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)

Triacylglycerol metabolism has been studied in kidney cortex tubules from starved rats, prepared by collagenase treatment. Triacylglycerol was determined by a newly developed fully enzymic method. Incubation of tubules in the absence of fatty acids led to a decrease of endogenous triacylglycerol by about 50% in 1h. Addition of albuminbound oleate or palmitate resulted in a steady increase of tissue triacylglycerol over 2h. The rate of triacylglycerol synthesis was linearly dependent on oleate concentration up to 0.8mm, reaching a saturation at higher concentrations. Triacylglycerol formation from palmitate was less than that from oleate. This difference was qualitatively the same when net synthesis was compared with incorporation of labelled fatty acids. Quantitatively, however, the difference was less with the incorporation technique. Gluconeogenic substrates, which by themselves had no effect on triacylglycerol concentrations, stimulated neutral lipid formation from fatty acids. Glucose and lysine did not have such a stimulatory effect. Inhibition of gluconeogenesis from lactate by mercaptopicolinic acid likewise inhibited triacylglycerol formation. This inhibitory effect was seen with oleate as well as with oleate plus lactate. When [2-(14)C]lactate was used the incorporation of label into triacylglycerol was found in the glycerol moiety exclusively. Addition of dl-beta-hydroxybutyrate (5mm) to the incubation medium in the presence of oleate or oleate plus lactate led to a significant increase in triacylglycerol formation. In contrast with the gluconeogenic substrates, dl-beta-hydroxybutyrate had no stimulatory effect on fatty acid uptake. The results suggest that renal triacylglycerol formation is a quantitatively important metabolic process. The finding that gluconeogenic substrates, but not glucose, increase lipid formation, indicates that the glycerol moiety is formed by glyceroneogenesis in the proximal tubules. The effect of ketone bodies seems to be caused by the sparing action of these substrates on fatty acid oxidation. The decrease of triacylglycerol in the absence of exogenous substrates confirms previous conclusions that endogenous lipids provide fatty acids for renal energy metabolism.
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PMID:Triacylglycerol metabolism in isolated rat kidney cortex tubules. 737 17

We have shown previously that phosphatidic acid esterified to polyunsaturated fatty acids is mitogenic for primary cultures of mouse mammary epithelial cells embedded within collagen gels. We hypothesized that this mitogenic competence resulted from the ability of this phospholipid to activate multiple signal transduction pathways in mammary epithelium. A closer examination of this hypothesis was undertaken by examining the effect of exogenous phosphatidic acid on phosphoinositide (PI) hydrolysis and its intracellular metabolism to diglyceride, an activator of protein kinase C. For assays of phosphoinositide-specific phospholipase C activation, mammary epithelial cells from virgin Balb/c mice were isolated by collagenase dissociation of mammary glands and cultured on the surface of Type I collagen-coated culture dishes. Phosphatidic acid (PA) stimulated a sustained increase in inositol phosphates and caused inositol phospholipid depletion when added to cells in which inositol phospholipids were prelabeled with 3H-myoinositol. This effect was specific for PA among phospholipids tested. Neither lineoleic acid, that can be released from PA, nor prostaglandin E2 affected PI hydrolysis. When mammary epithelial cells were cultured inside collagen gels in the presence of exogenous PA or phosphatidylcholine (PC) radiolabeled with 3H-glycerol, PA was found to persist intracellularly and be dephosphorylated to diglyceride (an activator of protein kinase C) to a greater extent than PC, a nonmitogenic phospholipid. In contrast to PA, epidermal growth factor (EGF) only slightly stimulated PI hydrolysis, showing that these two different growth-promoting factors do not actively couple to the same signal transduction pathways in mammary epithelial cells. These results show that PA may activate multiple pathways in mammary epithelial cells either directly or via its metabolism to diglyceride.
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PMID:Multifunctional phosphatidic acid signaling in mammary epithelial cells: stimulation of phosphoinositide hydrolysis and conversion to diglyceride. 777 98

