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

---

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

Human mammary epithelial cells from reduction mammoplasties were serially propagated in vitro from single cells and/or cell clusters using the NIH 3T3 cell feeder layer technique. In seven passages 46 cell population doublings, corrected for plating efficiency were achieved. The plating efficiency of epithelial cells in the primary culture was 0.2%. During subsequent passages it rose to 10-12% and decreased sharply towards the end of the culture life. In the third and fourth passages temporal prevalence of luminal cells was observed. The critical conditions for prevalence of the luminal phenotype were found to be the initial dissociation and optimum seeding density during subculturing. In primary cultures, after optimum dissociation of 0.15 cm3 mammary tissue with 0.05% collagenase A (Boehringher-Mannheim) in Eagle's MEM for 16 h at 37 degrees C, the yield on day 13 was 20 large colonies of 8-10 mm diameter. About 30% of the epithelial cells, which stained positively for the luminal cell marker cytokeratin 19, occupied colony centres. The remaining 70% were actin positive myoepithelial cells at the periphery. In subsequent passages, when using the optimum seeding density of 2 x 10(5) cells per 60 mm culture dish, the proportion of luminal cells gradually increased to 90% on day 35 in the fourth passage. A sudden rise in the proportion of rapidly growing myoepithelial cells to 65% was observed in the fifth passage. In the sixth and seventh passage small colonies were formed, most of which contained at least one keratin-19-positive (luminal) cell. Cells of human breast carcinomas are considered to be of luminal origin. Therefore, the described approach can be useful in studies of cell and molecular biology of mammary carcinomas.
...
PMID:Temporal in vitro expansion of the luminal lineage of human mammary epithelial cells achieved with the 3T3 feeder layer technique. 1093 Jan 12

Our study examined the expression of AP-1 family members in keratinocytes derived from the rat-4NQO model of oral carcinogenesis in which extremes of epithelial differentiation and tumour cell aggressiveness are evident. The constitutive expression of JunB was diminished in the undifferentiated, more aggressive tumour phenotype compared with the well-differentiated, less aggressive keratinocytes, whereas the expression of other AP-1 family members (c-jun, junD, c-fos, fra1, fra2 and fosB) was either very weak or variable. After transfection of the undifferentiated keratinocytes with junB cDNA, clonal populations were isolated that expressed similar levels of JunB protein as the well-differentiated cells. Both untransfected and transfected cell lines were keratin negative and vimentin positive. Increased expression of JunB in the transfected cells resulted in up-regulation of c-Jun and Fra1 and an enhanced AP-1 activity as demonstrated by transcriptional activation of the prototypic AP-1 dependent promoter, MMP-1. JunB transfected cells grew more quickly than vector-only controls and were refractory to the growth inhibitory effects of TGF-beta1. Over-expression of JunB resulted in the elevated expression of the AP-1 dependent proteinase, MMP-9, whereas the expression of the AP-1 independent enzyme, MMP-2, was unaffected. JunB transfected keratinocytes were highly invasive in an in vitro assay of tumour cell invasion compared with vector controls. The results indicate that increased expression of JunB above baseline levels in undifferentiated rat keratinocytes does not alter epithelial differentiation but enhances the malignant phenotype in vitro, possibly by altering the dynamics of the AP-1 complex.
...
PMID:Overexpression of JunB in undifferentiated malignant rat oral keratinocytes enhances the malignant phenotype in vitro without altering cellular differentiation. 1126 71

