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)

Several peptides were isolated from tryptic digests of insoluble calf aorta matrix by chromatography. Reductive pyridylethylation of a tryptic 15 kDa pool released fragments deriving from the C-terminus of type III collagen. A 50-residue peptide Tc(III) was shown by sequence analysis to be the C-terminal peptide from the alpha 1(III)-chain, containing a helical and non-helical region of equal sizes. The peptide was further digested with collagenase to give Colc(III), comprising the complete C-terminal non-helical region of alpha 1(III) including a hydroxylysine in position 16c. The peptide Tc(III) x TN(III) was isolated, demonstrating covalent cross-linking between the C-terminal non-helical region of one type III molecule and the N-terminal helical cross-linking region of another. Its digestion with cyanogen bromide yielded the small fragments alpha 1(III)CB3B* and alpha 1(III)CB3C, confirming TN(III) as an N-terminal helical crosslink site. Sequence analysis of both Tc(III) x TN(III) and its collagenase-derived cross-linked peptide Colc(III) x TN(III) established the 4D-staggered alignment of adjacent collagen III molecules. The cross-link structure of both peptides was mainly dihydroxylysinonorleucine with a small amount of hydroxylysinonorleucine, indicating that the lysine residues involved in formation of the cross-links are both hydroxylated. No pyridinoline or histidinohydroxylysinonorleucine cross-links were found within the non-reduced C-telopeptide region of type III collagen.
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PMID:Cross-link analysis of the C-telopeptide domain from type III collagen. 880 38

To determine whether G proteins are involved in the regulation of mouse placental lactogen-I (mPL-I) and/or mPL-II secretion before midpregnancy, mouse placental tissue from day 7 of pregnancy was dispersed with collagenase, cells were fractionated on a percoll gradient, and the purified trophoblast cells were cultured in a serum-free medium with cholera toxin (CTX) or pertussis toxin (PTX) which modulate the activities of distinct G proteins for 5 days. CTX inhibited both mPL-I and mPL-II secretion, but PTX inhibited mPL-I secretion and stimulated mPL-II secretion in a time- and dose-dependent manner. Addition of both CTX and PTX additionally inhibited mPL-I secretion but did not affect mPL-II secretion. 8-Bromo cAMP, which increases intracellular cAMP accumulation, inhibited both mPL-I and mPL-II secretion similarly to CTX. In contrast, H8, an inhibitor of cAMP-dependent protein kinase A, stimulated both mPL-I and mPL-II secretion. Addition of PTX and H8 synergistically stimulated mPL-II secretion. These findings suggest that G proteins play important roles in regulation of mPL-I and mPL-II secretion before midpregnancy.
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PMID:Regulation of mouse placental lactogen secretion by G proteins before midpregnancy. 890 70

New assays were compared with tritiated thymidine (3HTdR) and trypan blue dye exclusion for evaluating hepatocyte viability on cytodex 3 beads and in microcapsules, the matrices used in bioartificial liver support systems. They were the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 5 min qualitative assay and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 -sulfophenyl)-2H -tetrazolium, inner salt (MTS) 1 h quantitative assay. Both tetrazolium salts are cleaved in active mitochondria, the reaction occurring, therefore, in living cells only. After bathing at 39 degrees C with MTT for 5 min, porcine or rat hepatocytes on cytodex 3 beads were detached by collagenase while those in microcapsules were released by citrate treatment or passage through a fine needle. Cell viability was determined directly by microscope. The MTT 5 min metabolic inclusion test and MTS 1 h quantitative assay results correlated closely with those obtained by the 3HTdR, trypan blue dye exclusion, and fluorescein diacetate (FDA) methods. Both the new assays are sensitive, accurate, simple, and time-saving.
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PMID:Simple and reliable methods to assess hepatocyte viability in bioartificial liver support system matrices. 912 74

