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)

Human skin fibroblasts in culture have previously been shown to synthesize genetically distinct procollagens type I and type III. In the present study, cultured human skin fibroblasts were incubated under conditions optimized for synthesis of these procollagens in medium containing [3H]proline. The newly synthesized type I and type III 3H-labeled procollagens in the culture medium were then isolated as native proteins by DEAE-cellulose chromatography, or by gel filtration and SDS-polyacrylamide slab gel electrophoresis under denaturing conditons after limited pepsin proteolysis. The chromatographic procedures were optimized to yield reliable and reporducible results with good recoveries. The isolated procollagens were identified by cyanogen bromide peptide mapping and characterized by cleavage with highly purified collagenase synthesized by human skin fibroblasts. Assay of the relative synthesis of type I/III procollagens by normal human skin fibroblasts using DEAE-cellulose chromatography indicated that 80% of the procollagen in the medium was type I while the remaining 20% consisted of type III. When the ratio of newly-synthesized type I/III collagens was estimated by gel filtration or using SDS-polyacrylamide slab gel electrophoresis after limited pepsin proteolysis, relatively fewer type III collagen alpha-chains were recovered. This observation suggests that some of the type of the type III collagen molecules are in a conformation which is less resistant to digestion by pepsin than the triple-helix of type I procollagen. The coefficient of variation for the relative synthesis of type I and type III procollagens by control cultures was relatively small (16%), indicating that the phenotypic expression of type I and type III procollagen genes, under optimized culture conditions, is under a relatively tight control. The results further suggest that the optimized methodology developed for assay of the relative synthesis of type I and type III procollagens and collagens by cultured human skin fibroblasts can be utilized in studies on collagen aberrations in acquired and inherited diseases of connective tissue.
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PMID:Collagen biosynthesis by human skin fibroblasts. II. Isolation and further characterization of type I and type III procollagens synthesized in culture. 741 91

We previously observed that a retinoid analog can protect against liver parenchymal damage and liver fibrosis, whereas it accelerates liver fibrosis which is not accompanied by any parenchymal damage. To elucidate these conflicting effects, we examined the effects of retinoid in 3T3 L1 preadipocytes as a model of liver stellate cells. Retinoids, including all-trans retinol, all-trans and 9-cis retinoic acids, enhanced the cell growth and the expression of the type I procollagen gene as well as its peptide synthesis, while reducing collagenase activities. Although no retinoid enhanced the transforming growth factor (TGF)-beta 1 mRNA, retinoids may stimulate collagen production through activating TGF-beta, as was recently reported. These results help explain the observation in the liver fibrosis model with no parenchymal damage. In contrast, we also found that interferon (IFN) alpha beta and gamma inhibited cell growth and down-regulated markedly type I procollagen as well as TGF-beta 1 mRNA, suggesting that they suppress by acting directly on extracellular matrix-producing cells.
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PMID:Modulation of collagen synthesis and degradation by retinoids and cytokines in 3T3 L1 preadipocytes. 752 59

Using immunocytochemistry and Northern blot analysis, we investigated the role of cell morphology and reorganization of the cytoskeleton in the expression of transforming growth factor-beta 1 (TGF-beta 1) in human dermal fibroblasts. Disruption of the cytoskeleton was induced by three different agents--trypsin, ethyl-eneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or cytochalasin--and was confirmed by staining with rhodamine-labeled phalloidin. Immunocytochemical staining with antibodies specific for TGF-beta 1 revealed a cell-shape-related induction of TGF-beta 1. Northern blot analysis of total RNA showed a significant increase in the expression of TGF-beta 1 mRNA as early as 4 h and peaking at 12 h after disruption of the cytoskeleton. Quantitative analysis of TGF-beta 1 mRNA expression at 4 h after treatment with trypsin, EGTA, or cytochalasin C showed increases of 2.6-, 3.3-, and 2.6-fold, respectively. Disruption of the cytoskeleton by trypsin, EGTA, or cytochalasin C increased mRNA for collagenase by 3.8-fold, 2.3-fold, or 2.5-fold, respectively. The expression of mRNA for tissue inhibitor of metalloproteinases I (TIMP-I) also showed a 3.2-fold increase by trypsin, a 3.6-fold increase by EGTA, and a 2.5-fold increase by cytochalasin C. Cell-shape-related induction of TGF-beta 1, collagenase, and TIMP-I genes appears to be selective, as the levels of mRNA for fibronectin and type I procollagen were not significantly altered. These data suggest that gene expression of TGF-beta 1, collagenase, and TIMP-I is governed by the status of the cytoskeleton microfilament organization, which may be a mechanism of gene regulation during cell division, migration, and differentiation, events fundamental to wound healing.
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PMID:Alteration in cell morphology triggers transforming growth factor-beta 1, collagenase, and tissue inhibitor of metalloproteinases-I expression in normal and hypertrophic scar fibroblasts. 752 43