Brown adipose tissue and collagenase-isolated brown adipocytes were investigated in rats by means of 1H and 13C nuclear magnetic resonance spectroscopy. After chloroform-methanol extraction of brown adipose tissue, proton and natural abundance 13C spectra of the chloroform fraction showed resonances attributable to triglycerides, and were qualitatively similar to those of the corresponding fraction of white adipose tissue. By means of quantitative analysis of 1H spectra, fatty acid unsaturation and polyunsaturation in triglycerides were found to be lower in brown than white adipose tissue; moreover, unsaturation parameters decreased in triglyceride fatty acids of brown adipose tissue upon norepinephrine administration or cold acclimatization of rats, and were affected by the age of donors. The molar percentage of mono- and polyunsaturated C18 fatty acids in triglycerides was determined from 13C spectra and found to change in the early post-natal period. Isolated, agarose-embedded brown adipocytes from 4-day-old rats showed a number of peaks in the carbohydrate region of 1H spectra that were not present in spectra of white adipocytes and almost disappeared in brown fat cells of older animals. These peaks could be restored by insulin exposure. Natural abundance 13C spectra of isolated brown adipocytes were resolved enough to allow unambiguous assignment of resonances to carbons of fatty acids, glycerol, glucose, ethanolamine, and choline. Calculation of the mono- to polyunsaturated fatty acids ratio in the cells was also performed. Nuclear magnetic resonance spectroscopy is a useful tool for the investigation of brown adipose tissue and adipocytes therefrom.
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PMID:Magnetic resonance spectroscopy investigations of brown adipose tissue and isolated brown adipocytes. 789 17

GH deficiency impairs lipid metabolism in adults, but little is known about the direct effect of GH on adipose tissue in humans. First, the in vitro response of fat cells to GH in five GH-deficient adults was studied; second, it was investigated whether 6-month recombinant human GH (rhGH) administration modifies this response. Biopsies of fat were obtained from the periumbilical region before and after rhGH administration. The response of the collagenase-isolated fat cells to various concentrations of GH was assessed by glycerol release, measured by bioluminescence. Before treatment, GH induced a lipolytic activity from the adipocytes, which became significantly higher after 6 months of treatment. Thus, this study provides evidence for an intrinsic lipolytic activity of GH in GH-deficient adults and for its improvement after long term rhGH administration.
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PMID:Response of fat cells to growth hormone (GH): effect of long term treatment with recombinant human GH in GH-deficient adults. 820 Sep 42

This study examined the influence of dietary essential fatty acids on the cooperativity of isolated adipocytes and stromal-vascular cells in the biosynthesis of prostaglandins. Sprague-Dawley rats were fed either a diet rich in essential fatty acids (20% corn oil) or a diet poor in essential fatty acids (20% tallow) for 4 wk. Preparations containing primarily adipose cells (adipocytes) or stromal-vascular cells (nonfat cells) were obtained from epididymal fat pads by collagenase digestion and repeatedly washed. Prostaglandin production was evaluated in basal and epinephrine-stimulated media after incubation with either adipocytes or adipocytes+nonfat cells. Prostaglandin E2 and prostacyclin production by adipocytes+nonfat cells was dose-dependent with epinephrine stimulation in cells from rats fed both diets. Both prostaglandin E2 and glycerol release in response to epinephrine (10-100 mumol/L) stimulation from adipocytes or from adipocytes+nonfat cells were significantly higher for cells from corn oil-fed rats than for cells from tallow-fed rats. Regardless of dietary treatment, epinephrine-stimulated prostaglandin E2 and prostacyclin release from adipocytes+nonfat cells was much greater than from adipocytes. These results suggest that a diet high in essential fatty acids precipitates a higher prostaglandin E2 secretion and that nonfat cells potentiate the secretion of prostaglandin E2 and prostacyclin by adipocytes regardless of diet.
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PMID:The cooperation of adipocytes and stromal cells in the secretion of prostaglandins by rat adipose tissue is not influenced by diet. 832 May 61