Cryopreservation is an effective method of islet storage and may facilitate clinical trials of islet transplantation. It was the aim of the present study to evaluate the in vitro viability of cryopreserved rat islets, including the response to nonglucose secretagogues and glucose oxidation. After pancreatic digestion via intraductal injection of collagenase, 75- to 200-micron Wistar rat islets were handpicked and cultured in RPMI 1640 (glucose 11.1 mmol/L) and randomized into two groups: control (cultured 20 to 24 hours at 37 degrees C) and cryopreserved (after 20 to 24 hours of culture at 37 degrees C, islets were cryopreserved according to Rajotte's protocol: freezing velocity, -0.25 degree C/min; thawing velocity, 200 degrees C/min). In the two groups, we evaluated recovery, insulin content per islet, staining viability (ethidium bromide/orange acridine; semiquantitative scoring, measuring the viable area of the islet from 0 = less viable to 3 = more viable), insulin secretion after glucose and nonglucose secretagogues, and oxidation of D-[U-14C]glucose. The results for the control group were always higher for the following: recovery (95.4% +/- 1.2% v 83.0% +/- 2.1%, P = .00), insulin content (2,203.9 +/- 335.2 v 1,443.3 +/- 171.8 microU/islet, P = .03), insulin secretion after 5.5 mmol/L glucose (61.3 +/- 8.0 v 28.3 +/- 3.4 microU/islet/90 min, p = .00), 16.7 mmol/L glucose (151.4 +/- 16.1 v 98.7 +/- 14.1 microU/islet/90 min, p = .03), 10 mmol/L L-leucine +10 mmol/L L-glutamine (125.6 +/- 27.9 v 56.8 +/- 6.4 microU/islet/90 min, P = .05), and 10 mmol/L L-arginine (202.5 +/- 27.5 v 128.8 +/- 14.2 microU/islet/90 min, P = .01), and glucose oxidation at 5.5 mmol/L (12.5 +/- 1.1 v 7.9 +/- 0.6 pmol/islet/120 min, P = .00) and at 16.7 mmol/L (26.1 +/- 2.6 v 14.3 +/- 1.6 pmol/islet/120 min, P = .00). No significant differences in staining viability were found between groups (2.35 and 2.48, respectively, P = .55). However, cryopreserved and control islets showed a significant increase in insulin secretion and glucose oxidation after increasing the glucose concentration from 5.5 to 16.7 mmol/L. We conclude that when glucose is increased, cryopreserved islets keep the capacity to increase insulin secretion, but cryopreservation produces a significant decrease in several islet viability characteristics. This decrease may be due to a decline of beta-cell number per islet and/or a decrease in the content of insulin per beta cell.
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PMID:Cryopreservation: in vitro results in rat pancreatic islets. 918 99

The transcription factors Fos, Jun, and Ets regulate the expression of human stromelysin-1 and collagenase-1 genes. Recently, we found that ERG, an Ets family member, activates collagenase-1 gene but not stromelysin-1 by physically interacting with c-Fos/c-Jun. Interestingly, ERG binds to stromelysin-1 promoter and represses its activation by ETS2. Here, to investigate the molecular mechanism of this regulation, we have used an in vitro protein-protein interaction assay and studied the transcription factor interactions of ETS2. We found that ETS2 could weakly associate with in vitro synthesized ETS1, c-Fos, and c-Jun and strongly with c-Fos/c-Jun complex and ERG via several distinct ETS2 domains including the C-terminal region that contains the DNA-binding domain. Strikingly, these interactions were stabilized in vitro by DNA as they were inhibited by ethidium bromide. Both the N-terminal region, comprising the transactivation domain, and the C-terminal region of ETS2 associated with ERG and, interestingly, the interaction of ERG through the transactivation domain of ETS2 was DNA-independent. The DNA-dependent interaction of ETS2 with c-Fos/c-Jun was enhanced by specific DNA fragments requiring two Ets-binding sites of the stromelysin-1 promoter. Using the two hybrid system, we also demonstrated that ETS2 interacts with c-Jun or ERG in vivo.
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PMID:The Ets transcription factors interact with each other and with the c-Fos/c-Jun complex via distinct protein domains in a DNA-dependent and -independent manner. 933 86

Phosphoprotein appears to play an important role in the mineralization of dentin during tooth development and remineralization after demineralization by dental caries. To better understand this role, we describe the extraction and characterization of phosphoprotein from immature, human root apex dentin during and after EDTA demineralization. The extraction procedure included dissociation of the demineralized dentin matrix by guanidine hydrochloride (Gdn.HCl) followed by subsequent digestion with cyanogen bromide (CNBr) and collagenase. Characterization of these extracts included 'Stains-All' staining of SDS polyacrylamide gels (SDS-PAGE) and amino acid, protein and phosphorus analyses. The ability of these matrices to remineralize was determined by TEM and measuring calcium levels in the remineralized tissue by atomic absorption spectroscopy. The staining of SDS-PAGE gels and amino acid analysis showed that an intact phosphophoryn was extracted from the dentin of the immature apices during EDTA demineralization and that it had an apparent Mr approximately 140,000. In the subsequent extracts and digests, the phosphoprotein has a range of molecular weights, some of which may have been degraded products of the intact phosphoprotein. A greater quantity of phosphoprotein was found in the EDTA-demineralized dentin matrices than in dentin after Gdn.HCl, CNBr and collagenase digests. These EDTA-demineralized matrices also remineralized to a greater extent than those dissociated with Gdn.HCl. The differences in both the quantity and the quality, as defined by the amino acid residue profile, of the phosphoprotein in the sequential extracts of the root apex dentin may be important in affecting the ability of this tissue to remineralize.
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PMID:Phosphoprotein analysis of sequential extracts of human dentin and the determination of the subsequent remineralization potential of these dentin matrices. 970 61

