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)

The level of unscheduled DNA synthesis in the parenchymal cells from hyperplastic nodules and from the entire liver of rats fed N-2-fluorenylacetamide was studied and compared with that of normal liver cells. Measurements of unscheduled DNA synthesis were carried out by the use of a primary liver cell culture system. Livers were perfused with collagenase, the cells from individual hyperplastic nodules, and/or from the whole liver aspirated and plated onto plastic Petri dishes. Simultaneous histochemical measurements of beta-glucuronidase were carried out in the cultured cells as an aid in distinguishing functional cell types. The cells from hyperplastic nodules obtained from the liver during carcinogen feeding survived much longer than normal liver cells in culture. The level of unscheduled DNA synthesis was determined radioautographically after exposing cells to ultraviolet light and incubating with [3H]thymidine. [3H]Thymidine labeling was variable among individual nodules or animals and fluctuated as a function of the number of days in culture. In general, however, the level of unscheduled DNA synthesis in the cells from hyperplastic nodules was always higher than or similar to that of normal liver cells. Thus, the cells of hyperplastic nodules are not more readily transformed into the malignant state than normal cells as a result of their lowered DNA repair mechanisms.
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PMID:Unscheduled DNA synthesis in cells from N-2-fluorenylacetamide-induced hyperplastic nodules of rat liver maintained in a primary culture system. 123 66

The expression of a novel regenerating (reg) gene has been reported previously in the regenerating islets of a surgical model of diabetes in rats. We exposed collagenase-isolated rat islets for three days to nutrient and non-nutrient growth factors in minimally supplemented RPMI medium (2.7 mmol/l glucose, 2% fetal calf serum), and investigated the relationship between reg gene expression and islet cell replication. RNA was prepared from half of the islets by homogenisation in guanidinium isothiocyanate followed by phenol/chloroform extraction. Northern/dot blot analyses were used to semi-quantify reg mRNA. Islet cell replication was estimated by culturing the remaining islets in radiolabelled thymidine to determine de novo DNA synthesis. Thymidine uptake was stimulated by the following factors: 11 mmol/l glucose (50% increase); 10% amino acids (126% increase); 10% fetal calf serum (39% increase); 100 ng/ml insulin (45% increase); 250 ng/ml growth hormone (65% increase); 1.5 nmol/l aldosterone (29% increase); 2 U/ml platelet derived growth factor (116% increase). The results are expressed as a percentage of the thymidine incorporated into control islets cultured in minimal RPMI (1118 +/- 100 (SD) cpm/microgram protein, n = 15). Increased islet cell replication was paralleled in each case by a clear rise in reg mRNA expression compared to controls. Furthermore, the rank order for reg gene expression was the same as that for thymidine uptake (r = 0.90). The present findings suggest a clear association between reg gene expression and islet cell replication in vitro, and are the first to demonstrate reg gene expression in response to individual growth factors.
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PMID:Expression of an islet regenerating (reg) gene in isolated rat islets: effects of nutrient and non-nutrient growth factors. 137 94

A three-dimensional culture model for isolated murine pelage hair follicles in a type I collagen gel has been utilized to study the effects of selected growth factors on follicle cell proliferation and release of collagenolytic factors. Cultured follicle organoids differentially express cytokeratins 6 and 14 in a pattern suggesting they contain cells of the outer root sheath, inner root sheath and follicle matrix. Using incorporation of [3H]thymidine as a measure of proliferation, follicle organoids show a peak of DNA synthesis between day 1 and 5 of culture, depending on plating density, and then have a low rate of DNA synthesis. Thymidine incorporation is stimulated by transforming growth factor-alpha (TGF-alpha) in a dose-dependent response. Only peripheral cells presumably of the outer root sheath, incorporate thymidine in basal or stimulated conditions. TGF-beta 1 and TGF-beta 2 inhibit constitutive cell proliferation and oppose growth stimulation by TGF-alpha. Hair follicles lyse the collagen gel matrix when exposed to certain cytokines. Epidermal growth factor (EGF) and TGF-alpha stimulate gel lysis, but TGF-beta 1, TGF-beta 2 and cholera toxin do not. Other skin-derived cells, such as interfollicular epidermal cells, dermal fibroblasts, or combinations thereof, do not lyse gels in this culture model even when exposed to growth factors. Combinations of EGF or TGF-alpha with TGF-beta 1 or TGF-beta 2 are synergistic for collagenase release. These cytokines stimulate release of multiple species of matrix metalloproteinases, but the 92-kDa and 72 kDa type IV procollagenases and their activated derivatives predominate on zymograms. In cytokine-stimulated follicles, both peripheral and centrally located cells in the organoids express the 72-kDa type IV collagenase and a similar immunostaining pattern is present in developing follicles in vivo. Thus growth factors appear to work in concert for certain hair follicle responses and in opposition for others. These combined actions may play a role in different phases of hair follicle development that require cell replication and invasion into the deeper dermis.
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PMID:Growth factors specifically alter hair follicle cell proliferation and collagenolytic activity alone or in combination. 196 9

