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 direct effects of human GH (hGH), ovine pituitary PRL (oPRL), and human chorionic somatomammotropin [placental lactogen (hPL)] on the endocrine pancreas were studied in isolated pancreatic islets maintained in tissue culture. Islets of Langerhans were isolated by collagenase treatment of pancreatic tissue obtained from adult NMRI mice and adult or newborn Wistar rats. The islets were maintained for up to 3 weeks in petri dishes containing tissue culture medium RPMI 1640 supplemented with newborn calf serum or normal human serum. The release of insulin during culture and the islet content of insulin, glucagon, and DNA after culture were determined. The DNA synthesis in the newborn rat islets was evaluated by the incorporation of [methyl-3H]thymidine into islet cell DNA. In mouse islets, 1 micrograms/ml hGH, oPRL, or hPL markedly stimulated insulin release during a 2-week culture period and caused a significant increase in the insulin content in the islets after culture. While hGH did not affect the DNA content in adult mouse islets, an increase was observed in adult rat islets after 2-3 weeks of culture. In islets isolated from 3- to 5-day-old rats cultured for 2 weeks with hGH, there was a 30-40% higher DNA content than that found without hGH. Correspondingly, a significant stimulation of the incorporation of [methyl-3H]thymidine could be demonstrated 24 h after the addition of hGH, oPRL, or hPL. hCG and porcine ACTH had no effect. In conclusion, these results indicate that GH and related hormones have a direct stimulatory effect on both the insulin production and DNA synthesis in isolated islets of Langerhans. Whether the effect is directly on the beta-cell or mediated via locally produced growth factors remains to be determined.
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PMID:Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets. 627 41

We have used cultured trypsin-collagenase-dispersed cells from uteri of 21-day-old rats to investigate the mechanism of control of uterine motility by the beta-adrenergic receptor. After 5 to 7 days in RPMI 1640 the cells started to assume some of the morphological characteristics of smooth muscle cells. When cultures were incubated with 45Ca2+ for 3 h then washed free of isotope and incubated in medium with unlabeled Ca2+, efflux from the prelabeled intracellular pools was linear for up to 60 min. The potent beta-adrenergic agonist isoproterenol had a rapid effect on the rate of efflux and increased it almost sevenfold. Isoproterenol's effect was blocked by propranolol and could be duplicated by the addition of 8-bromo-adenosine 3',5'-cyclic monophosphate or cholera toxin. The cultured myometrial cells had adenylate cyclase properties similar to those of intact muscle strips when these were determined by the conversion of radioactive substrate (alpha-32P-ATP) to 32P-cAMP using a broken-cell preparation. Adenylate cyclase was sensitive to stimulation by GTP and by isoproterenol in the presence but not in the absence of GTP. Adenylate cyclase was also sensitive to stimulation by Ca2+ in the absence of GTP. We conclude that the primary cultures had the properties expected of smooth muscle cells including beta-adrenergic receptors that were coupled to a physiologically important function, Ca2+ flux. The beta-adrenergic receptor's effect on Ca2+ flux was cAMP mediated, and the divalent cation may also regulate its rate of flux by an effect on Ca2+-sensitive cAMP production.
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PMID:beta-Adrenergic catecholamine-dependent properties of rat myometrium primary cultures. 630 58

Knowledge of protective effects of corticosteroids on traumatized cells prompted us to test the potential benefit of islet cryopreservation in the presence of hydrocortisone. Neonatal murine islets were isolated by collagenase, followed by 2- to 3-day tissue culture. Precryopreservation glucose-stimulated (50-500 mg/dl) insulin release was 25-388% above basal (mean = 113%) in 18/20 fresh islet preparations. Subsequent freezing was done in RPMI 1640 medium plus 10% (v/v) heat-inactivated fetal calf serum and 10% (v/v) Me2SO with or without 1 mg/ml hydrocortisone at 0.25 degrees C per minute in a programmed freezing system, to -80 degrees C, and stored for greater than 60 days at -196 degrees C. Thawing, by transfer to room air, was followed by dilution, 4x (v/v), in 4 degrees C RPMI plus 10% protein, after which glucose-stimulated insulin release was reassessed, showing 56-280% response over basal in 3/8 steroid-treated preparation and 20-220% response in 3/10 control preparations. Basal insulin release was 0.72 ng/microgram protein/hr in fresh islets (N = 20) and 0.22 ng/microgram protein/hr after freeze-thawing. We conclude that functional islet survival by this method is approximately 30% and that hydrocortisone did not improve viability.
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PMID:Murine islet cryopreservation and corticosteroids: functional studies. 634 52

