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)

Polyhydroxyethylmethacrylate (PHEMA) based microcarriers with different bulk structures were prepared by a phase inversion polymerization technique. PHEMA surfaces were further modified chemically by glow-discharge treatment, and biologically by covalent attachment of fibrinogen and collagen. Hepatocytes were isolated from young male Wistar rats using an in situ portal vein collagenase perfusion technique. Freshly isolated hepatocytes were seeded at 6 x 10(5) cells/mL and microcarrier concentration was 10 g/L. Stationary microcarrier cultures were carried out in standard (nontissue culture) polystyrene petri dishes in a humidified 5% CO2 incubator at 37 +/- 0.5 degrees C. Cell attachment was followed by light microscopy by taking samples from the culture medium every 30 min. Urea and protein syntheses by microcarrier-attached hepatocytes were determined by standard techniques. Nonswellable (highly cross-linked) hydrophilic PHEMA microcarriers did not support cell attachment and viability. However, swellable (low cross-linked) PHEMA microcarriers (pretreated in FBS) allowed high attachment and cell spreading. PHEMA microcarriers treated in dimethylaminoethylmethacrylate (DMAEMA) glow-discharge plasma also improved the cell attachment characteristics of the PHEMA microcarriers. The highest attachment efficiencies (immobilization yields) were observed with the biologically modified PHEMA microcarriers, especially modified with fibronectin. Metabolic activity, as estimated by urea and protein syntheses, was also higher in these microcarriers.
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PMID:Hepatocyte immobilization on PHEMA microcarriers and its biologically modified forms. 134 12

Studies were conducted to assess the mitogenic effect of lysosomal hydrolases, enzymes known to have an association with allergen- or ozone-induced airway hyperreactivity, on bovine tracheal myocytes in culture. Addition of purified human placental beta-hexosaminidase and partially purified bovine liver beta-glucuronidase resulted in the doubling of cell count after 4 d of incubation in medium M199 with 0.4% FBS. Unstimulated cells remained quiescent without a significant increase of cell count. Lysosomal hydrolases also selectively enhanced 3H-thymidine incorporation four to seven times more than that in vehicle-treated cells or cells treated with endotoxin, a common contaminant of purified enzymes. Ovalbumin (glycoprotein control), pronase, and lysozyme caused a modest but statistically insignificant increase (up to twofold) in 3H-thymidine incorporation. Elastase, collagenase and dialyzed E. coli beta-glucuronidase had no effect. The mitogenic effect of hydrolases was equally seen in quiescent, serum-depleted cells as well as in those maintained in medium with 10% FBS, suggesting that it was independent of serum factors. The effect of lysosomal hydrolases was inhibited by exposure to yeast mannan, and mannosylated human serum albumin had a mitogenic effect, suggesting the involvement of a mannose receptor. We conclude that lysosomal hydrolases may play a role in the development of the hyperplasia/hypertrophy of respiratory smooth muscle.
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PMID:Mitogenic effect of lysosomal hydrolases on bovine tracheal myocytes in culture. 183 69

Tracheal smooth muscle cells (TSMCs) were isolated from dog trachea in order to analyze the direct effects of growth factors and hormones on cell proliferation and muscarinic receptor (mAchR) expression. Dissection and dissociation of tracheal smooth muscle tissue with a collagenase I, deoxyribonuclease I and elastase IV mixture resulted in high yield and viability of TSMCs. A screen of growth factors, hormones, and serum concentration for the stimulation of cell growth, revealed that insulin-like growth factor, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, or hydrocortisone alone at the concentration used was not necessary or sufficient to stimulate growth of TSMCs in the primary culture with DMEM/F-12 containing 1% FBS. The regulation of cell surface mAchR expression in response to serum and cell growth in primary culture of TSMCs has been examined. In the presence of 1% serum, TSMCs withdraw from the cell cycle and express high levels of cell surface mAchRs. Exposure of quiescent TSMCs to 10% serum results in a loss of surface mAchRs. In addition, insulin-like growth factor, insulin or transferrin could stimulate the expression of mAchRs on TSMCs cultured in DMEM/F-12 containing 1% FBS. The results demonstrated that low serum concentration culture system may provide a useful model to elucidate the expression of mAchRs in the culture of TSMCs.
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PMID:Muscarinic receptor expression in the primary culture of tracheal smooth muscle cells. 207 1