Thymic epithelial cells were grown in defined medium without unknown serum factors and without concurrent growth of other cell types. Thymic tissue was obtained from 1- to 4-wk-old mice, disaggregated, and incubated in a mixture of collagenase-dispase-DNAse. The resulting organoids were seeded on collagen-coated flasks. The culture medium consisted of DME-F12 with low or high concentration of Ca2+ supplemented with insulin, epidermal growth factor, cholera toxin, hydrocortisone, and transferrin. Under these conditions, explants attached to the substrate within 2 d, and expanding epithelioid monolayer islets emerged from the organoids during the following days. [3H]Thymidine incorporation revealed a growth fraction of the cells close to 5%. By omitting either epidermal growth factor, insulin, or cholera toxin from the medium, pronounced reduction in sizes of islets and in [3H]thymidine incorporation was found. Throughout the culture period, the islets appeared as continuous sheets of polygonal cells. The epithelial nature of the expanding cell islets was confirmed by demonstration of cytokeratins and of desmosomes. Ultrastructural evaluation of early cultures revealed clusters of epithelial cells intermixed with lymphocytes, and late cultures showed a typical pattern of stratified keratinizing epithelium. However, squamous metaplasia was avoided by the use of low Ca2+ medium, which also proved essential for cell transfer. MHC class II antigen was detected on the majority of the cultured cells, and culture supernatants contained co-mitogenic activity for thymocytes and GM-colony stimulating activity.
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PMID:Short-term cultivation of murine thymic epithelial cells in a serum-free medium. 238 45

Collagenase dissociation, performed on 40 human breast cancers, yielded between 1 million and 50 million cells from less than 1 g of tissue from each tumor. Approximately 60% of cells (mean) was considered viable as judged by trypan blue exclusion and phase microscopy. On subsequent flow cytometric analysis, 20 cancers (50%) were considered diploid, three were tetraploid, and the remainder, hyperdiploid. Thymidine labeling (TLI) and flow cytometry following mechanical dissociation also were performed on 23 of these 40 tumors. Among this group of 23 cases, the median percentage of S-phase cells obtained by collagenase dissociation was 5.4, by TLI was 5.7, and by mechanical dissociation was 9.7. There was excellent correlation between the percentage of S-phase cells obtained by collagenase and TLI (r = 0.847, p = 0.0001) but only fair correlation between the percentage of S-phase cells obtained by mechanical dissociation and TLI (r = 0.597, p = 0.0027). The percentage of S-phase cells obtained by either collagenase or mechanical dissociation predicted whether a tumor was above or below median TLI in 19 of 23 cases (p = 0.0018). Estrogen receptor positivity or negativity did not predict whether a tumor was above or below median TLI (r = 0.283, p = 0.130) or above or below median S-phase fraction following collagenase dissociation (r = 0.218, p = 0.182), nor did quantitative estrogen receptor correlate significantly with TLI (r = 0.283, p = 0.13) or S-phase fraction (r = 0.218, p = 0.18).
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PMID:A method for dissociation of viable human breast cancer cells that produces flow cytometric kinetic information similar to that obtained by thymidine labeling. 632 21