To improve the in vitro growth of Plasmodium falciparum we attempted to cultivate its erythrocytic stages on monolayers of functionally active hepatocytes. Hepatocytes from Swiss Albino mice were isolated by perfusing the liver with a collagenase solution and were co-cultured with a liver epithelial cell type in RPMI 1640 medium supplemented with 10% human umbilical cord serum. The results show that the presence of hepatocytes improves both the multiplication rates of three strains of P. falciparum already in cultivation and the proliferation of freshly isolated strains. Of nine primary isolates tested, only three could be adapted in the standard conditions, whereas all grew readily in the presence of hepatocytes. After two to three weeks of culture with feeder cells, all the strains could be maintained continuously in standard conditions. Similar results were obtained using hepatocytes from another rodent species. Growth was also improved using the supernatant from hepatocyte cultures. No improvement resulted from the use of two human hepatoma cell lines, one rat hepatoma, human embryonic lung fibroblasts, human liver fibroblasts and rat liver epithelial cells as feeder layers. From these results it appears that better culture media can be designed and that the effect of hepatocytes is probably related to the specific functions exhibited by these cells. Hepatocytes may act either by removing toxic substances, particularly lactic acid in the Krebs and Cori cycles, and/or supplying nutrients essential to the parasite.
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PMID:Hepatocytes as feeder-layers for in vitro cultivation of Plasmodium falciparum blood-stages. 638 21

It was the aim of the present study to investigate the significance of culture before and after freeze-thawing of isolated mouse pancreatic islets. To evaluate the impact of culture before freezing (5 degrees C/min; 2 M dimethyl sulfoxide), islets were frozen either directly after isolation or after 2, 4, or 7 days of culture in medium RPMI 1640. The culture period after thawing was 7 days. Islets immediately frozen exhibited virtually no (pro)insulin biosynthesis and also a severe inhibition of glucose-stimulated insulin release. The precultured (2-7 days), frozen islets synthesized and released insulin at rates comparable to those of nonfrozen, cultured islets. Studies of the effects of culture after freeze-thawing were performed after a 3-day culture period prior to freezing. The (pro)insulin biosynthetic rates did not differ between islets cultured for 0-7 days after thawing. There was an apparent increase of glucose-stimulated insulin release when the islets were cultured for more than 2 days after thawing. It may be that the decreased viability of islets frozen immediately after isolation was due to minor cell damage induced by the collagenase incubation. During culture the islets may recover and become more resistant to freeze-damage. The beneficial effect of culture after thawing may reflect the loss of damaged cells, which otherwise would influence the results of the viability tests.
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PMID:The significance of culture for successful cryopreservation of isolated pancreatic islets of Langerhans. 638 13

In order to study the hormonal characteristics of human prolactin-secreting pituitary adenomas, in vitro monolayer and suspension cultures from human pituitary glands were established. Optimal conditions for cultures included enzymatic dispersion into viable single-cell suspensions with the use of 1% collagenase in phosphate-buffered saline solution. After pelleting the dispersed cells by centrifugation (800 rpm for 10 minutes), they were cultured in RPMI medium that contained 20% fetal calf serum and then incubated at 37degrees C in 5% CO2. Cells were subcultured weekly at a ratio of one plate to two. In an attempt to establish whether bromocriptine has a direct inhibitory effect on pituitary secretion of prolactin (PRL), variable doses of bromocriptine were added to duplicate plates. The addition of bromocriptine to the culture medium induced suppression of PRL within 7 days. In conclusion, this study demonstrated that either monolayer of suspension cultures of human PRL secreting adenomas can be established, and that bromocriptine in doses of 1 ng/plate or more has a direct inhibitory effect on the secretion of PRL.
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PMID:Monolayer and suspension culture of human prolactin-secreting pituitary adenoma. 743 30