We have developed monoclonal antibody 5109 against a unique highly acidic sequence in type II collagen. When paired with previously reported monoclonal antibody 9A4, 5109 can be used as the capture antibody in an ELISA assay for the neoepitope generated by collagenase-cleavage of type II collagen. The assay detects the sequence ZGlyGluX(759)GlyAspAspGlyProSerGlyAlaGluGlyProX(771)GlyProGlnGly(775) where Z is a variable length polypeptide, X is proline or hydroxyproline, and Gly(775) corresponds to C-terminal amino acid of the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epitope with a K=1.2x10(-8) M independently of hydroxylation of X(759). When X(771) is proline, the sequence is 90x more sensitively detected by this ELISA than when it is hydroxyproline. Type II collagen of human articular cartilage was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline(771) averaged 81% hydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine of control individuals of 50-70 years of age, we failed to detect the presence of the collagen fragment in a majority (8/10) of specimens. The two controls with measurable levels averaged 123 pM. In a similar age cohort of osteoarthritic patients, the majority (9/10) showed measurable values of urinary collagen fragments averaging 312 pM. This assay can be used for monitoring type II collagen metabolism in patients with osteoarthritis.
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PMID:Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA. 1115 May 34

To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63+/-78 x 10(8) total cells with a viability of 69.2+/-25.3 %, but our combined method had a yield of 167+/-31 x 10(8) total cells with a viability of 87.9+/-4.4% (mean +/- SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387+/-89 x 10(8) hepatocytes with a viability of 87.1+/-6.9% and a purity of 93.6+/-2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 10(10) porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 - 500 microm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 10(8) porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.
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PMID:Mass preparation of primary porcine hepatocytes and the design of a hybrid artificial liver module using spheroid culture for a clinical trial. 1179 50

There is uncertainty in the literature regarding the number and location of fibronectin binding sites on denatured collagen. Although most attention has focused on a single site near the collagenase-sensitive region of each alpha chain, there is evidence for additional sites in other regions. We treated bovine type I collagen with cyanogen bromide, labeled the resulting mixture with fluorescein, and separated the peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent bands were excised from the gel and dialyzed exhaustively to remove detergent. Titration of eight distinct fluorescent-labeled fragments with the 42-kDa gelatin-binding fragment of fibronectin caused increases in anisotropy that were fully reversible with unlabeled gelatin. By fitting the dose responses it was possible to calculate apparent K(d)'s whose values ranged between 1 and 4 microM. The largest fragment, alpha(2)-CB3,5, composing about 2/3 of the alpha(2) chain, when further digested with endoproteinase Lys-C, yielded at least three additional subfragments that also bound with similar affinities. Thus, there appear to be at least 14 distinct fibronectin binding sites of similar affinity in bovine type I collagen, five on each of the alpha(1) chains and four on the alpha(2) chain. Experiments with several synthetic peptides failed to reveal the exact nature of the binding site.
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PMID:Type I collagen contains at least 14 cryptic fibronectin binding sites of similar affinity. 1241 94

Hepatic stellate cells play a key role in the pathogenesis of hepatic fibrosis. In this study, we investigate the inhibitory effect of butein on the activation and proliferation of rat primary cultured hepatic stellate cells. Possible cytotoxic effects were measured on stellate cells and hepatocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of butein on the production of collagen and smooth muscle alpha-actin proteins were examined at the same concentration, by western blot. The effects of butein on alpha1(I) collagen, tissue inhibitor of metalloproteinase-1, and metalloproteinase-13 gene expression in activated stellate cells were investigated by measuring mRNA levels using reverse transcription polymerase chain reaction. The effect of butein on DNA synthesis was also determined. Butein, at a concentration of 1 microg mL(-1), reduced DNA synthesis without affecting cell viability, and downregulated smooth muscle alpha-actin and type-I collagen expression, and alpha1(I) collagen and tissue inhibitor of metalloproteinase-1 mRNA expression, while treatment with butein induced metalloproteinase-13 mRNA expression. These findings suggest that butein is a potent inhibitor of stellate cell transformation.
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PMID:Butein suppresses myofibroblastic differentiation of rat hepatic stellate cells in primary culture. 1272 40


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