Stromal cells derived from collagenase-digested benign hyperplastic adult prostates were isolated and grown in culture. Androgen and oestrogen receptor status were determined and growth in response to mibolerone (a synthetic androgen) and oestradiol-17 beta was measured. In addition, the ability of oestrogens to regulate the androgen receptor in stromal cells was investigated. [3H]Thymidine incorporation into DNA was stimulated by mibolerone in primary and secondary cultures, but sensitivity was lost with subsequent passages. Androgen stimulation of [3H]thymidine incorporation was consistently inhibited by the anti-androgen cyproterone acetate. Oestradiol-17 beta also stimulated [3H]thymidine incorporation into DNA, and this effect was inhibited by the anti-oestrogen tamoxifen. Sensitivity to oestradiol was lost with subsequent passages. A combination of mibolerone and oestradiol was not synergistic in increasing [3H]thymidine incorporation into DNA, but maximal stimulation occurred at 100-fold lower concentrations of mibolerone and oestradiol when the two hormones were applied in combination. Specific high-affinity [3H]mibolerone- and [3H]oestradiol-binding sites were demonstrated by radioligand binding in intact cells. The affinity for oestradiol binding to its receptor exceeded that quantified for mibolerone binding to the androgen receptor, whilst the number of oestradiol-binding sites was approximately tenfold less than that quantified for mibolerone. Treatment with oestradiol down-regulated the number of [3H]mibolerone binding sites 1.7-fold (P < 0.005) as early as day 2 after oestradiol treatment. In conclusion, we successfully cultured stromal cells derived from hyperplastic prostates which retained sensitivity to androgen and oestrogen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Androgen and oestrogen responsiveness of stromal cells derived from the human hyperplastic prostate: oestrogen regulation of the androgen receptor. 753 Feb 86

The effect of surface roughness on osteoblast proliferation, differentiation, and protein synthesis was examined. Human osteoblast-like cells (MG63) were cultured on titanium (Ti) disks that had been prepared by one of five different treatment regimens. All disks were pretreated with hydrofluroic acid-nitric acid and washed (PT). PT disks were also: washed, and then electropolished (EP); fine sandblasted, etched with HCl and H2SO4, and washed (FA); coarse sandblasted, etched with HCl and H2SO4, and washed (CA); or Ti plasma-sprayed (TPS). Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by brightfield and darkfield microscopy, cold field emission scanning electron microscopy, and laser confocal microscopy, while chemical composition was mapped using energy dispersion X-ray analysis and elemental distribution determined using Auger electron spectroscopy. The effect of surface roughness on the cells was evaluated by measuring cell number, [3H]thymidine incorporation into DNA, alkaline phosphatase specific activity, [3H]uridine incorporation into RNA, [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagenase-digestible protein (NCP), and [35S]sulfate incorporation into proteoglycan. Based on surface analysis, the five different Ti surfaces were ranked in order of smoothest to roughest: EP, PT, FA, CA, and TPS. A TiO2 layer was found on all surfaces that ranged in thickness from 100 A in the smoothest group to 300 A in the roughest. When compared to confluent cultures of cells on plastic, the number of cells was reduced on the TPS surfaces and increased on the EP surfaces, while the number of cells on the other surfaces was equivalent to plastic. [3H]Thymidine incorporation was inversely related to surface roughness. Alkaline phosphatase specific activity in isolated cells was found to decrease with increasing surface roughness, except for those cells cultured on CA. In contrast, enzyme activity in the cell layer was only decreased in cultures grown on FA- and TPS-treated surfaces. A direct correlation between surface roughness and RNA and CDP production was found. Surface roughness had no apparent effect on NCP production. Proteoglycan synthesis by the cells was inhibited on all the surfaces studied, with the largest inhibition observed in the CA and EP groups. These results demonstrate that surface roughness alters osteoblast proliferation, differentiation, and matrix production in vitro. The results also suggest that implant surface roughness may play a role in determining phenotypic expression of cells in vivo.
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PMID:Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). 754 45

