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 objective of this study was to determine whether DNA synthesis induced in the livers of female rats treated with ethinyl estradiol (EE) was due to direct effects of this synthetic estrogen on hepatocytes. Hepatocytes, obtained by collagenase perfusion from female Lewis rats, were cultured in serum-free medium containing low or no phenol red and supplemented with insulin, transferrin, and selenium. When present at 10-15 microM for the initial 30 h of culture, EE caused a subsequent 2-2.7-fold increase in hepatocyte DNA synthesis. Pretreatment of the hepatocytes with EE during the first 30 h of culture caused an EE concentration-dependent enhancement of their subsequent DNA synthetic response to epidermal growth factor (EGF). Pretreatment with EE shifted the EGF dose-response curve, causing a dramatic enhancement of the response to EGF beginning at 2 ng EGF/ml. The response to a saturating (25 ng/ml) dose of EGF was also greatly enhanced. Determination of the effect of EE on hepatocyte surface EGF receptors revealed that the increased responsiveness of DNA synthesis to EGF was accompanied by a twofold increase in EGF receptor number per cell. These results indicate that EE has direct, growth-related effects on hepatocytes which may contribute to liver growth induced in vivo by this tumor promoter.
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PMID:Effects of the liver tumor promoter ethinyl estradiol on epidermal growth factor-induced DNA synthesis and epidermal growth factor receptor levels in cultured rat hepatocytes. 278 53

Rat hepatocytes prepared by collagenase digestion or EDTA dissociation were examined in culture for comparison of culture stability and morphology, and retention of selected adult rat liver characteristics. Cells prepared by EDTA perfusion followed by Percoll centrifugation were deemed to form confluent monolayer cultures more rapidly and monolayers remained intact for up to 21 days without signs of nonparenchymal cell growth or loss of primary hepatocyte appearance. The spectrally determined cytochrome P-450 content remained constant through eight days in culture. Collagenase-prepared cells contained an identical amount of P-450 but within 72 hr lost greater than 80% of the spectrally detectable P-450. Glutathione (GSH) content was higher in the EDTA-prepared hepatocytes and remained constant with only a modest effect of transferrin and selenium (T/S) supplementation, while GSH levels in collagenase-prepared cells increased, thereafter decreased with time in culture and was dependent on T/S supplementation. Cells prepared with EDTA also displayed an increase in GSH efflux rate in response to chronic GSH depletion by ethacrynic acid. gamma-Cystathionase (CNase) activity was retained at initial levels in EDTA-prepared hepatocytes supplemented with T/S and declined only about 25% in unsupplemented cells. Collagenase-prepared cells lost 75% of CNase activity by 72 hr. The established marker of hepatocyte neoplastic transformation, gamma-glutamyl transpeptidase (GGT), increased rapidly in collagenase-prepared cells. The accumulation of GGT was slowed by T/S supplementation. GGT activity did not increase in EDTA-prepared hepatocytes. Evaluation of morphological and biochemical criteria suggest that hepatocytes prepared without collagenase present superior model systems for the study of biochemical events through more extended culture times.
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PMID:Rat hepatocytes prepared without collagenase: prolonged retention of differentiated characteristics in culture. 285 31

Corpora lutea were removed from regularly cycling dairy cows, dissociated with collagenase and cultured for 8 or 10 days in Ham's F-12 medium. In Exp. 1 treatment with insulin, or an insulin-transferrin-selenium combination (ITS), increased progesterone production from basal levels on Day 4 of culture to 234% (P less than 0.01) above controls on Day 10. LH alone increased progesterone production 45% above controls on Day 10 (P greater than 0.05). When LH was combined with insulin or ITS, progesterone production was stimulated to an average of 1802% (P less than 0.01) above controls on Day 10 of culture. Transferrin or selenium without insulin did not allow LH to stimulate progesterone synthesis. In Exp. II, LH alone or LH plus gentamicin or penicillin-streptomycin increased progesterone production from basal levels on Day 2 steadily to an average of 468% (P less than 0.01) above controls (no antibiotics) by Day 8 of culture. The addition of amphotericin-B, alone or in combination with the other antibiotics, inhibited all LH-stimulated progesterone synthesis, but did not affect basal progesterone levels. We conclude that insulin is essential for maximal steroidogenesis in a bovine luteal cell culture system, and that LH-stimulated progesterone production is inhibited in the presence of amphotericin-B, but is not inhibited by gentamicin or penicillin-streptomycin. The elimination of amphotericin-B, coupled with the addition of insulin to the cell culture system increased the responsiveness of the cells to LH. These culture conditions represent the first report in which LH increased total progesterone production for 10 days, maintaining luteal function in a chemically-defined culture system.
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PMID:Effects of antibiotics and medium supplements on steroidogenesis in cultured cow luteal cells. 327 87