This study investigated the direct effects of hydrocortisone (HS), corticotropin-releasing factor (CRF), and adrenocorticotropin (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-stimulated secretion of follicle-stimulating hormone (FSH) from dispersed pig pituitary cells in vitro. Pig pituitaries were dispersed into cells with collagenase, DNAase, and hyaluronidase and then cultured in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) pretreated with dextran-coated charcoal for 3 days. Cells were preincubated with steroids, CRF, or ACTH before GnRH was added. HS did not affect basal FSH secretion after 72 h of incubation. Treatment of pituitary cells with increasing concentrations (0.001-800 micrograms/ml) of HS for 72 h resulted in a dose-dependent decrease in GnRH-stimulated FSH release. HS pretreatment did not cause a change in cellular FSH content. Increasing duration (6-72 h) of treatment with HS (200 micrograms/ml) led to a time-dependent decrease in GnRH-stimulated FSH release, achieving statistical significance by 12 h. Porcine ACTH had no influence on basal and GnRH-stimulated FSH secretion. CRF decreased GnRH-stimulated FSH secretion in a dose-dependent manner, and the inhibitory effect required preincubation (6-18 h) with CRF. HS inhibited the FSH secretory responses to phospholipase C, melittin, and 8-bromo-cAMP but did not affect the response to 1,2-dioctanoyl-sn-glycerol and ionophore A23187. These results indicate that both cortisol and CRF can act directly on pig pituitary to inhibit FSH responsiveness to GnRH.
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PMID:Actions of corticotropin-releasing factor or cortisol on follicle-stimulating hormone secretion by isolated pig pituitary cells. 839 May 96

Hepatic zonation of cholesterol and glycerolipid synthesis was investigated in regenerating rat livers 24 hr after partial hepatectomy. Tritiated acetate and [U-14C]glycerol were injected into rats' peritoneal cavities for a short-term labeling study. Periportal and perivenous hepatocytes were isolated by digitonin collagenase perfusion. Cholesterol synthesis was significantly higher in periportal hepatocytes of the sham-operated livers (periportal/perivenous = 1.67; p < 0.05). Twenty-four hours after partial hepatectomy, cholesterol synthesis was selectively decreased by 40% (p < 0.01) in periportal hepatocytes. Consequently, hepatic zonation of cholesterol synthesis was abolished in regenerating livers. To study the cholesterol homeostasis on a long-term basis, we substituted deuterated water (25% enriched) for drinking water for 5 days to label newly synthesized cholesterol in a steady state. This procedure clearly demonstrated the net negative cholesterol balance 24 hr after partial hepatectomy. However, the newly synthesized cholesterol contributed equally to the cellular cholesterol pool in both zones. The synthesis of glycerolipids, whether measured from tritiated acetate or [U-14C]glycerol, was significantly increased without apparent zonation in the regenerating livers (twofold increase in phospholipid, and threefold to sevenfold increase in triacylglycerol). We concluded that hepatic zonation of cholesterol synthesis is caused by higher de novo synthesis in periportal hepatocytes, which is abolished in regenerating livers. No zonation of glycerolipid synthesis exists in normal and regenerating livers.
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PMID:Zonation of cholesterol and glycerolipid synthesis in regenerating rat livers. 842 26

Mesothelial cells (MC) obtained from the human omentum are a good alternative to the use of endothelial cells (EC) as a covering for vascular prostheses of polytetrafluoroethylene (PTFE), given the antithrombogenic properties and good behavior in vitro of mesothelial cells. We studied the behaviour of mesothelial cells seeded on PTFE prostheses with an interposed fibroblastic matrix for seeding. The mesothelial cells were extracted from 30-40 g fragments of human omentum by enzymatic digestion with collagenase. The cells extracted were seeded onto small disks of PTFE to which a matrix composed of fibroblastic cells had been fixed with 5% glycerol after the fibroblasts reached convergence. Interposition of a fibroblastic matrix fixed with glycerol notably improved the adherence of the seeded mesothelial cells and the stability and durability of the cell layer formed on the prosthetic surface. The effectiveness of seeding mesothelial cells was confirmed by labelling the cells with 111In-oxine. This showed that once the cell layer had formed (24 h after seeding), the fibroblastic matrix favoured the maintenance of a stable layer of mesothelial cells 4 hours after uptake of the radioactive substance.
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PMID:Use of a fibroblastic matrix improves the results of mesothelial-cell seeding on vascular prostheses of polytetrafluoroethylene. 857

Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.
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PMID:Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase. 862 83

Tamoxifen and its analogues 4-hydroxytamoxifen, toremifene, 4-hydroxytoremifene, clomifene and droloxifene were tested for clastogenic effects in a human lymphoblastoid cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase and in a cell line containing only the viral vector (Ho1). MCL-5 or Ho1 cells were incubated with 4-hydroxytamoxifen, 4-hydroxytoremifene, clomifene or droloxifene and the incidence of micronuclei estimated. With MCL-5 cells there was an increase in micronuclei with 4-hydroxytamoxifen, 4-hydroxytoremifene and clomifene but not with droloxifene. With Ho1 cells only 4-hydroxytamoxifen and 4-hydroxytoremifene caused an increase in micronuclei. MCL-5 cells were incubated with tamoxifen, 4-hydroxytamoxifen, toremifene, droloxifene, clomifene or diethylstilbestrol (0.25-10 microg/ml) for 48 h and subjected to 3 h treatment with vinblastine (0.25 microg/ml) to arrest cells in metaphase. The incidence of cells with chromosomal numerical aberrations (aneuploidy) was increased in cells treated with tamoxifen, 4-hydroxytamoxifen, toremifene, clomifene and diethylstilbestrol but not droloxifene. The frequency of cells with structural abnormalities (excluding gaps) was increased in cells treated with tamoxifen and toremifene but not 4-hydroxytamoxifen, clomifene, droloxifene or diethylstilbestrol. The clastogenic activities of tamoxifen (35 mg/kg), toremifene (36.3 mg/kg), droloxifene (35.2 mg/kg) and diethylstilbestrol (25 mg/kg) were compared in groups of four female Wistar rats. Each chemical was dissolved in glycerol formal, administered as a single dose by gavage and hepatocytes isolated by collagenase perfusion 24 h later. The cells were cultured in the presence of epidermal growth factor (40 ng/ml) for 48 h, colchicine (10 microg/ml) being added for the final 3 h of incubation. At least 100 chromosomal spreads were examined from each animal for the presence of numerical and structural abnormalities. The incidences of aneuploidy following treatment were: tamoxifen 81%, toremifene 46%, droloxifene 9.6%, diethylstilbestrol 45.7%, vehicle control 5.3%. The incidences of chromosomal structural abnormalities excluding gaps were: tamoxifen 4.3%, toremifene 0.8%, droloxifene 0.5%, diethylstilbestrol 0.8%, control 0.5%. The incidence of chromosomal structural aberrations excluding gaps in the treated animals was not statistically significantly different from controls except in the tamoxifen-treated group. Tamoxifen (35 mg/kg per os) and toremifene (36.3 mg/kg per os) were dosed to rats for 4 weeks and chromosomal spreads made from hepatocytes. The incidences of aneuploidy were: tamoxifen 94%, toremifene 57%, control 6.5%. The incidences of chromosomal aberrations excluding gaps were: tamoxifen 12%, toremifene 1%, control 0.5%. The incidence of tamoxifen-induced chromosomal structural abnormalities was significantly elevated compared with control levels. The results demonstrate that tamoxifen and toremifene are the only two drugs tested in the study that cause chromosomal structural and numerical aberrations in vitro and tamoxifen is the only drug that induces both these effects in rat liver cells stimulated to divide in culture following oral dosing. Since chromosomal mutations require cell division for their manifestation and tamoxifen is the only compound of those tested that causes hyperplasia in the rat liver, chromosomal aberrations and aneuploidy in the rat liver would only be expected to occur following treatment with tamoxifen alone, although aneuploidy could be induced by toremifene in conjunction with a promoter such as phenobarbitone.
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PMID:Clastogenic and aneugenic effects of tamoxifen and some of its analogues in hepatocytes from dosed rats and in human lymphoblastoid cells transfected with human P450 cDNAs (MCL-5 cells). 905 22


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