The present study was performed to investigate the effects of endotoxins from periodontopathic bacteria on collagen metabolism. Endotoxins were extracted from Bacteroides gingivalis 381 and Bacteroides intermedius ATCC 25611 using the hot-phenol method. A commercially available endotoxin from Escherichia coli 0111: B4 was also used as a control. Human gingival fibroblasts (Gin-1, ATCC CRL 1292) were maintained with DMEM containing 10% FBS. When the fibroblasts became confluent they were exposed to each of the endotoxins in various concentrations (0, 5, 10, 15, 20 micrograms/ml). The effects of these endotoxins on the collagen metabolism of the fibroblasts were assessed on the basis of collagen synthesis and collagenase activity. The former was assessed by 3H-proline incorporation and bacterial collagenase digestable protein. The latter was assessed by fibril assay. The results were as follows: There was no change in glucose consumption, cell viability or morphology under the light microscope when the concentration of endotoxin was 20 micrograms/ml or less. 3H-proline incorporation into protein and collagen synthesis were inclined to decrease. Endotoxin from B. gingivalis resulted in an acceleration of collagenase activity. There findings indicate that the endotoxins from these bacteria might affect collagen metabolism early in gingivitis in vivo.
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PMID:[Effects of endotoxins from periodontopathic bacteria on the collagen metabolism of cultured normal human gingival fibroblasts]. 256 64

Fibroblast growth factor-2 (FGF-2) is a potent autocrine mitogen for fetal epiphyseal growth plate chondrocytes and exhibits a transient nuclear translocation during G1 of the cell cycle. We have characterized an intracellular binding protein (FGFBP) for FGF-2 that undergoes a juxtanuclear localization coincident with the nuclear translocation of the growth factor. Chondrocytes were isolated from the proliferative zone of the ovine fetal proximal tibial growth plate at 50-130 days gestation by collagenase digestion and were maintained in monolayer at early passage number. Cells were growth restricted by serum starvation for 48 h, and the synchronized culture was restarted into the cell cycle in the presence of 2% FBS. Cells were removed between 4-26 h of incubation, and fractions representing the plasma membrane, cytoplasm, nuclear membrane, and nuclear contents were separated by differential centrifugation. FGFBPs were separated using FGF-2 affinity chromatography. Ligand blot analysis using 125I-labeled FGF-2 showed that a FGFBP of 46-48 kDa (represented by a double band) was present on the nuclear membrane at mid to late G1, and Western blot showed this to be immunologically related to a part of the extracellular domain of the high affinity FGF receptor 1 (FGFR1). Immunocytochemistry with intact cell cultures showed that this protein underwent a juxtanuclear distribution through mid to late G1. Immunoprecipitation was performed to monitor newly synthesized FGFR1 migration throughout the cell cycle. Synchronized cells were cultured in medium containing 35S-labeled methionine/cysteine, and the cellular compartments were separated before immunoprecipitation using an antibody raised against the extracellular domain of FGFR1. Newly synthesized FGFR1-related proteins appeared throughout G1 and migrated multidirectionally within the cell; intact receptor of 125-145 kDa accumulated at the plasma membrane, while both intact receptor and truncated FGFR1 of 46-48 kDa were detected on the nuclear membrane, but not within the nucleus. Cells were incubated with protamine sulfate to prevent the binding of endogenous, cell membrane-associated FGF-2 to high affinity FGFRs and their subsequent internalization. This did not alter the juxtanuclear accumulation of truncated FGFR1 in late G1, suggesting that this was not derived from the plasma membrane. The truncated FGFR1 may mediate the nuclear translocation of FGF-2 during late G1.
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PMID:Perinuclear localization of an intracellular binding protein related to the fibroblast growth factor (FGF) receptor 1 is temporally associated with the nuclear trafficking of FGF-2 in proliferating epiphyseal growth plate chondrocytes. 889 82