Selenium is an essential dietary trace element which has anticancer properties. Among its effects in rats, selenium has been shown to inhibit the development of carcinogen-induced mammary tumors by interfering with the postinitiation, promotion phase of carcinogenesis. We studied the effects of selenium on the growth of rat mammary tumor cells in primary culture. Our objective was to determine whether selenium had any direct influence on cell growth which might explain its influence on tumor development. Rat mammary tumors were induced by N-nitrosomethylurea. Tumor epithelium was prepared by collagenase dispersion and the cells were separated by Ficoll gradient centrifugation. The tumor epithelium was grown in primary culture using a defined serum-free medium. The addition of low concentrations of sodium selenite, less than 1.0 micrograms/ml, stimulated tumor cell proliferation. Protein synthesis and the production of type IV collagen increased within the first hour of exposure, prior to any measurable increase in DNA synthesis. Concentrations of selenite greater than 1.0 micrograms/ml inhibited cell proliferation, the synthesis of protein, and the replication of DNA in a dose-related manner. These studies demonstrated that selenium has the potential to influence the postinitiation phase of rat mammary tumorigenesis by directly altering the growth of tumor cells, possibly through the regulation of protein synthesis.
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PMID:Influence of selenium on the growth of N-nitrosomethylurea-induced mammary tumor cells in culture. 403 31

Primary tubular epithelial cells were isolated from renal cortex following enzymatic dissociation with collagenase. These cells were then grown in chemically defined media containing insulin, transferrin, selenium, tri-iodothyronine and either fibronectin or laminin. The tubular epithelial cells were studied ultrastructurally and compared to another epithelial cell type present in the renal cortex, the glomerular epithelial cell. In contrast to the constant morphology of glomerular epithelial cells grown in chemically defined media, tubular epithelial cell morphology depended on whether the cells were placed in fibronectin or laminin and on the age of the donor animal used for culture. Primary tubular cells grown in laminin formed colonies; cells grown from young animals were rounded, whereas cells grown from adult animals were flattened. Primary tubular cells grown in fibronectin were flattened regardless of age, but cells from young animals formed colonies while those from adult animals formed a monolayer. Despite these differences in gross morphology, scanning and transmission electron microscopy revealed similar ultrastructural features in primary tubular cells from young and adult animals grown in fibronectin or laminin. Quantitative adhesion studies demonstrated that secondary subcultured tubular cells adhered equally well to dimeric and multimeric forms of fibronectin, but not to laminin. Quantitative colony growth studies of subcultured secondary tubular cells showed that laminin supports colony formation of trypsinized tubular cells, while previous work has demonstrated that fibronectin supports colony formation of glomerular cells. These results are consistent with the hypothesis that different extracellular matrix molecules are involved in colony formation of different cell types, with fibronectin stimulating growth of glomerular cells and laminin supporting growth of tubular cells.
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PMID:Effect of the extracellular matrix molecules fibronectin and laminin on the adhesion and growth of primary renal cortical epithelial cells. 614 66

Renal cell cultures were initiated using fresh autopsy material from two individuals with cystinosis, ages 5 and 8 yr. Cells obtained from collagenase treated autopsy material were grown in a selective kidney medium containing Coon's modified F12, 2.5% fetal bovine serum, transferrin, insulin, selenium, hydrocortisone, PGE1, and fibronectin. These cells had an epithelial appearance, formed domes, and were periodic acid-Schiff positive. Both tight junctions and microvilli were seen by electron microscopy. Fibroblasts had a cloning efficiency of zero in the selective medium and grew poorly compared to their growth in Coon's F12 with 10% fetal bovine serum. The lysosomal cystine content of the renal cells was greatly elevated and comparable to that of fibroblasts from cystinotic patients. Renal cell lysosomal cystine levels were only partially reduced by exposure to either pantethine or the aminothiol, cysteamine. However, exposure to either compound effectively depleted cystinotic cultured fibroblasts of their lysosomal cystine. Study of cultured renal material may have practical significance in pharmacologic considerations.
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PMID:Renal cell culture using autopsy material from children with cystinosis. 669 73

