Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Basic fibroblast growth factor (basic FGF) is a potent mitogen for chondrocytes in vitro and is present in developing cartilage in vivo. Studies of intracellular basic FGF localization in other cell types have revealed a transient nuclear presence. We have examined ovine fetal growth plate chondrocytes for the presence of intracellular basic FGF by immunocytochemistry. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 75 and 80 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. In non-synchronized cell cultures 58 +/- 6% of cells (mean +/- s.d., n = 3) demonstrated cytoplasmic staining for immunoreactive basic FGF. Of these cells, 18 +/- 3% also exhibited strong nuclear staining. Chondrocytes were growth-restricted and restarted into the cell cycle with 2% (vol/vol) fetal calf serum. The timing of S phase was followed by nuclear labelling of nuclei with [3H] thymidine followed by autoradiography, or by the incorporation of [3H] thymidine into trichloroacetic acid-precipitable DNA in parallel cultures. A cytoplasmic presence of immunoreactive basic FGF did not appear, following immunocytochemistry, until the second half of G1 with 97% of cells immunopositive 2 hr prior to S phase. Nuclear staining for basic FGF appeared 2 hr before peak nuclear labelling index, and 56% of cell nuclei were immunopositive. Following entry into S phase cytoplasmic and nuclear basic FGF immunostaining rapidly disappeared. When these experiments were repeated with or without the presence of anti-basic FGF antibody or heparin, the presence of the antibody significantly reduced peak [3H] thymidine incorporation into DNA during S phase while exposure to heparin increased this. However, the proportion of cells demonstrating cytoplasmic or nuclear staining for immunoreactive basic FGF, and the time of onset of staining, were unaltered. Incubation of cells with suramin blocked subsequent DNA synthesis and no intracellular basic FGF was visualized. Cell-conditioned culture medium, extracellular matrix and cytoplasm from synchronized cultures of chondrocytes were taken at time points throughout the cell cycle and assessed for basic FGF content by radioimmunoassay. Basic FGF was detectable in each compartment and steadily rose throughout the second half of G1 to reach maximum values around the S phase. The accumulation of basic FGF in medium, matrix and cytoplasm was blocked by the presence of cycloheximide.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cell cycle-dependent localization of immunoreactive basic fibroblast growth factor to cytoplasm and nucleus of isolated ovine fetal growth plate chondrocytes. 145 27

Recent studies suggest that proteolytic enzymes are involved in the degradation of extracellular matrix components of the renal glomerulus. In the present study, the effects of feeding 3 different protein diets on glomerular cysteine proteinase and metalloproteinase activities to healthy rats for 6 weeks were examined. The diets contained 5, 20, or 60% casein and were made isocaloric by starch. On sacrifice, the glomeruli were isolated by differential sieving. Proteolytic activities were measured using fluorogenic substrates and were expressed per glomerular DNA content. Body weight was virtually unchanged by the amount of protein ingested, whereas kidney weight was closely correlated with dietary protein content (5%: 1,625 +/- 324; 20%: 2,110 +/- 326; 60%: 2,705 +/- 910 mg). Activity of cathepsin B, the most abundant cysteine proteinase in the glomerulus, decreased with protein loading (5%: 1,498 +/- 110; 20%: 1,321 +/- 82; 60%: 914 +/- 84 pmol/min/micrograms DNA). The same pattern emerged with cathepsin L (5%: 869 +/- 71; 20%: 846 +/- 70; 60%: 517 +/- 83 pmol/min/micrograms DNA) and cathepsin H (5%: 498 +/- 45; 20%: 478 +/- 55; 60%: 330 +/- 39 pmol/min/micrograms DNA). The differences between the 20 and 60% groups were statistically significant for all 3 cathepsins measured. The intraglomerular activity of the metalloproteinase collagenase declined significantly with the amount of protein ingested (5%: 233 +/- 14; 20%: 189 +/- 13; 60%: 137 +/- 11 microU/micrograms DNA). Gelatinase activity also fell as protein intake increased (5%: 183 +/- 18; 20%: 115 +/- 10; 60%: 94 +/- 11 F/micrograms DNA).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of dietary protein on glomerular proteinase activities. 146 85

A complementary DNA clone synthesized from the chicken junD mRNA, containing 5'- and 3'-untranslated sequences, was inserted in the retroviral expression vector RCAS to yield the construct JD. A second RCAS construct (DDDD) contained only the coding domains of JunD. DDDD did not transform upon primary transfection, but JD produced small numbers of transformed cell foci in chicken embryo fibroblast cultures. The virus recovered from these foci, JDV, was moderately transforming for chicken fibroblasts and weakly oncogenic in the animal. Its genome was rearranged, showing evidence for two recombination events. The first crossover was located between 5'-untranslated and coding sequences of junD and incorporated part of the 5'-untranslated region into an open reading frame. The second crossover occurred between junD and gag. The two crossovers generate a single open reading frame of 2064 nucleotides that encodes an 85 kilodalton protein in which sequences in the amino-terminal region of JunD are duplicated. This gag junD reading frame was recloned and then reconstituted into a replication-defective but transformation-competent retrovirus, indicating that the Gag-JunD fusion protein is the effector of transformation. A construct containing this rearranged coding sequence of JunD in Rc/RSV transactivated the collagenase promoter in chicken cells. Southern blot analysis of several independently isolated JunD transformants and deletion analysis of JDV indicated that duplication of a domain in the amino-terminal region of JunD is crucial for transformation and transactivation.
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PMID:A rearranged junD transforms chicken embryo fibroblasts. 147 71

