RESULTS: Overall, gene expression profiling showed only very limited similarities between SW1353 cells and PHCs at the transcriptional level. Similarities were predominantly seen with respect to catabolic effects after IL-1beta treatment. In both cell systems matrix metalloproteinase-1 (MMP-1), MMP-3 and MMP-13 were strongly induced by IL-1beta, without significant induction of MMP-2. IL-6 was also found to be up-regulated by IL-1beta in both cellular models. On the other hand, intercellular mediators such as leukemia inhibitory factor (LIF) and bone morphogenetic protein-2 (BMP-2) were not induced by IL-1beta in SW1353 cells, but significantly up-regulated in PHCs. Bioinformatical analysis identified nuclear factor kappa-B (NFkappaB) as a common transcriptional regulator of IL-1beta induced genes in both SW1353 cells and PHCs, whereas other transcription factors were only found to be relevant for individual cell systems. CONCLUSION: Our data characterize SW1353 cells as a cell line with only a very limited potential to mimic PHCs, though SW1353 cells can be of value to study the induction of protease expression within cells, a phenomenon also seen in chondrocytes.

Aged↗
PubMed2003

YKL-39 (chitinase 3-like protein 2), but not YKL-40 (chitinase 3-like protein 1), is up regulated in osteoarthritic chondrocytes.

OBJECTIVE: To investigate quantitatively the mRNA expression levels of YKL-40, an established marker of rheumatoid and osteoarthritic cartilage degeneration in synovial fluid and serum, and a closely related molecule YKL-39, in articular chondrocytes. METHODS: cDNA array and online quantitative polymerase chain reaction (PCR) were used to measure mRNA expression levels of YKL-39 and YKL-40 in chondrocytes in normal, early degenerative, and late stage osteoarthritic cartilage samples. RESULTS: Expression analysis showed high levels of both proteins in normal articular chondrocytes, with lower levels of YKL-39 than YKL-40. Whereas YKL-40 was significantly down regulated in late stage osteoarthritic chondrocytes, YKL-39 was significantly up regulated. In vitro both YKLs were down regulated by interleukin 1beta. CONCLUSIONS: The up regulation of YKL-39 in osteoarthritic cartilage suggests that YKL-39 may be a more accurate marker of chondrocyte activation than YKL-40, although it has yet to be established as a suitable marker in synovial fluid and serum. The decreased expression of YKL-40 by osteoarthritic chondrocytes is surprising as increased levels have been reported in rheumatoid and osteoarthritic synovial fluid, where it may derive from activated synovial cells or osteophytic tissue or by increased matrix destruction in the osteoarthritic joint. YKL-39 and YKL-40 are potentially interesting marker molecules for arthritic joint disease because they are abundantly expressed by both normal and osteoarthritic chondrocytes.

Adipokines↗
PubMed2001

MELAS as an example of a mitochondrial disease.

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) is a disease mainly due to a mutation at position 3243 (A --> G) in the leucine tRNA gene in mitochondrial DNA. Symptoms of the disorder are complex and the exact pathogenesis is not understood. A review of the literature on the subject is presented.

Journal Article↗
PubMed2001

Human mitochondria in health, disease, ageing and cancer.

In every human cell there are hundreds of mitochondria, which are required for oxidative phosphorylation as well as many other metabolic processes. Each mitochondrion contains approximately 5 mitochondrial DNA molecules. These circular DNAs of 16.5 kb in size contain only 39 genes. Mutations in mitochondrial DNA are responsible for many diseases. Alterations in these molecules may also play a role in ageing and in tumour formation.

Journal Article↗
PubMed2000

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris.

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).

Blotting, Western↗
PubMed2000

Truncation of the amino-terminus of the recombinant aggrecan rAgg1mut leads to reduced cleavage at the aggrecanase site. Efficient aggrecanase catabolism may depend on multiple substrate interactions.