An in vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzymes DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transferring the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7-10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and gamma-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.
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PMID:Normal rat kidney proximal tubule cells in primary and multiple subcultures. 879 58

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.
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PMID:Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma. 960 Mar 41

Much progress has been made in the understanding of the pathogenesis of alcoholic liver disease, resulting in improvement of prevention and therapy, with promising prospects for even more effective treatments. The most successful approaches that one can expect to evolve are those that deal with the fundamental cellular disturbances resulting from excessive alcohol consumption. Two pathologic concepts are emerging as particularly useful therapeutically. Whereas it continues to be important to replenish nutritional deficiencies, when present, it is crucial to recognize that because of the alcohol-induced disease process, some of the nutritional requirements change. This is exemplified by methionine, which normally is one of the essential amino acids for humans, but needs to be activated to S-adenosylmethionine (SAMe), a process impaired by the disease. Thus, SAMe rather than methionine is the compound that must be supplemented in the presence of significant liver disease. Indeed, SAMe was found to attenuate mitochondrial lesions in baboons, replenish glutathione, and significantly reduce mortality in patients with Child A or B cirrhosis. Similarly, polyenylphosphatidylcholine (PPC) corrects the ethanol-induced hepatic phospholipid depletion as well as the decreased phosphatidylethanolamine methyltransferase activity and opposes oxidative stress. It also deactivates hepatic stellate cells, whereas its dilinoleoyl species (DLPC) increases collagenase activity, resulting in prevention of ethanol-induced septal fibrosis and cirrhosis in the baboon. Clinical trials with PPC are ongoing in patients with alcoholic liver disease. Furthermore, enzymes useful for detoxification, such as CYP2E1, when excessively induced, become harmful and should be downregulated. PPC is one of the substances with anti-CYP2E1 properties that is now emerging. Another important aspect is the association of alcoholic liver disease with hepatitis C: a quarter of all patients with alcoholic liver disease also have markers of HCV infection, with an even higher incidence in some urban areas but, at present, no specific therapy is available since interferon is contraindicated in that population. However, in addition to antiviral medications, agents that oppose oxidative stress and fibrosis should also be tested for hepatitis C treatment since these two processes contribute much to the pathology and mortality associated with the virus. In addition to antioxidants (such as PPC, silymarin, alpha-tocopherol and selenium), anti-inflammatory medications (corticosteroids, colchicine, anticytokines) are also being tested as antifibrotics. Transplantation is now accepted treatment in alcoholics who have brought their alcoholism under control and who benefit from adequate social support but organ availability is still the major limiting factor and should be expanded more aggressively. Finally, abstinence from excessive drinking is always indicated; it is difficult to achieve but agents that oppose alcohol craving are becoming available and they should be used more extensively.
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PMID:Alcoholic liver disease: new insights in pathogenesis lead to new treatments. 1072 99

The in vitro growth and developmental pattern of caprine preantral follicles cultured in agar gel was observed. Preantral follicles 50 to 150 microm in diameter were isolated from prepuberal goat ovaries by treatment with collagenase and DNase. The isolated preantral follicles were cultured in agar gel for up to 14 days. A group of 10 follicles in different developmental stages was cultured in a culture well coated with 0.6% agar gel and filled with DMEM medium supplemented with FCS (10%), hypoxanthine (2 mmol/mL), dbcAMP (2 mmol/mL), FSH (100 ng/mL), insulin-transferrin-selenium (ITS) (50 ng/mL), IGF-1 (50 ng/mL), hydrocortisone (40 ng/mL) and antibiotics. Follicle viability was determined under an inverted phase-contrast microscope according to morphological and histological criteria, and follicle growth was assessed by their size and appearance. The results showed that the three-dimensional structures and forms of follicles were basically maintained intact during culture. Primary follicles developed into secondary follicles and a few of them into antral follicles. A large portion of secondary follicles entered the antral stage, and oocytes also acquired growth. The formation of theca lamina and zona pellucida was observed. The survival capacity of secondary follicles was greater than primary follicles. The survival rates for primary and secondary follicles were 11.36% (5/44) and 71.16% (53/74), respectively. During in vitro development the follicles demonstrated dominance. This experiment revealed the preliminary characteristics of the in vitro development of caprine preantral follicles.
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PMID:In vitro development of caprine ovarian preantral follicles. 1107 Nov 38


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