The differential expression of hundreds of tightly, transcriptionally controlled genes in isolated human colorectal cancer and respective normal mucosa from two patients was analyzed by the cDNA macroarray technique. mRNA prepared from the colorectal cancer tumors was compared with 588 genes spotted onto the filter. Case A showed down-regulation of the expression of cell-cycle-related genes including cyclins, cyclin-dependent kinase (CDK) 2, and CDK-activating kinase, as compared with normal mucosa from the same patient. The tumors showed up-regulation of expression of angiogenesis-related genes such as type II cytoskeletal 8 keratin, metalloproteinase subtypes, VEGF, and bFGF, to over 5-fold the levels in normal mucosa. Thus, colorectal carcinoma tissues are characterized by the upregulation of molecules related with angiogenesis. These results suggest that angiogenesis-related molecules are suitable candidates for target-based therapies for colorectal cancer patients. In case B, the largest difference in expression between the tumor and mucosal tissues was observed in the MMP-1 gene. In contrast to the first case, there was no increase in expression of angiogenesis-related molecules or decrease in expression of cell-cycle-regulatory molecules. The expression profile was quite different between these two patients. This approach may eventually provide a mean of selecting target-based drugs in individual colon cancer patients.
...
PMID:Upregulated expression of angiogenesis genes and down regulation of cell cycle genes in human colorectal cancer tissue determined by cDNA macroarray. 1129 27

The study of the capacity of 310 strains of microorganisms from different taxonomic groups (40 bacilli, 43 yeast, 105 streptomycetes, 12 micromycetes) to hydrolyze collagen and keratin allowed to establish that the highest level of collagenase (KA) and keratinase (KerA) activity is inherent in representatives of streptomycetes. Two strains of Streptomyces sp.--1349 and 1382 with the highest KA and KerA indices--1.9 and 1.85 un./mg of protein, respectively, have been chosen. It has been established that collagenase activity in the medium without adding the inducers decreases 4.76 times, while that of keratinase--5.71 times, i.e. the above enzymes are inducible. The investigation of the spectrum of activities has demonstrated that the both strains possess low level of the general proteolytic and elastase activities and high level of collagenase and keratinase activities. Partial purification of the enzyme complex of Streptomyces sp. 1349 by the successive precipitation by ammonium sulphate with 30, 60 and 80% saturation and a single precipitation by ammonium sulphate with 80% saturation helped to increase the level of KA 5.6-5.9 times, and that of KerA--4.2-4.5 times.
...
PMID:[Screening of collagenase and keratinase producers]. 1194 44

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.
...
PMID:Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta. 1202 Oct 45

The schemes of isolation and purification of collagenolytic enzymes of Streptomyces sp. 1349 and keratinolyte enzymes of Streptomyces sp. 1382, which include fractionation by ammonium sulphate separation on TSK-gels: ion-exchange chromatography on Toyopearl DEAE-650(M) and gel-filtration on Toyopearl HW-50, as well as highly efficient liquid chromatography. The purified enzyme preparations proved to be proteases of serine type (collagenase 2 and keratinases) as well as metalloproteases (collagenases 1 and 3). It has seen established that collagenases are enzymes of broad specificity, which are active in respect of proteins of both globular and fibrillar nature. And vice versa, keratinases are proteolytic enzymes of narrow specificity which hydrolyze native keratin. Molecular masses of purified enzyme preparations, from the data of SDS-PAAG are approximately 30-40 kDa (collagenases 1-3) and about 15-20 kDa (keratinases 1 and 2). It is shown that the charged aminoacid residues (about 85%) prevail in enzyme molecules. The enzymes are distinguished by pH- and thermooptima.
...
PMID:[Purification and physico-chemical properties of Streptomyces sp. 1349 collagenase and Streptomyces sp. 1382 keratinase]. 1520 50

