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)

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.
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PMID:Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation. 1120 68

Coronary microvascular endothelial cells exert (patho)physiological effects on the function of cardiac myocytes, which may be studied experimentally using pure cell populations. As an essential pre-requisite to the investigation of cells from gene-modified mice, we studied the phenotypic properties of coronary microvascular endothelial cells isolated from normal mice, and biochemically characterized the superoxide production by these cells. Microvascular endothelial cells were isolated from devitalized mouse ventricular tissue after sequential digestion with collagenase, trypsin and DNase. Coronary microvascular endothelial cells were separated from cardiac myocytes and other cells by differential centrifugation, plating and culture. Mouse coronary microvascular endothelial cells showed an irregular "cobblestone" morphology at confluence, were >98% positive for CD31 by FACS analysis, and were also positive for VE-cadherin and endothelial-type nitric oxide synthase (eNOS) by confocal microscopy. The cells took up fluorescently labelled, acetylated low-density lipoprotein, but were negative for a alpha -smooth muscle actin, desmin and cytokeratin. Unlike human endothelial cells, mouse coronary microvascular endothelial cells only weakly expressed von Willebrand factor. Immunoblotting showed that the mouse cells expressed components of a phagocyte-type NADPH oxidase. They exhibited NADPH-dependent O(2)(-)-generating activity, which was increased by angiotensin II but completely inhibited by diphenyleneiodonium. Thus, mouse coronary microvascular endothelial cells express both eNOS and NADPH oxidase, interactions between which may play a role in endothelial cell pathophysiology.
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PMID:Phenotypic properties and characteristics of superoxide production by mouse coronary microvascular endothelial cells. 1144 17

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.
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PMID:Variability in glucose transporter-1 levels and hexokinase activity in human melanoma. 1182 56

Recently, increasing evidence suggests that alpha inhibin or related proteins may be a functional regulator in the ovary, which is independent of hetero-dimer inhibin. In our previous study, it was demonstrated that human inhibin alpha-N-terminal fragment Tyr-1-32 (P(33)) significantly inhibited progesterone production by rat corpus luteal cells in vitro, and stimulated luteal functional regression and apoptosis in vivo. In the present work, the action of P(33) on apoptosis was further studied in vitro in cultured rat CL cells. Gel electrophoretic analysis for detection of oligonucleosomal DNA fragmentation, AO-EB or PI assays and flow cytometry were used to observe the action of P(33) on the occurrence of spontaneous apoptosis by collagenase-DNase dispersed CL cells, obtained from PMSG-hCG induced pseudopregnant rats. The results showed that P(33) (1 microg/ml) stimulated spontaneous apoptosis of CL cells. The inhibitor of tyrosine protein kinase, genistein (50 microg/ml),inhibited P(33) enhanced spontaneous apoptosis. RNA and protein synthesis inhibitors cycloheximide (Cyx,50 microg/ml) and actinomycin D(Act D,50 microg/ml) did not protect the cells from apoptosis stimulated by P(33). The results suggest that P(33) stimulates spontaneous apoptosis in cultured rat CL cells with the involvement of tyrosine specific protein kinase system. This work provides further evidence for the hypothesis that alpha inhibin or related protein might be a functional regulator in the ovary.
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PMID:[Effect of human inhibin alpha-fragment 1-32-Tyr(P33) on apoptosis of cultured rat corpus luteal cells]. 1197 81

Alternative approaches to overcome the shortage of donors for liver transplantation may be the use of hepatocytes for bioartificial devices or transplantation. Therefore, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of hepatocytes represents a formidable challenge. Aim of this study was to obtain a liver homologous acellular matrix (HAM) able to support viability and metabolic functions of rat hepatocytes in primary culture. HAMs were prepared by sequential incubation of rat liver slices in deoxycholic acid and DNase solutions. Dispersed rat hepatocytes were obtained by collagenase digestion and mechanical disaggregation. Isolated hepatocytes were seeded on uncoated and collagen- or HAM-coated tissue culture plastic wells. Cultures were examined by scanning electron microscopy (SEM), and the viability of hepatocytes and their ability to produce albumin and urea were assessed. The viability of freshly dispersed hepatocytes was about 98%. Hepatocytes seeded on HAM exhibited a significantly higher viability and a markedly lower apoptotic rate than those grown on plastic or collagen. Accordingly, albumin and urea nitrogen productions were significantly higher in HAM-cultured hepatocytes. SEM showed that hepatocytes seeded on HAM displayed a clustered organization, and were well anchored to the matrix and morphologically stable. Taken together, these findings indicate that HAM strongly improves viability and functional activity of rat hepatocytes cultured in vitro.
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PMID:Acellular liver matrix improves the survival and functions of isolated rat hepatocytes cultured in vitro. 1537 76