Cirrhotic livers are considered to regenerate less actively than normal livers after hepatic resection. Little is known about the mechanisms responsible for impaired capacity of regeneration in cirrhotic liver. In the present study, we investigated the effect of phorbol ester on hepatocyte proliferation in healthy and cirrhotic hepatocytes, using one of the phorbol esters, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which has a direct effect on activation of protein kinase C (PKC). Cirrhosis was established by the administration of carbon tetrachloride and phenobarbital to rats. Healthy and cirrhotic hepatocytes were isolated from Wistar male rats by a two-step collagenase perfusion technique. DNA synthesis was estimated by [3H]thymidine incorporation into DNA and by autoradiographic nuclear labeling index. [3H]Thymidine incorporation was measured 24 hr after hepatocytes were stimulated by appropriate reagents. TPA (50 nM) stimulated [3H]thymidine incorporation in healthy hepatocytes (control vs TPA, 991 +/- 247 vs 2569 +/- 766 mean +/- SEM cpm/microgram DNA; P < 0.05), whereas TPA (50 nM) failed to stimulate in cirrhotic hepatocytes (control vs TPA, 1144 +/- 184 vs 1304 +/- 187 cpm/microgram DNA; NS). Staurosporine, a specific PKC inhibitor, suppressed [3H]thymidine incorporation in TPA-stimulated healthy hepatocytes (806 +/- 263 cpm/microgram DNA; P < 0.05); however, it had no effect on cirrhotic hepatocytes (1295 +/- 180 cpm/microgram DNA; NS). An autoradiographic nuclear labeling index exhibited the same results with [3H]thymidine incorporation. We conclude that TPA stimulates hepatocyte proliferation in healthy rat hepatocytes but has no effect on cirrhotic hepatocytes.
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PMID:Impaired phorbol ester-induced hepatocyte proliferation in cirrhosis. 772 25

The effects of fluoride on bone tissue are now well documented by in vivo histological studies performed on both human and animal bone biopsies and demonstrating an increase in osteoblast (OB) population. In order to elucidate whether the mechanism of action of fluoride on osteoblasts was direct or indirect, 14 three-week-old Sprague-Dawley rats were selected. Seven animals received 100 ppm fluoride as sodium fluoride (NaF) in drinking water for one month. The other animals, which did not receive fluoride, were considered as controls. At the end of the experiment, femurs and vertebrae were excised and osteoblastic cells were obtained after collagenase digestion separately from each animal. The osteoblastic cells derived from control and NaF-treated rats were exposed in vitro to 10(-5) M NaF. Alkaline phosphatase (AP) activity was measured, and the cellular proliferation was assessed by 3H-thymidine incorporation. Thymidine incorporation and AP activity were significantly higher in osteoblastic cells derived from NaF-treated rats than in cells obtained from control rats (p = 0.05 and p < 0.01, respectively). In contrast, the osteoblast proliferation and activity were not modified after in vitro exposure to NaF in cells derived from control and NaF-treated rats. In conclusion, the function of osteoblasts was not modified after in vitro exposure to fluoride. In contrast, given in vivo to rats for one month, fluoride has a mitogenic effect on osteoblasts and stimulates their activity. These data emphasize the hypothesis that fluoride may act either on osteoprogenitor cells or through an indirect mechanism mediated by a cofactor.
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PMID:Fluoride increases rat osteoblast function and population after in vivo administration but not after in vitro exposure. 826 46

I cultured human adult endothelial cells derived from cadavers. Iliac arteries of cadavers were resected at autopsy and their endothelial cells were yielded sterile by digestion of 0.1% collagenase solution. I tried culture of endothelial cells of 72 cadavers at autopsy and 14 cases of them were successful. They were observed with phase-contrast microscopy, transmission electron microscopy and immunofluorescence microscopy, which showed polygonal monolayered cells, Weibel-Palade bodies and Factor VIII antigen in these cells. I also examined the doubling time (2-4 weeks) of these cells. 3H-Thymidine leveling index showed the differentiation of effects between fibroblast growth factor and endothelial cell growth factor on human adult endothelial cells, while no differentiation between them on human umbilical vein endothelial cells. Also, I observed apolipoprotein E in the cells, but not in umbilical vein endothelial cells. It established the procedure of their culture method.
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PMID:[Culture of human adult endothelial cells derived from cadavers of autopsy]. 832 Nov 86


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