Bone proteins in alveolar bone of mandibles from young adult rabbits (3-month-old) were extracted with 4.0 M guanidine hydrochloride (GuHCl), followed by 0.5 M ethylenediaminetetraacetate, and again with 4.0 M GuHCl (G2-ext). The proteins in the G2-ext were fractionated on a gel-filtration column, followed by an anion-exchange column in the presence of 7.0 M urea. A 28-kDa protein was isolated from the G2-ext. The purified 28-kDa protein showed intense staining with silver on SDS-PAGE slab-gel under reducing conditions. This protein was digested with bacterial collagenase, and a 19-kDa fragment appeared on the gel. However, the protein was not susceptible to reduction with cyanogen bromide. The protein did not bind to hydroxyapatite crystals in the presence of 7.0 M urea, and also did not bind to some lectins. On SDS-PAGE under non-reducing conditions, the protein migrated as two bands; a new band appeared at approximately the 85-kDa region in addition to the original 28-kDa band. The amino acid compositions of the protein were similar to those of the alpha 1-pN-propeptide of type I procollagen obtained from other tissues.
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PMID:Characteristics of a 28-kDa collagenous protein extracted with guanidine from EDTA-demineralized rabbit alveolar bone. 815 87

Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast-specific bone matrix protein that is thought to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H-proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady-state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast-enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally-derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.
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PMID:Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells. 822 74

We have previously shown that mouse L.P3 cells secrete fibronectin and a novel protein, gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). Here, we screened and characterized cell-adhesive proteins in the conditioned media (CM's) of a series of cell lines growing in a protein- and lipid-free synthetic medium (P3 cell lines). Although cell-attachment activity of the CM's ranged from undetectable to highly significant, fractionation with affinity columns revealed the presence of significant cell-attachment activity in all CM's. Using cell-attachment assay and immunoassay on blotted filters, various cell-adhesive proteins were detected in the CM's, and most of them were identified as fibronectin, laminin, vitronectin, and collagen. GNCP-like proteins were detected in the CM's of HeLa.P3, JTC-16.P3, L.P3, and JTC-12.P3. There was no relationship between the origin of the cell lines and the cell-adhesive proteins secreted. GNCP purified from L.P3-CM was separated into 120-, 140-, 150-, and 160-kDa proteins on SDS-PAGE, which were judged to be a type of mouse type I procollagen from the following results: (1) they were digested by collagenase, (2) pepsin treatment converted the 150- and 160-kDa proteins into 120- and 140-kDa proteins, (3) they were recognized by anti-type I collagen antiserum, and (4) amino-terminal sequence of the pepsin-digested 140-kDa protein had significant homology with type I collagen. GNCP showed half-maximum activity of cell attachment at 0.03 micrograms/ml, indicating that GNCP was a cell-adhesive protein with an extremely high specific activity compared to other known cell-adhesive proteins.
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PMID:Characterization of cell-adhesive proteins secreted by cell lines growing in protein- and lipid-free synthetic medium: mouse L.P3 cells secrete a procollagen molecule with potent cell-attachment activity. 850 60

We have developed quantitative immunoassays for the intact, trimeric amino-terminal propeptide of human type I procollagen (PINP) and its Col1 domain. Intact PINP was isolated from the pleural fluids of cancer patients by a combination of ion-exchange, gel-filtration, and reversed-phase chromatographies. The amino-terminal Col1 domain of PINP was isolated after bacterial collagenase treatment of the heat-denatured trimeric propeptide. For the intact PINP assay we used a polyclonal antibody with only 1.2% cross-reaction with the monomeric Col1 domain. In human serum, this assay detects only one peak of PINP antigenicity that has the size of known intact PINP. Under similar conditions, an assay for the Coll domain of PINP recognized two circulating antigens. The biological relevance was further verified in wound fluid. Interassay and intraassay CVs were 3.1-9.3% for values within the reference intervals (mean +/- 2SD) for intact PINP in serum, which were 19-84 microg/L for women and 20-76 microg/L for men.
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PMID:Immunoassay for intact amino-terminal propeptide of human type I procollagen. 866 88

