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 attachment of rat hepatocytes to polystyrene-adsorbed serum protein is relatively insensitive to inhibitors such as dextran sulphate, cycloheximide, colchicine and cytochalasin B, and enzymes like trypsin and neuraminidase, but it is strongly dependent on divalent cations. Mg2+ supports attachment better than Ca2+, but a combination of both is required for maximal attachment. The attachment is very temperature-sensitive, with a biphasic Arrhenius plot indicating an activation energy of 123 kJ/mol above 34 degrees C and 374 kJ/mol below 34 degrees C. The adsorbed attachment-promoting serum factor is inactivated by trypsin, or by Ca2+-dependent proteases which contaminate commercial preparations of collagenase. The adsorbed factor is resistant to treatment with glutaraldehyde, neuraminidase and heating to 90 degrees C, whereas the same factor in the unadsorbed state (in serum) is destroyed by heating to 70 degrees C. The factor in serum is unable to compete with the adsorbed factor for cell binding, hence it would appear that adsorption to polystyrene induces the active, heat-resistant conformation of the factor.
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PMID:Effect of temperature and divalent cations on the substratum attachment of rat hepatocytes in vitro. 74 33

Spontaneously contracting myocytes were isolated from ventricles of the adult rat heart. Hearts were perfused retrogradally via the aorta for 30 minutes at 37 degrees C with Ca2+-free phosphate-buffered saline containing collagenase and hyaluronidase. The venticles were divided into pieces and incubated 15 minutes with the enzymes. Dislodged cells were decanted, diluted with cold buffer and allowed to settle. The washed cells were then sedimented through 3% Ficoll. This procedure yielded approximately 50 mg of protein from 1 gm of heart. Viability measured by trypan-glue exclusion is 90-95%. Approximately 80% of the cells were beating. Scanning electron microscopic studies suggest that the isolated myocytes are morphologically intact. The cells oxidize glucose, pyruvate, citrate and palmitate to CO2 and synthesize protein and RNA. Uptake of glucose, 2-deoxyglucose, leucine and taurine was saturable. Glucose uptake was stimulated by insulin. The cells retained LDH and CPK as well as their capacity to oxidize substrates after 24 hours at 4 degrees C or 4 hours at 37 degrees C. After 24 hours at 4 degrees C the cells resume contracting when returned to room temperature. The procedure reported here for the isolation of spontaneously contracting, adult, rat heart myocytes provides cells with a high index of viability and greater yield than previously reported methods. The cells retain metabolic activity and withstand storage for longer periods than other described preparations.
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PMID:Isolation and characterization of myocytes from the adult rat heart. 91 20

A procedure is described for the isolation and culture of large numbers of follicle cell-free Xenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenace of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morpological and functional characteristics of oocytes for periods up to 3 weeks.
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PMID:Defined nutrient medium for the in vitro maintenance of Xenopus laevis oocytes. 98 65

A method is described for the preparation of isolated bovine parathyroid cells. Fresh, minced bovine parathyroid tissue is incubated with 2 mg/ml collagenase and 50 mug/ml DNAse for 1 h at 37 C with periodic pipetting. Parthyroid cells are separated from contaminating fat cells and red cells by low speed centrifugation. The resulting cell preparation is indistinguishable from parathyroid cells in intact bovine glands by light and electron microscopy. Hormone release is linear for at least 3 h at both high (2.0 mM) and low (0.5 mM) calcium concentrations and is inversely proportional to divalent cation concentration between 0.3 mM and 1.0 mM calcium as been observed previously both in vivo and in vitro. As in previous studies, hormone release is also stimulated up to 2-fold by 10(6) M (-)isoproterenol, an effect blocked by 10(-6)M (-)propranolol, suggesting a beta-adrenergic effect. Such a cell preparation should be useful for studying hormone binding and effects on cyclic nudleotides and cellular transport phenomena in both normal amd abnormal tissue.
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PMID:Preparation of viable isolated bovine parathyroid cells. 100 Dec 55

A method is described for the dissociation of mouse ovaries and the isolation of oocytes free of somatic cells by agitating pieces of ovary in collagenase and deoxyribonuclease in a calcium and magnesium free salt solution. This method yielded about 50% of the growing oocytes from immature mice. The utilization of exogenously administered 14C-labelled energy sources by oocytes in various growth stages was determined by measurement of evolved 14CO2. Little or no evolution of 14CO2 was detected from oocytes of any size incubated in 14C-glucose, lactate or succinate. The production of 14CO2 from 14C-pyruvate increased logarithmically when plotted against increasing oocyte volume with a plateau occurring after occytes reached a volume of 65,500 mum3 (50 mum diameter). Thus, the pattern of energy metabolism for oocyte maturation and early egg cleavage, wherein glucose and lactate are not utilized as efficiently as pyruvate, has been established by the earliest stages of oocyte growth.
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PMID:Analysis of mouse oogenesis in vitro. Oocyte isolation and the utilization of exogenous energy sources by growing oocytes. 100 46