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of alpha-smooth muscle (alpha-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, alpha-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor beta (TGFbeta) in FBS and PCS was determined and the effect of neutralizing anti-TGFbeta antibody was evaluated. It was determined that FBS contained more TGFbeta than PCS, but that essentially all the TGFbeta was latent in both sera. However, results obtained with anti-TGFbeta antibody show that active TGFbeta is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFbeta or an enzyme that is able to activate latent serum TGFbeta. Alternatively, sera may contain two different forms of latent TGFbeta, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds.
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PMID:Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro. 911 85

Recent studies indicate that vitamin D metabolites exert rapid effects on growth plate chondrocytes via changes in PG production and protein kinase C (PKC) activity. This suggests that these two products of vitamin D action may be interrelated. To test this hypothesis, we examined the effect of PGE2 on rat costochondral resting zone and growth zone cartilage cells and determined whether the effects of PGE2 are mediated by changes in the level of cAMP and/or PKC activity, whether there is a relationship between cAMP production and PKC activity, and whether cell maturation-specific effects are involved. Confluent, fourth passage resting zone and growth zone cartilage cell cultures were incubated in DMEM containing 10% FBS, 50 microg/ml vitamin C, and 1% antibiotics. The PGE2 concentration was varied from 0.007-15 ng/ml. Low concentrations of PGE2 caused a dose-dependent increase in cell number and [3H]thymidine incorporation and stimulated alkaline phosphatase specific activity. These effects were comparable in resting zone and growth zone cartilage cells at the same PGE2 concentrations. At higher concentrations, PGE2 caused a general increase in the synthesis of collagenase-digestible protein and noncollagenase-digestible protein in resting zone cartilage cells and of collagenase-digestible protein in growth zone cartilage cells, resulting in a net increase in the percent collagen synthesis for both cell types. cAMP production was increased over the entire range of chondrocyte response. Prevention of cAMP metabolism with the protein kinase A inhibitors H-8 and H-89 blocked the PGE2-dependent inhibition of PKC in resting zone cartilage cells in a dose-dependent manner. H-8 alone had no effect on PKC in resting zone cartilage cells, but stimulated PKC activity in growth zone cartilage cells; H-89 alone stimulated PKC activity in resting zone cartilage cells. These results suggest that low levels of PGE2 promote differentiation, whereas high doses promote an anabolic response; PGE2 increases cAMP production and PKC activity in a cell maturation-dependent manner; PGE2 exerts its effects via cAMP production and PKC activity; and regulation of PGE2-dependent PKC is via cAMP.
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PMID:The effect of prostaglandin E2 on costochondral chondrocyte differentiation is mediated by cyclic adenosine 3',5'-monophosphate and protein kinase C. 952 68

Coronary microvascular endothelial cells (CMECs) play an important role in many physiological processes. Porcine CMECs from large breed pigs have been isolated and successfully cultured. However, because micropigs offer research advantages over large breed pigs, micropig CMEC (MPCMEC) cultures may be useful as an alternative in vitro porcine model for cardiovascular studies. We isolated MPCMECs from six Panepinto micropigs using a simplified technique and developed a system for their successful culture. MPCMECs were isolated by collagenase digestion of left ventricular samples obtained using sterile techniques. Primary isolates of MPCMECs grew steadily in complete DMEM supplemented with 20% FBS, 4 mM MgSO(4), and 500 microM dibutyryl cAMP and reached confluence in 7-10 days. Endothelial origin was demonstrated by rapid (4-h) uptake of acetylated low-density lipoprotein, immunostaining for the presence of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), von Willebrand factor (vWf)-related antigen, vascular endothelial cadherin (VE-cadherin), endothelial nitric oxide synthase (eNOS), and by positive staining using two fluorescein isothiocyanate-labeled endothelial-specific lectins, Dolichos biflorus agglutinin and Ulex europaeus agglutinin-1. MPCMECs also exhibited immunostaining for alpha-smooth muscle actin. MPCMECs were successfully subcultured in the absence of dibutyryl cAMP and continued to express PECAM-1 and vWf, but not eNOS, to passage six. The typical morphology of subconfluent MPCMECs consisted of elongated cells that grew in a swirling, herringbone pattern.
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PMID:Cultivation and characterization of coronary microvascular endothelial cells: a novel porcine model using micropigs. 1220 52

