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)

An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox dishes in a modified Chee's medium containing 1 microM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.
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PMID:Characterization of a primary hepatocyte culture system for toxicological studies. 872 45

The Porphyromonas gingivalis collagenase-specific serum immunoglobulin A (IgA), IgM, and IgG responses from 20 patients with early-onset periodontitis (EOP), 20 patients with adult periodontitis, (AP), and 20 age- and sex-matched healthy controls were examined by immunoblot analysis. A recombinant collagenase antigen used for the immunoblot analysis was produced by using the plasmid pGEX-2T, which allows the fusion between the collagenase and glutathione S-transferase. There was no significant difference in collagenase-specific IgG antibody detection between samples from the EOP, AP, and control groups. In contrast, 85% of AP and EOP sera had collagenase-specific IgA antibodies, whereas only 20% of control sera showed collagenase-specific IgA reactivity. Plaque samples from all groups were assessed by PCR with primers complementary to the collagenase-encoding gene prtC. The results indicated that 90% of AP and EOP plaque samples and 10% of control samples were positive for P. gingivalis. All patients with collagenase-specific IgA antibodies were PCR positive. The results of the study indicate a nearly complete concordance (k = 0.856) between the presence of collagenase-specific IgA antibodies and PCR detection of P. gingivalis. By using PCR as the "gold standard," the sensitivity and specificity of the IgA immunoblot test were 94.7 and 90.9%, respectively. Therefore, the recombinant collagenase is a potential candidate for use in the serodiagnosis of periodontitis.
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PMID:Serodiagnosis of Porphyromonas gingivalis infection by immunoblot analysis with recombinant collagenase. 888 Apr 90

Through its ability to bind extracellular matrix constituents and growth factors the small leucine-rich chondroitin/dermatan sulfate proteoglycan decorin which is present in many types of connective tissues may play an important biological role in remodeling and maintenance of extracellular matrices during inflammation, fibrosis, and cancer growth. In this study we investigated the known binding of decorin to human collagen XIV. This binding was unaffected when the small collagenous moiety of collagen XIV was removed with collagenase. Therefore, fragments covering the large noncollagenous domain NC3 of collagen XIV were expressed in Escherichia coli, each fused to a 26-kDa fragment of glutathione S-transferase. Using radioiodinated decorin as ligand for the immobilized fusion proteins, a binding site that interacted with the decorin core protein could be assigned to the NH2-terminal fibronectin type III repeat of collagen XIV. In addition, an auxiliary binding site located COOH-terminal to this fibronectin type III repeat interacted with the glycosaminoglycan component of decorin.
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PMID:Localization of a binding site for the proteoglycan decorin on collagen XIV (undulin). 925 49

The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pz-peptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the full-length enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the full-length collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.
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PMID:A study of the collagen-binding domain of a 116-kDa Clostridium histolyticum collagenase. 945 93

We have demonstrated that RRR-alpha-tocopheryl succinate (10 microg/mL vitamin E succinate (VES) treatment of estrogen receptor-negative MDA-MB-435 human breast cancer cells induces 9, 19, 51, and 72% apoptotic cells on days 1-4, respectively, after treatment, which involves transforming growth factor-beta signaling. Here, we show that VES-triggered apoptosis of MDA-MB-435 cells induced prolonged elevated expression of c-jun mRNA and protein (neither of which was caused by major increases in stability) and also induced enhanced activator protein-1 (AP-1) binding to the consensus DNA oligomer. Furthermore, VES treatments resulted in increased AP-1 transactivation activity, as measured with an AP-1 promoter/luciferase reporter construct and by the measurement of increased mRNA expression of the AP-1-dependent endogenous gene collagenase. Evidence of VES-induced involvement of the c-jun amino-terminal kinase in these AP-1-dependent events was suggested by data showing prolonged activity of this kinase, as measured by a kinase assay using glutathione S-transferase-c-jun as the substrate. The c-jun-dependent transcriptional activity was verified by cotransfection of a chimeric transcription factor having a galactose 4 DNA-binding domain coupled with the transactivation domain of c-jun plus the reporter plasmid 5X GAL4-luciferase. MDA-MB-435 cells infected with an adenovirus expression vector containing the TAM-67 sequence for dominant/negative-acting mutant c-jun or transiently transfected with c-jun antisense exhibited a 50-77% reduction in VES-mediated apoptosis as compared with control adenovirus-infected or control sense oligomer-transfected cells.
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PMID:RRR-alpha-tocopheryl succinate induction of prolonged activation of c-jun amino-terminal kinase and c-jun during induction of apoptosis in human MDA-MB-435 breast cancer cells. 972 17