A reproducible and original method for the preparation of chicken intestine epithelial cells from 18-day-old embryos for long-term culture was obtained by using a mechanical isolation procedure, as opposed to previous isolation methods using relatively high concentrations of trypsin, collagenase, or EDTA. Chicken intestine epithelial cells typically expressed keratin and chicken E-cadherin, in contrast to chicken embryo fibroblasts, and they increased cell surface MHC II after activation with crude IFN-gamma containing supernatants, obtained from chicken spleen cells stimulated with concanavalin A or transformed by reticuloendotheliosis virus. Eimeria tenella was shown to be able to develop until the schizont stage after 46 hr of culture in these chicken intestinal epithelial cells, but it was not able to develop further. However, activation with IFN-gamma containing supernatants resulted in strong inhibition of parasite replication, as shown by incorporation of [3H]uracil. Thus, chicken enterocytes, which are the specific target of Eimeria development in vivo, could be considered as potential local effector cells involved in the protective response against this parasite.
...
PMID:Chicken primary enterocytes: inhibition of Eimeria tenella replication after activation with crude interferon-gamma supernatants. 1552 84

The accuracy of DNA ploidy measurements of paraffin-embedded tissues is limited by the lack of resolution and the inability to identify the DNA diploid population unequivocally in bimodal DNA histograms. A multi-parameter DNA flow cytometric method has been developed that enables the simultaneous detection of neoplastic and stromal cells in samples from dewaxed 50 microm sections or 2 mm diameter punches of archival tissue blocks. The method combines heat pretreatment in sodium citrate buffer and subsequent enzymatic dissociation with a collagenase/dispase mixture. Cells were simultaneously stained for keratin (FITC), vimentin (R-PE), and DNA (PI) before flow cytometric analysis. The method was applied to 12 paraffin-embedded cervical carcinomas and four colorectal carcinomas. In all cervical cancers, distinct keratin-positive and vimentin-positive cell populations were observed. While the exclusive vimentin-positive cell fractions always yielded unimodal DNA content distributions, bimodal distributions were observed for the keratin-positive cell fractions in nine cervical carcinomas, whereas one cervical carcinoma showed three distinct G0G1 populations. Coefficients of variation of the G0G1 peaks ranged from 1.70% to 4.79%. Average background, aggregate, and debris values were 14.7% (vimentin-positive fraction) and 33.8% (keratin-positive fraction). Flow sorting confirmed that the exclusively vimentin-positive cell fractions represent different normal stromal and infiltrate cells that can serve as an internal ploidy reference enabling discrimination between DNA hypo-diploid and DNA hyper-diploid tumour cell subpopulations. The neoplastic origin of the keratin-vimentin co-expressing cells from two cervical carcinomas was confirmed by genotyping of flow-sorted samples revealing loss of heterozygosity (LOH) of 6p. This improved method obviates the need for fresh/frozen tumour tissue for high-resolution DNA ploidy measurements and enables the isolation of highly purified tumour subpopulations for subsequent genotyping.
...
PMID:High-resolution multi-parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin-embedded tissues. 1582 70

Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest Km among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.
...
PMID:Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. 1600 Jul 44

The paper determines the action specificity concerning the bonding type of collagenases of Strepomyces sp. 1349 and keratinases of Streptomyces sp. 1382. Experimental data obtained evidence for the wide specificity of the obtained enzymatic drugs. It has been established that both collagenases and keratinases display high specificity in respect of the bonds made by the residues of hydrophobic amino acids. Collagenases of Streptomyces sp. 1349, as to their wide action specificity, differed from collagenase of Clostridium histolyticum described in literature, that was confirmed by the results of double immunodiffusion in agar by Ouchterloni. Preparations of streptomycete collagenase obtained by the authors did not interact with serum obtained for highly purified collagenase of C. histolyticum of the firm "Merck". Investigation of the feather keratin lysates by the fractions of keratinases 1 and 2 have shown the difference in the content of amino acids released after hydrolysis that may be determined by different specificity of the enzymes action. Cysteine in the amount of 3.8% was also found in keratin lysate of the enzymatic fraction 1, that may evidence for the capacity of the fraction 1 to break the disulphide bonds in keratin molecule.
...
PMID:[Substrate specificity of collagenase of Streptomyces sp. 1349 and keratinase of Streptomyces sp. 1382]. 1601 11


<< Previous 1 2 3 4 5 Next >>

---