Aggrecanase cleavage at the Glu(373)-Ala(374) site in the interglobular domain of the cartilage proteoglycan aggrecan is a key event in arthritic diseases. The observation that substrates representing only the aggrecanase cleavage site are not catabolized efficiently by aggrecanase prompted us to investigate the requirement of aggrecanase for additional structural elements of its substrate other than the actual cleavage site. Based on the recombinant substrate rAgg1mut we constructed deletion mutants with successively truncated N- or C-termini of the interglobular domain. Catabolism by aggrecanase activities induced in rat chondrosarcoma cells, porcine chondrocytes, and by human recombinant ADAMTS4 showed a gradually decreasing catabolism of progressively shortened, N-terminal deletion mutants of the substrate rAgg1mut. A reduction to 32 amino acids N-terminal to the aggrecanase site resulted in a decrease of at least 42% of aggrecanase cleavage products as compared with the wild-type substrate. When only 16 amino acids preceded the Glu(373)-Ala(374) site, aggrecanase cleavage was completely inhibited. In contrast, C-terminal deletions did not negatively affect aggrecanase cleavage up to the reduction to 13 amino acids C-terminal to the cleavage site. Unlike aggrecanase(s), membrane type 1-matrix metalloprotease (MT1-MMP), able to cleave rAgg1mut both at the aggrecanase and the MMP site, was insensitive to N-terminal deletions regarding aggrecanase cleavage, indicating that the importance of the N-terminus is characteristic for aggrecanase(s). Taken together, the results demonstrate that the amino-terminus of rAgg1mut, containing the MMP site, plays an important role for efficient cleavage by aggrecanase(s), possibly by serving as a further site of interaction between the enzyme and its substrate.

ADAM Proteins↗
PubMed1999

A human putative Suv3-like RNA helicase is conserved between Rhodobacter and all eukaryotes.

We have cloned and sequenced a cDNA of the human homologue of the Saccharomyces cerevisiae Suv3 putative RNA helicase which is indispensable for mitochondrial function in yeast. The human Suv-3-like protein has a typical mitochondrial leader sequence. Northern blot data and analysis of ESTs in the data banks indicate that this human gene (SUPV3L1) is expressed in practically all tissues, though at different levels. Sequence homology analysis has shown a strong conservation of the protein in a number of eukaryotic organisms -- plants, mammals and fungi, but no close homologues exist in bacteria with the exception of the purple bacterium Rhodobacter sphaeroides. This gene is thus ubiquitously present in all eukaryotic organisms.

Amino Acid Sequence↗
PubMed1998

Differential expression of aggrecanase and matrix metalloproteinase activity in chondrocytes isolated from bovine and porcine articular cartilage.

The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (MMP) catabolism in this domain of aggrecan. Our studies have shown that (i) there are species differences in the expression of latent versus active MMP activity on the aggrecan IGD; (ii) interleukin-1alpha exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; however (vi) the physiological (tissue) inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity. Collectively, these studies show that aggrecanase and MMP catabolism of the aggrecan IGD are independent and uncoupled.

Animals↗
PubMed1998

Membrane type 1 matrix metalloproteinase (MT1-MMP) cleaves the recombinant aggrecan substrate rAgg1mut at the 'aggrecanase' and the MMP sites. Characterization of MT1-MMP catabolic activities on the interglobular domain of aggrecan.

The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704-709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor 'aggrecanase' catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269-20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the 'aggrecanase' site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS...) and BC-14 (anti-FFGV...) to distinguish between 'aggrecanase' and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong 'aggrecanase' activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for 'aggrecanase' activity in vivo.

Aggrecans↗
PubMed1998

Yeast nuclear PET127 gene can suppress deletions of the SUV3 or DSS1 genes: an indication of a functional interaction between 3' and 5' ends of mitochondrial mRNAs.

Saccharomyces cerevisiae nuclear genes SUV3 and DSS1 encode putative RNA helicase and RNase II, respectively, which are subunits of the mitochondrial degradosome (mtEXO): a three-protein complex which has a 3' to 5' exoribonuclease activity and plays a major role in regulating stability of mitochondrial RNA. Lack of either of the two gene products results in a respiratory negative phenotype, while on the molecular level it causes a total block of mitochondrial translation, loss of the in vitro exoribonuclease activity and changes in stability and processing of many mtRNAs. We have found that the yeast nuclear gene PET127 present on a low or high copy number vector can effectively suppress the effects of the SUV3 or DSS1 gene disruptions. Since the product of the PET127 gene is involved in processing of the 5' ends of mitochondrial mRNAs, we suggest that there is a functional coupling between the 5' and 3' ends of mitochondrial mRNAs.

Blotting, Northern↗
PubMed1997

Utilization of a recombinant substrate rAgg1 to study the biochemical properties of aggrecanase in cell culture systems.