The retinoblastoma susceptibility gene product (RB) is a transcriptional modulator. One of the targets for this modulator effect is the AP-1 binding site within the c-jun and collagenase promoters. The physical interactions between RB and c-Jun were demonstrated by co-immunoprecipitation of these two proteins using anti-c-Jun or anti-RB antisera, glutathione S-transferase affinity matrix binding assays in vitro, and electrophoretic mobility shift assays. The C-terminal site of the leucine zipper of c-Jun mediated the interaction with RB. Although the B-pocket domain of RB alone bound to c-Jun, a second c-Jun binding site in the RB was also suggested. Mammalian two-hybrid-based assay provided corroborative evidence that transactivation of gene expression by RB required the C-terminal region of c-Jun. We conclude that RB enhances transcription activity mediated through the AP-1 binding site. Adenovirus E1A or human papillomavirus E7 inhibits RB-mediated transcription activity. These data reveal that the interactions between these two distinct classes of oncoproteins RB and c-Jun may be involved in controlling cell growth and differentiation mediated by transcriptional regulation.
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PMID:Recruitment of the retinoblastoma protein to c-Jun enhances transcription activity mediated through the AP-1 binding site. 1002 57

To study growth regulation in the beginning of carcinogenesis, we established a novel ex vivo model for co-cultivation of normal and putatively initiated hepatocytes. Rats received the genotoxic hepatocarcinogen N-nitrosomorpholine (NNM). This led to the appearance of hepatocytes expressing placental glutathione S-transferase (G(+) cells). These cells exhibited elevated rates of cell replication and apoptosis, as known from further advanced preneoplasia; G(+) cells were considered initiated. At days 20-22 post-NNM treatment their frequency was maximal (1-2%); approximately 40% were still single and 60% were arranged in mini foci. At this time-point liver cells were isolated by collagenase perfusion and cultivated. G(+) cells, identified by immunostaining of the culture-plates, were present at the same percentage as in vivo, excluding selective loss, enrichment or spontaneous expression of the G(+) phenotype. In untreated cultures G(+) hepatocytes showed significantly higher rates of replicative DNA synthesis than normal G(-) cells. Application of the hepatomitogen cyproterone acetate (CPA) elevated DNA replication preferentially in G(+) cells. Transforming growth factor beta1 (TGF-beta1) suppressed replicative DNA synthesis which was more pronounced in G(+) than in G(-) hepatocytes. Combined treatment with CPA and TGF-beta1 had no effect on G- cells, but considerably inhibited DNA replication in G(+) cells. This suggests that the effects of TGF-beta1 predominated in G(+) hepatocytes. We conclude that putatively initiated G(+) hepatocytes, both in vivo and in culture, exhibit higher basal rates of DNA replication than normal G(-) hepatocytes and an over-response to mitogens and growth inhibitors. Therefore, G(+) cells show (i) nearly identical behaviour in intact liver and in primary culture and (ii) inherent defects in growth control that are principally similar although somewhat less pronounced than in later stages of carcinogenesis. The present ex vivo system thus provides a novel and useful tool to elucidate biological and molecular changes during initiation of carcinogenesis.
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PMID:Initiated rat hepatocytes in primary culture: a novel tool to study alterations in growth control during the first stage of carcinogenesis. 1060 37

The role of transforming growth factor alpha (TGFalpha) and prostaglandins (PGs) in the preferential growth of preneoplastic liver cells was studied. Rats received the genotoxic hepatocarcinogen N-nitrosomorpholine (NNM); placental glutathione S-transferase (GSTp) was used as a marker to identify preneoplastic foci. Preneoplastic foci expressing TGFalpha (TGFalpha(+)) grew more rapidly than TGFalpha negative (TGFalpha(-)) ones. Almost all tumours studied were positive for TGFalpha. The key enzymes of prostaglandin synthesis, cyclooxygenase I (Cox-1) and II (Cox-2), were present in all unaltered and preneoplastic cells and tended to decrease in the later stages of hepatocarcinogenesis. Immunostaining revealed that cultures of hepatocytes, isolated from NNM-treated livers by collagenase perfusion, contained 1-2% GSTp-positive (GSTp(+)) and 9% TGFalpha(+) hepatocytes; 0.6% of the cells were GSTp(+)/TGFalpha(+). Cox-1 and Cox-2 were present in all cells. DNA replication was almost exclusively associated with expression of TGFalpha. GSTp(+) hepatocytes showed a 3- to 4-fold higher probability of TGFalpha expression and of DNA synthesis than GSTp-negative (GSTp(-)) cells. PGE(2) or PGF(2alpha) increased expression of TGFalpha and DNA replication in GSTp(-) cells but not in GSTp(+) cells. PGA(2) and PGJ(2) decreased DNA synthesis in TGFalpha(+) cells without an obvious effect on the intracellular levels of TGFalpha. The Cox-2 inhibitor SC236 suppressed DNA replication preferentially in GSTp(+) cells; this inhibition was reversed by PGE(2)/F(2alpha). Indomethacin had no effect. These results suggest the following conclusions. (i) Growth regulation of preneoplastic GSTp(+) cells in culture exhibits distinct differences from GSTp(-) cells and elevated expression of TGFalpha contributes to their growth advantage. (ii) TGFalpha renders preneoplastic hepatocytes sensitive to suppression of DNA synthesis by PGA(2)/J(2). (iii) SC236, a Cox-2 inhibitor, may have preventive value in hepatocarcinogenesis.
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PMID:Role of transforming growth factor alpha and prostaglandins in preferential growth of preneoplastic rat hepatocytes. 1147 Jul 56