Effects and mechanisms of FR167653, 1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4] triazin-2-yl]-2-phenylethanedione sulfate monohydrate, a dual inhibitor of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), on rat adjuvant arthritis (AA) was investigated. Complete Freund's adjuvant was used to induce AA in rats. Secondary paw swelling of AA rats was measured, and polyarthritis index was scored. Synoviocytes were separated by the method of collagenase and DNase digestion. Synoviocytes proliferation was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. TNF-alpha, IL-1 and interleukin-10 (IL-10) production of synoviocytes was measured with ELISA. The expression of IL-10 mRNA of synoviocytes was determined using RT-PCR. There were significant secondary inflammatory reactions in AA rats, which accompanied with the decrease of body and immune organs weight simultaneously. The administration of FR167653 (4, 12, 36 mg/kg, subcutaneously (s.c.)) inhibited the inflammatory response and restored the weight of body and immune organs of AA rats. Synoviocytes proliferation of AA rats significantly increased, and the levels of TNF-alpha and IL-1 in supernatants of synoviocytes in AA rats were also elevated compared with the sham group. The administration of FR167653 (4, 12, 36 mg/kg, s.c.) reduced the above changes significantly. In contrast to TNF-alpha and IL-1, IL-10 production and the level of its mRNA of synoviocytes in AA rats were apparently decreased. FR167653 (4, 12, 36 mg/kg, s.c.) markedly increased IL-10 in synoviocytes at protein and transcription level. The results indicated that FR167653 had a beneficial effect on rats AA due to modulating inflammatory cytokines production of synoviocytes, which played a crucial role in pathogenesis of this disease.
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PMID:Effects and mechanisms of FR167653, a dual inhibitor of interleukin-1 and tumor necrosis factor, on adjuvant arthritis in rats. 1545 15

Goat preantral follicles were cultured to investigate the effects of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the in vitro growth and viability of oocytes. Preantral follicles were isolated mechanically and enzymatically (using collagenase and DNase) from prepuberal goat ovaries. The working medium was composed of Defined Eagle's Minimum Essential Medium (DMEM) supplemented with HEPES (20 mM), 10% fetal calf serum (FCS), hypoxanthine (2 mM), dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) (2 mM), penicillin (75 ng/ml) and streptomycin (50 ng/ml). The culture medium consisted of the working medium with follicle stimulating hormone (FSH) (100 ng/ml) and hydrocortisone (40 ng/ml) added. In the experiment, goat preantral follicles were cultured for 9 days in the culture medium and in the culture medium supplemented with either IGF-I (100 ng/ml), EGF (50 ng/ml), bFGF (50 ng/ml) or IGF-I (100 ng/ml)+EGF (50 ng/ml). The results indicated that IGF-I (100 ng/ml) effectively maintained the survival of oocytes and promoted their growth; EGF (50 ng/ml) enhanced the survival rate of oocytes but had a negative effect on oocyte growth; bFGF (50 ng/ml) stimulated oocyte survival but had no obvious effect on their growth while IGF-I (100 ng/ml) and EGF (50 ng/ml) in combination had a greater effect on both survival and growth rate of oocytes than IGF-I or EGF alone. The supplementation of IGF-1 and EGF to the culture medium is recommended in the culture of goat preantral follicles.
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PMID:Regulation of in vitro growth of preantral follicles by growth factors in goats. 1576 Jun 65

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.
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PMID:FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles. 1672 44

During mammalian follicle development, a fluid-filled antrum develops in the avascular centre of the follicle. We investigated the hypothesis that follicular fluid contains osmotically-active molecules, sufficiently large so as to not freely escape the follicular fluid. Such molecules could generate an osmotic differential and thus recruit fluid from the surrounding vascularised stroma into the antrum. Follicular fluid was collected from bovine follicles classified histologically as healthy (n = 4 pools) or atretic (n = 4 pools). Dialysis of the follicular fluid at 300 kDa or 500 kDa resulted in a reduction in colloid osmotic pressure of 35% and 60%, respectively, in fluid from healthy follicles and 29% and 80% from atretic follicles. Digestion of follicular fluid with Streptomyces hyaluronidase, chondroitinase ABC or DNase 1 followed by dialysis resulted in reductions in osmotic pressure of 43%, 53% and 43% respectively for fluids from healthy follicles and 34%, 20% and 31% for atretic follicles. Digestion with collagenase I, proteinase K, heparanase 1 or keratanase had no significant effect on the osmotic pressure of follicular fluid of healthy follicles. Ion exchange and size exclusion, Western blotting and ELISA identified the proteoglycans versican and inter-alpha trypsin inhibitor and the glycosaminoglycan hyaluronan in follicular fluid. We conclude that these molecules or aggregates of them are of sufficient size to contribute to the osmotic potential of follicular fluid and thus recruit fluid into the follicular antrum. DNA may also contribute but it is probably not a component that is regulated for this role.
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PMID:Formation of ovarian follicular fluid may be due to the osmotic potential of large glycosaminoglycans and proteoglycans. 1681 38

This study was conducted to culture in vitro caprine pre-antral follicles for determining the competence of growth and maturation of oocytes and establishing a suitable culture system for oocyte maturation from pre-antral follicles. Two different culture methods (microdrop and agar gel clot) were employed to culture caprine pre-antral follicles. The pre-antral follicles were isolated from prepubertal goat ovaries by treatment with collagenase and DNase. The isolated pre-antral follicles were cultured in basic culture medium for 9 days (for growth). And oocytes were cultured in maturation culture medium for another 2 days for maturation. The result demonstrated that the growth rate of oocytes cultured in microdrops was significantly (p < 0.05) higher than that in agar gel clots, whereas the viability of oocytes in microdrops was considerably (p < 0.05) lower than that in agar gel clots. The oocytes grew over 150 microm in diameter, and two of 151 oocytes cultured in microdrops yielded morphologically abnormal first polar bodies. However, the size of oocytes cultured in agar gel approached to 120 microm in diameter and no polar body was produced.
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PMID:In vitro growth and maturation of caprine oocytes. 1698 51


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