Extracellular matrix is the principal component of the fibrous caps of atherosclerotic plaques and intimal hyperplastic lesions of reconstructed arteries. Interstitial collagen form an important part of the matrix, and the balance between collagen synthesis and degradation by interstitial collagenase (matrix metalloproteinase-1, MMP-1) may determine whether plaques rupture or vessels develop stenosis. We examined type I procollagen gene expression in human atherosclerotic and restenotic carotid arteries using in situ messenger RNA (mRNA) hybridization and the expression of MMP-1 and its endogenous inhibitor (tissue inhibitor of metalloproteinases-1, TIMP-1) by immunohistochemistry. Compared with normal arteries, atherosclerotic plaques bed increased expression of immunoreactive MMP-1 and TIMP-1 with modest increase of type 1 procollagen mRNA. Early restenotic lesions (< 1.5 years) contained abundant type I procollagen mRNA but little immunoreactive MMP-1 and TIMP-1. Late restenotic lesions (> 4 years) resembled atheroma and exhibited increased immunoreactive MMP-1 and TIMP-1 as well as abundant type I procollagen mRNA. Compared with atherosclerotic plaques, type I procollagen is increased and MMP-1 is decreased in early restenotic lesions. MMP-1 and TIMP-1 expressions are upregulated in lesions with a clear atheroma. These findings suggest that the balance between proteolysis and matrix synthesis may influence both the stability of atheromatous plaques and the development of restenotic lesions.
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PMID:Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy. 877 33

The effects of topical tretinoin on collagen synthesis and degradation were studied in 29 volunteers. The subjects applied 0.1% tretinoin cream on their non-sun-exposed abdominal skin once a day for 1 week (n = 10) (experiment 1) or twice a day for 2 weeks (n = 8) (experiment 2) or once a day for 2 months (n = 11) (experiment 3). After the treatments, suction blisters were induced and amino-terminal propeptides of type I and III procollagens (PINP, PIIINP, respectively) (experiments 1 and 3) and carboxy-terminal propeptide of type I procollagen (PICP) (experiment 2) were assayed as an index of de novo collagen synthesis by radioimmunoassays. Matrix metalloproteases 2 (MMP-2) and 9 (MMP-9) were assayed by the zymography method in experiment 2. In experiment 3, histology, immunohistochemistry of type I and III procollagens, tenascin, mRNA levels of type I collagen alpha 1-chain [alpha 1 (I)], interstitial collagenase (MMP-1), MMP-2, MMP-9 by slot-blot analysis and the levels of alpha 1 (I) collagen mRNA by a quantitative polymerase chain reaction method were studied. The proportional area of elastic fibres visualized in Verhoeff-stained sections was analysed by computerized digital image analysis. The results indicated that treatment with topical tretinoin does not markedly induce de novo synthesis of collagen in vivo or affect matrix metalloproteases. In the immunohistochemical staining, tenascin was increased in the papillary dermis. As it has been suggested that tretinoin could counteract the atrophogenic effect of corticoids on the dermis, the effect of a combination of betamethasone-17-valerate (once a day) and tretinoin (once a day) on the propeptide levels was also studied. Betamethasone alone caused a 60% decrease in the concentrations of PINP and PIIINP, and a similar decrease was found after the combination treatment, indicating that topical tretinoin administered during short treatment periods does not counteract the inhibitory effect of a potent corticoid on collagen propeptides.
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PMID:Effect of topical tretinoin on non-sun-exposed human skin connective tissue: induction of tenascin but no major effect on collagen metabolism. 921 22

Understanding the mechanisms responsible for photodamage to the skin is most important for dermatology. 3-D cultures have been used as tools to mimic the in vivo situation for several years. We irradiated such a system containing human dermal fibroblasts cultured in collagen gels, a well-known model considered to be a dermal equivalent, which reproduces the interaction between cells and the surrounding extracellular matrix. The effects of solar irradiation (315-800 nm) on the steady-state levels of the mRNAs of extracellular matrix components (type I and III collagens) and their degrading enzymes (interstitial collagenase, MMP-1 and stromelysin 1, MMP-3) were measured. Exposure to low levels of solar radiation (0-10 J cm-2 in the UVA, i.e. suberythemal UVA doses) caused a transient decrease in type I procollagen mRNA, an increase in MMP-mRNA, and no change in type III procollagen mRNA steady-state levels. These results describe the early changes in the connective tissue of the skin following exposure to low-level solar stimulation, and may help explain the long-term changes in photodamaged skin.
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PMID:Effects of simulated solar radiation on type I and type III collagens, collagenase (MMP-1) and stromelysin (MMP-3) gene expression in human dermal fibroblasts cultured in collagen gels. 959 12


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