Recent studies suggest that proteolytic enzymes located within the glomerulus are involved in the degradation of extracellular matrix components. In the present investigation glomerular proteinase activities were followed in a variety of non-immune-mediated renal diseases as well as during different dietary manipulations. Azocaseinolysis was significantly reduced in the obese Zucker rat compared with lean littermates (pH 5.4:8.9 +/- 0.4 vs 11.4 +/- 0.7; pH 7.4:5.8 +/- 0.7 vs 9.3 +/- 0.6 arb. U/mg protein). When the glomerular proteolytic capacity was measured in old rats, again a significant decline in proteolysis was observed (pH 5.4:9.8 +/- 0.8 vs 17.7 +/- 0.8; pH 7.4:6.4 +/- 0.7 vs 11.7 +/- 0.5 arb. U/mg protein). In Goldblatt hypertensive rats the unclipped kidney, which is exposed to high blood pressure, revealed lower glomerular azocaseinolytic activity compared with the contralateral clipped kidney (pH 5.4:8.1 +/- 0.4 vs 12.9 +/- 0.5 arb. U/mg protein). In parallel, the cathepsin B content was also diminished in glomeruli from kidneys exposed to hypertension. When proteinases were followed in glomeruli from intact kidneys of rats fed protein-modified diets (fraction of casein 0.05, 0.20 or 0.60) a significant fall in the activities of cysteine proteinases, e.g. cathepsin B (casein 0.05:1,498 +/- 110 vs casein 0.60:914 +/- 84 microU/micrograms DNA), as well as metalloproteinases, e.g. collagenase (casein 0.05:233 +/- 14 vs casein 0.60:137 +/- 11 microU/micrograms DNA), occurred. These data indicate that in both early and late stages of glomerulosclerosis, proteolytic activities within the glomerulus tend to be reduced, which could allow extracellular matrix accumulation. Moreover, changes in dietary protein intake resulted in profound alterations of glomerular proteinases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of glomerular proteinases in the evolution of glomerulosclerosis. 149 56

Two clones were isolated by screening a shrimp hepatopancreas cDNA library with a DNA fragment obtained by PCR amplification using two oligonucleotides based on the partial protein sequence of Penaeus vanameii chymotrypsin purified earlier. One of these clones, PVC 7 contains a complete cDNA coding for a serine protease. The deduced amino acid sequence shows the existence of a 270 residue-long preproenzyme containing a highly hydrophobic signal peptide of 14 amino acids. This suggests the existence of a putative zymogen form of the enzyme containing a 30 amino acid-long peptide which is cleaved to give a mature protein of 226 residues. A highly preferred codon usage is observed for this protein. The other obtained cDNA was found to encode the less predominant variant of the protein. Sequence alignments show that shrimp chymotrypsin is highly homologous with crab collagenase (77% homology taking into account the same amino acid at the same position, and 83% homology taking into account amino acids with conserved function) and that it is more similar to mouse trypsin (41% homology of strictly conserved amino acids) than to hornet chymotrypsin (35% homology).
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PMID:Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda). 151 90

In situ hybridization was used to demonstrate serum amyloid A (SAA) gene expression in arthritic joint tissue from lentivirus-infected sheep and goats. SAA gene transcription occurs in isolated individual synovial cells and occasional giant cells but not in uninfected or uninflamed synovial tissue. These findings support the notion (derived from in vitro observations) that at least one member of the SAA gene/protein family may function as an autocrine collagenase inducer in inflammatory arthritis. Since we used heterologous DNA probes derived from human clones, our findings also support the growing evidence for interspecies conservation of SAA genes.
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PMID:Serum amyloid A gene transcription in synovial cells during retroviral arthritis. 151 61

To clarify the contribution of c-fos DNA to bone formation, the effect of constitutive expression of the c-fos gene in collagen synthesis was examined by introducing c-fos DNA into osteoblastic MC3T3-E1 cells. The [3H] proline incorporation into the collagenase digestible protein(CDP) and the percent collagen synthesis were significantly decreased in the c-fos transfectants which constitutively express c-fos mRNA as compared with control transfectants. Transcription of type 1(alpha 1) collagen gene was also specifically decreased in the c-fos transfectants. This indicates that constitutive expression of c-fos DNA interferes with bone formation by inhibiting collagen synthesis in osteoblasts.
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PMID:Constitutive expression of c-fos gene inhibits type 1 collagen synthesis in transfected osteoblasts. 154 Jan 82