N-nitrosomorpholine (NNM) is a hepatotoxic and hepatocarcinogenic agent. This agent was administered in the form of drinking water which contained 200 mg of NNM/liter. Its time-dependent intake profile showed four phases over 20 weeks, followed by a fifth phase where only water was supplied. Most frequently, hepatocellular carcinoma appeared between the end of phase IV and the beginning of phase V. At 5 weeks of NNM administration, foci of altered hepatocytes (FAH) containing 100-1000 hepatocytes could be isolated together with free hepatocytes by the collagenase perfusion method. When these foci were grown on the William's Medium E containing hormonally defined medium, they were able to survive approximately twice as long as normal hepatocytes At 10 weeks of NNM administration, few FAH were isolated together with free hepatocytes. The hepatocytes which had been placed under extended chemical stress showed increased heat tolerance (7 to 8 h) at 43 degrees C, while normal hepatocytes could survive 3 to 4 h. At the neoplastic phase spanning the end of the 20 weeks of the NNM administration and water phase, the rats bearing hepatocellular carcinoma entered the terminal stage, where observable tumor masses could be isolated from the tumor bearing liver and tested for ex vivo growth in tissue culture. After stabilization of the isolated primary hepatoma cells through 10 passages of propagation on William's Medium E or minimal Eagle's medium containing 10% FBS, their gene expression profile was analyzed by DNA microarray and compared with the profile of normal hepatocytes. The comparison revealed that upregulation involved ribosome-dependent protein synthesis, including 40S ribosomal proteins (S4, S7, S18, S20), 60S ribosomal proteins (L6, L21, L32, L37, P1), initiation factor 4A, and elongation factor 1alpha.
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PMID:Hepatotoxin N-nitrosomorpholine-induced carcinogenesis in rat liver: ex vivo exploration of preneoplastic and neoplastic hepatocytes. 1264 35

Recent studies suggest that human adipose tissue contain pluripotent cells similar to bone marrow-derived stromal cells (BSCs). Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that BSCs differentiate into a variety of cell lineages both in vitro and in vivo). In the present study, we extend this approach to characterize adipose-derived stromal cells (ASCs)). These cells derived from human are sometimes called processed lipoaspirate (PLA) cells. ASCs were prepared from inguinal fat pads of GFP transgenic mice after extensive washing with PBS and treatment with collagenase. After the primary culture in control medium (DMEM + 10% FBS), the cells were incubated in either chondrogenic medium (DMEM + 1% FBS + insulin + ascorbate 2-phosphate + TGF-beta 1) or osteogenic medium (DMEM + 10%FBS + dexamethasone + ascorbate-2-phosphate + beta-glycerophosphate) for two to four weeks. Chondrogenic differentiation was assessed by Alcian blue staining, while osteogenic differentiation was by von Kossa and Alkaline phosphatase staining. ASCs incubated in chondrogenic medium induced Alcian blue positive cells. Incubation with osteogenic medium became positive for von Kossa and Alkaline phosphatase staining. No osteochondrogenic differentiation was observed in cells incubated with control medium. This cell population can be easily identified through fluorescence microscope, it should be an ideal source of ASCs for further experiments of stem cell biology and tissue engineering.
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PMID:Chondrogenic and osteogenic differentiation of adipose-derived stem cells isolated from GFP transgenic mice. 1532 83


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