Cultures of perivenous (PV) and periportal (PP) hepatocytes could provide suitable in vitro models for studying the zone-specific hepatotoxic potential of xenobiotics. However, it is not known whether cultured PP and PV hepatocytes keep their phenotypes when the microcirculation of the liver changes. This question has been studied by culturing rat hepatocytes at 13 and 4% (v/v) O(2), respectively, mimicking the acinar oxygen gradient. PP and PV adult rat hepatocytes were isolated by digitonin-collagenase in situ perfusion and cultured on plastic Falcon and gas-permeable Petriperm dishes in Williams' E medium and kept at 13 and 4% (v/v) O(2), respectively. Cultures at 20% (v/v) O(2) on plastic dishes served as a control. Two types of cultures were studied, namely conventional cultures either unsupplemented or supplemented with 30 mM pyruvate. The activities of glutamine synthetase (GS) and glutathione S-transferase (GST) were measured in freshly isolated PP and PV hepatocytes and all cultures. The heterogeneous expression of GS (PV>PP), observed in freshly isolated hepatocytes, was kept for at least 4 days in culture. Total, Mu and Alpha class GST activities were predominantly expressed in PV freshly isolated cells. However, no beneficial effect could be observed in culture by exposing the cells to their specific in vivo oxygen concentration. The best maintenance of GST PV predominance in culture was observed in Petriperm dishes at 20% (v/v) O(2), as well in pyruvate-supplemented as unsupplemented cultures. PV GST predominance was thus kept in particular when the highest oxygen concentration was used and made available to the cells through the gas-permeable membranes. The results on GS PV predominance support these findings.
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PMID:Effect of oxygen concentration on the expression of glutathione S-transferase activity in periportal and perivenous rat hepatocyte cultures. 1156 68

To study the biological role of p73 alpha, a member of the p53 tumor suppressor family, we performed a yeast two-hybrid screen of a human cDNA library. Using a p73 alpha fragment consisting of amino acids 49-636 as bait, we found that p73 alpha is functionally associated with the human homologue of mouse and hamster homeodomain-interacting protein kinase 2 (HIPK2). The hamster homologue, also known as haHIPK2 or PKM, was used for further characterization of interactions between HIPK2 and members of the p53 protein family. Systematic yeast two-hybrid assays indicated a physical interaction between the oligomerization domains of p73 alpha and p53 (amino acid regions 345-380 and 319-360, respectively) and amino acid region 812-907 of haHIPK2. This region of haHIPK2 includes a PEST sequence, an Ubc9-binding domain, and a partial speckle retention sequence and is identical to amino acid residues 846-941 of human HIPK2 (hHIPK2). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro and immunoprecipitation assays in vivo. HIPK2 colocalized with p73 and p53 in nuclear bodies, as shown by confocal microscopy. Overexpression of HIPK2 stabilized the p53 protein and greatly increased the p73- and p53-induced transcriptional repression of multidrug-resistant and collagenase promoters in Saos2 cells but had little effect on the p73- or p53-mediated transcriptional activation of synthetic p53-responsive and p21WAF1 promoters. Stable expression of HIPK2 in U2OS cells enhanced the cisplatin response of sub-G(1) and G(2)/M populations, and it also increased the apoptotic response to cisplatin and adriamycin as demonstrated by fluorescence-activated cell sorter and 4',6-diamidino-2-phenylindole-staining analyses. HIPK2 potentiated the inhibition of colony formation by p73 and p53. These results suggest that physical interactions between HIPK2 and members of the p53 family may determine the roles of these proteins in cell cycle regulation and apoptosis.
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PMID:Identification and characterization of HIPK2 interacting with p73 and modulating functions of the p53 family in vivo. 1192 30


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