In vitro, rheumatoid arthritis (RA) synovial cells display several of the characteristics of neoplastic and virally transformed cells. The recent observation that synovial cell cultures, derived from collagenase digests of synovial membranes from RA patients, proliferate in serum-free medium suggests that these cells have the capacity to synthesize those factors essential for their growth. Direct immunocytochemical staining and Western analysis have identified transforming growth factor-beta (TGF-beta) band and basic fibroblast growth factor (FGF) in the cytoplasm of RA and normal synovial cells in long-term culture. Greater amounts of each growth factor were found in RA, as compared with normal synovial cell lysates. Western analysis identified a single TGF-beta band in RA and normal synovial cell lysates. Four bands were identified by Western analysis on RA synovial cell lysates probed with monoclonal antibodies recognizing bFGF, whereas only two bands (which co-migrated with human native recombinant bFGF) were identified in normal cell lysates probed with these antibodies. Gene expression analysis using PCR identified mRNA transcripts encoding TGF-beta 1 and FGF-2 (bFGF), but not TGF-beta 2 in all cell cultures studied. Taken together, these data indicate that cultured synovial cells co-express TGF-beta 1 and multiple isoforms of hFGF. These data further strengthen the concept that both polypeptide growth factors are involved in the regulation of synovial cell growth.
DNA Cell Biol 1992 Apr
PMID:Regulation of synovial cell growth by polypeptide growth factors. 156 59

We describe a reproducible method for growing small intestinal epithelium (derived from the suckling rat intestine) in short-term (primary) cultures. Optimal culture conditions were determined by quantitative assays of proliferation (i.e. changes in cellularity and DNA synthesis). Isolation of the epithelia and, significantly, preservation of its three-dimensional integrity was achieved using a collagenase/dispase digestion technique. Purification of the epithelium was also facilitated by the use of a simple differential sedimentation method. The results presented below support the idea that proliferation of normal gut epithelium ex vivo is initially dependent upon the maintenance of the structural integrity of this tissue and upon factors produced by heterologous mesenchymal cells. Proliferation in vitro was also critically dependent upon the quality of the medium and constituents used. Cultures reached confluence within 10-14 days and consisted of epithelial colonies together with varying amounts of smooth-muscle-like cells. Cultures have been maintained for periods up to one month, but the longer-term potential for growth by sub-culturing has not been examined. Strategies for reducing the proliferation of these non-epithelial cells are also described.
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PMID:The development of a method for the preparation of rat intestinal epithelial cell primary cultures. 156 26

Gastrin-releasing polypeptide (GRP) has been implicated in the development of the human fetal lung. To determine whether GRP has a wider role in fetal development, its actions on DNA synthesis and cell replication by isolated epiphyseal growth plate chondrocytes obtained from ovine fetuses between 35 days gestation and near term (145 days) were examined. Chondrocytes were isolated using collagenase from the proximal tibia and cultured in monolayer. Synthesis of DNA was assessed from the incorporation of [3H]thymidine into previously growth-restricted cells after incubation in medium supplemented with GRP1-27 (40-1280 nM). Increase in cell number was assessed after incubation with test medium for 1 week. GRP caused a dose-dependent increase in both cell number and DNA synthetic rate compared to control incubations. Cell number was increased by 50% in the presence of a maximally effective 160 nM GRP and DNA synthesis by up to 800% utilizing chondrocytes obtained from animals of 75-80 days gestation. The mean (+/- SEM) half-maximal concentration of GRP for the stimulation of DNA synthesis was 97 +/- 12 nM (5 separate fetuses). Concentrations of GRP in excess of 160 nM caused a sharp reduction in both cell replication and DNA synthesis. To determine where within the cell cycle GRP exerted its mitogenic action, synchronized chondrocytes were transiently exposed to fetal bovine serum and cultured with GRP for increasing periods of time before pulse labeling with [3H]thymidine during S phase. GRP was as effective in stimulating DNA synthesis when present for the initial 4 h of G1 as when present for the entire G1 period. Since isolated fetal growth plate chondrocytes release insulin-like growth factor II (IGF II) and basic fibroblast growth factor (basic FGF) the possible mediation of GRP action by the release of these peptides or synergistic interactions were examined. Specific antibodies shown to negate the mitogenic actions of exogenous IGFs or basic FGF on chondrocytes did not alter GRP-stimulated DNA synthesis. The release of radioimmunoassayable IGF II by chondrocytes was not altered in the presence of GRP. Coincubation of GRP with submaximal concentrations of IGF I or basic FGF showed additive effects on DNA synthesis. When the actions of galanin were examined it was found to inhibit basal DNA synthesis by chondrocytes at a concentration of 167 nM. However, 66 nM or greater galanin was able to render 160 nM GRP inactive as a mitogen. These results suggest that GRP may potentially influence skeletal development in the ovine fetus and may interact with locally released peptide growth factors or other neuropeptides.
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PMID:Mitogenic action of gastrin-releasing polypeptide on isolated epiphyseal growth plate chondrocytes from the ovine fetus. 157 94


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