This paper describes the first report of the production and use of an artificial recombinant protein substrate to study "aggrecanase" activity. The substrate (rAgg1) is composed of the complete interglobular domain (IGD) of human aggrecan flanked by the "marker" sequences FLAGTM at the amino terminus and the human immunoglobulin G1 constant region at the carboxyl terminus. The expressed protein occurs as large multimolecular aggregates (>120 kDa) that, upon reduction, consist of a major isoform of 72 kDa (containing the IGD) and a minor 39-kDa species that through alternative splicing has had the IGD deleted. Using this recombinant substrate we developed a novel agarose cell culture system containing either rat chondrosarcoma or bovine chondrocytes that could be used in studies of the biochemical characterization of aggrecanase activities. These studies showed the following. (i) rAgg1 is a suitable substrate for aggrecanase proteolysis. (ii) Aggrecanase activity was specifically induced by exposing chondrocytes to retinoic acid. (iii) A considerable time period was required to synthesize and/or activate aggrecanase, with considerable differences in that found in rat chondrosarcoma versus bovine chondrocyte culture systems. (iv) Aggrecanase cleavage of the aggrecan IGD does not require the presence of the G1 or G2 globular domains or keratan sulfate post-translational modification in the IGD. (v) Aggrecanase is a diffusible activity that does not require association with the chondrocyte plasma membrane or immediate pericellular matrix for its action.

Aggrecans↗
PubMed1997

Expression of membrane type 1 matrix metalloproteinase in human articular cartilage.

OBJECTIVE: To detect the message for membrane type 1 (MT1) matrix metalloproteinase (MMP) in articular cartilage and chondrosarcoma cells, to study its expression in osteoarthritis (OA), and to determine whether interleukin-1beta (IL-1beta) influences its expression. METHODS: Reverse transcription-polymerase chain reaction methods were used to detect message. Cloning and sequencing were applied to confirm the sequence. Northern blotting was used to quantify the message for MT1-MMP and to compare its expression in OA cartilage with or without IL-1beta treatment. In situ hybridization was utilized to show MT1-MMP transcripts in cartilage and to study the influence of IL-1beta. RESULTS: The results show that MT1-MMP messenger RNA (mRNA) is expressed in chondrosarcoma cells and OA chondrocytes. Results of the in situ hybridization confirmed the expression in OA cartilage as well as in normal cartilage. The level of mRNA was not modulated following IL-1beta stimulation. CONCLUSION: This study shows that MT1-MMP is expressed by chondrocytes. The similarities in mRNA levels in OA and normal chondrocytes suggest that regulation of MT1-MMP mRNA may not be a primary factor in OA.

Blotting, Northern↗
PubMed1997

Complexity of IL-1 beta induced gene expression pattern in human articular chondrocytes.

The mRNA fingerprinting technique, differential display reverse transcription polymerase chain (DDRT-PCR), was used to detect changes in the overall pattern of gene expression in human articular knee chondrocytes-induced by interleukin-1 beta (IL-1 beta), the prototypical inducer of catabolic responses in degenerate joint diseases. One hundred different primer combinations generated approximately 10,000 different PCR fragments for IL-1 beta treated, as well as for untreated human chondrocytes, cultivated in alginate beads. This represented 53% of all expressed chondrocyte genes as based on statistical considerations. Side by side comparisons of differential display patterns originating from two different donor tissues yielded 44 reproducibly, differentially-displayed cDNA fragments, which were subcloned and sequenced. Sequence homology searches revealed sequence identities to the human necrosis factor alpha (TNF-alpha) and IL-1 regulated gene TSG-6, fibronectin, osteopontin, calnexin, and the DNA repair enzyme ERCC5. The differential expression was confirmed with Northern and quantitative PCR analyses. The known function of these genes and their known IL-1 responsiveness indicate that the employed model system reflects the pleiotropic effects of IL-1 on the overall gene expression in human articular chondrocytes and identifies genes involved in very different biochemical pathways. Twenty-seven cDNAs lacked sequence homologies to known genes and may represent novel genes.

Aged↗
PubMed1996

alpha(1,3)Fucosyltransferase expression in E-selectin-mediated binding of gastrointestinal tumor cells.

E-selectin recognizes the oncofetal antigen sialyl-Lewis X, which is highly expressed in adenocarcinoma. Five alpha(1,3)fucosyltransferases (FT) have been cloned that confer cell-surface expression of sialyl-Lewis X on transfected cells. We show here that 12/18 gastrointestinal-tumor cell lines bind specifically to immobilized E-selectin and that in sialyl-Lewis-X-positive cells binding is inhibited with a monoclonal antibody against sialyl-Lewis X. Using RT-PCR, we determined the expression of the alpha(1,3)fucosyltransferases III, IV, V, VI and VII in gastrointestinal tumor cells. Transcripts of FT IV and FT VII are abundantly expressed in all tested cells. Therefore no single fucosyltransferase could be correlated with the expression of sialyl-Lewis X and the ability of the tumor cells to bind to E-selectin. The data suggest that in gastrointestinal-tumor cells sialyl-Lewis X is necessary but not sufficient for E-selectin binding.

Base Sequence↗