Addition of estradiol-17beta in vitro to suspensions of isolated endometrial cells resulted in significant effects on glucose, water and electrolyte metabolism. Cells were prepared from uterine tissues of ovariectomized rats. In part, the procedures involved incubation with collagenase in Ca2+-, Mg2+-free, phosphate-buffered mammalian Ringer's solution, followed by restoration of divalent cations before gentle scraping of the endometrium from the underlying smoothmuscle. Cells were then disaggregated, washed, separated from coarse and fine debris, and incubated in an enriched medium for 2 h before the start of all experiments. Cellular integrity was established by measurement of electrolyte contents and by dye exclusion methods. Substantial production of 14CO2 from glucose-U-14C by the cell suspensions provided further evidence of cell viability. Estradiol-17beta, 10-9M, elicited significant increments in sodium and water contents within 2 h. Addition of estradiol-17beta, but not the alpha-epimer, also resulted in a significant increase in the yield of 14CO2 as early as 1.5 h, peaking at 2 h. The responses were dose-dependent between 10-10M through 10-8M. The stimulatory effect of estradiol-17beta at 10-9M was abolished in the presence of 3 times 10-6M cortisol or by cellular homogenization. Epithelial cells isolated from rat urinary bladder responded significantly to 6 times 10-9M aldosterone but not to estradiol-17beta, demonstrating specificity of the target site. These data lend further support to the suggestion that a primary action of estrogen in its target cell involves specific changes in the ionic and biochemical profile of the cytoplasm which may ultimately be communicated to the nucleus.
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PMID:Steroid hormone-responsive, isolated endometrial cells. 112 Apr 83

Human mast cells were obtained from adenoids and mesentery by enzymatic dispersion of the tissues with the enzyme collagenase. The digestion of the tissues resulted in a cell suspension which contained 1-2% mast cells. 37.3% (adenoids) and 33.4% (mesentery) of total histamine initially present in the tissues was recovered in the dispersed cell suspensions. More than 90% of the cells were viable. The adenoidal mast cells could be sensitized passively in vitro with homologous reaginic serum and released histamine after challenge with specific antigen. Both populations of mast cells were sensitive to the action of anti-human IgE; the reversed anaphylaxis with anti-IgE was higher in mesenteric mast cells. Both examined mast cell populations were sensitive to the challenge with polymyxin B, concanavalin A and ionophore A23187, however, histamine release was only up to 10% and 20% for adenoidal and mesenteric cells, respectively. Only mesenteric mast cells responded to the action of compound 48/80. Histamine release, induced by polymyxin B, was rapid (maximal release within 5 min), maximal in the presence of 3 mM extracellular calcium ions (but also occurred in the absence of the cation).
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PMID:Histamine secretion from human mesenteric and adenoidal mast cells. 128 67

A technique to record whole cell membrane current during stretching single cardiac myocytes was developed. Ventricular myocytes were dissociated by treating guinea-pig hearts with collagenase. One end of the cell was fixed either to a microglasstool tip or to a glass plate, while the other end was attached either to a microglasstool tip or to a suction pipette, which was mounted on a micromanipulator. A time-independent current showing a reversal potential of -15 +/- 4 mV (n = 7) was activated when the myocytes were stretched more than 20% of the length between two fixed point. The current gradually relaxed during the maintained stretch, and disappeared on releasing the stretch. We failed to detect any consistent change in either the L-type Ca2+ current or the inward rectifier K+ current. For comparison, current changes induced by inflating the cell using a hypo-osmotic solution were recorded. The inflation was not accompanied by any change in the time-independent current. Instead, the delayed rectifier K+ current was increased to 170 +/- 48% control by the 70% hypo-osmotic solution. Thus, the effect of mechanical stretch on the time-independent current is different from those of hypo-osmotic cell inflation. The stretch-induced time-independent current is compared with reported current changes induced by the intracellular microinjection of Ca2+.
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PMID:Effects of mechanical stretch on membrane currents of single ventricular myocytes of guinea-pig heart. 129 61

Hepatotoxic alkylation of mouse liver cells by acetaminophen is characterized by an early loss of ion regulation, accumulation of Ca2+ in the nucleus, and fragmentation of DNA in vitro and in vivo. Acetaminophen-induced DNA cleavage is accompanied by the formation of a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. These events unfold well in advance of cytotoxicity and the development of necrosis. The present study utilized cultured mouse hepatocytes and mechanistic probes to test whether DNA fragmentation and cell death might be related in a "cause-and-effect" manner. Cells were isolated by collagenase perfusion, cultured in Williams' E medium for 22-26 hr, and exposed to acetaminophen. Aurintricarboxylic acid, a general Ca(2+)-endonuclease inhibitor, and EGTA, a chelator of Ca2+ required for endonuclease activation, significantly decreased DNA fragmentation at 6 and 12 hr and virtually abolished cytotoxicity. N-Acetylcysteine also eliminated DNA fragmentation and cytotoxicity. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase-stimulated DNA repair, failed to alter the amount of DNA fragmentation at 6 hr but substantially increased acetaminophen cytotoxicity in hepatocytes at 12 hr. With the exception of when DNA repair was inhibited by 3-aminobenzamide, Ca2+ accumulation in the nucleus, DNA fragmentation, and hepatocyte death varied consistently and predictably with one another. Collectively, these findings suggest that unrepaired damage to DNA contributes to acetaminophen-induced cell death in vivo and may play a role in necrosis in vivo.
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PMID:Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: effects of Ca(2+)-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death. 131 Jan 69

A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.
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PMID:Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase. 131 76


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