Cortical collecting duct fragments were manually dissected from 6-wk-old Sprague-Dawley rats. The fragments were enzymatically digested (collagenase A) into single cells, washed, and resuspended in serum-free RPMI 1640. Individual cells were examined electrophysiologically using the whole cell patch-clamp technique. Two morphologically distinct cell types were present in the cell suspension. Small round cells that had a capacitance of 7 pF and larger oval cells with a capacitance of 29 pF were consistently observed. Whole cell electrophysiological examination revealed that the small round cells had virtually no plasma membrane ionic conductance, whereas both inward and outward currents were observed in the larger oval-type cells. Also, superfusion of 250 pM arginine vasopressin specifically increased the inward conductance of only the larger cells. The effect could be completely inhibited by 2 microM amiloride or 100 mumol of the Rp diastereomer of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a specific adenosine 3',5'-cyclic monophosphate inhibitor). These findings are consistent with the hypothesis that the larger cells are principal cells and the smaller cells are intercalated cells and directly demonstrate that an amiloride-sensitive whole cell conductance is readily observable in freshly isolated cortical collecting duct cells. Thus the whole cell configuration of the patch-clamp technique appears to be well suited for assessing cellular mechanisms that regulate the ionic conductances of cortical collecting duct cells.
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PMID:Whole cell sodium conductance of principal cells freshly isolated from rat cortical collecting duct. 757 11

We studied the effects of alloxan on insulin and glucagon secretion, islet insulin content, and morphology of human fetal islet-like cell clusters (ICCs). ICCs were derived after collagenase digestion and culture of pancreata from two fetuses. Culture medium (RPMI 1640) containing either 2.0 (low) or 11.1 (high) mM glucose was used during the alloxan exposure. Alloxan exposure lasted for 5 min at room temperature, with final concentrations of 0.3, 1, 3, 10, 30 and 100 mM. Medium samples were collected for hormone assays on days 0, 1, 2, 3, 6, and 10 and islet insulin contents were measured on day 10 after alloxan treatment. Electron microscopy of ICCs was done 24 h after the drug exposure. Control ICCs steadily increased their insulin secretion during the whole study period. Alloxan concentrations above 0.3 mM significantly (p < 0.01) decreased insulin secretion at the low glucose concentration. High glucose protected beta cells from alloxan toxicity. There was no difference in islet insulin contents between alloxan-treated and control cultures. Glucagon secretion by glucose media was not affected by alloxan exposure. All islet cells including beta cells remained intact in electron microscopy. The results suggest a block in insulin secretion by alloxan, but beta cells appear to recover at least partly in their insulin-secreting capacity.
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PMID:Effect of alloxan on the endocrine function of human fetal islet-like cell clusters--an in vitro study. 771 36

The immunogenicity of allogeneic cardiac valves (ACV) has not been previously demonstrated in vitro, though valve failure due to tissue degeneration has been attributed to adverse immunological reactions. A novel in vitro assay has been developed in a Brown Norway (BN; RT1n)-Lewis (RT1; donor-recipient) rat model system that demonstrates the immunogenicity of ACVs. A single cell suspension of viable cardiac valve conduit (CVC) cells was obtained by collagenase treatment of BN rat aortic valve conduits. Brown Norway rat CVC cells (5 x 10(4)) and Lewis responder lymphocytes (10(5)) were co-cultured in 96 well plates in RPMI 1640 plus 2.5% (v/v) non heat-inactivated Lewis rat serum and supplements with appropriate controls. Stimulation of responder lymphocytes by CVC cells was measured by 3H-thymidine incorporation into DNA. The counts obtained between 96-192 h of co-culture in the CVC cell/responder lymphocyte reaction were significantly higher (P < 0.05) than those of responder cell controls as assessed by analysis of variance. These results indicate the presence of potent immunostimulatory cells in viable ACVs and the possibility of using a sensitive and reproducible in vitro assay to evaluate ACV immunogenicity.
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PMID:In vitro immunogenicity of allogeneic cardiac valves. 782 53

Allogeneic islet transplantation in Type I diabetic patients is considerably hampered by the variable outcome of islet isolation and purification. After collagenase digestion of the pancreas, islet isolation is traditionally performed under hypothermic conditions in physiological solutions such as Hanks and RPMI. The University of Wisconsin solution (UWS) has been shown superior for hypothermic preservation of the pancreas. We, therefore, compared the UWS and RPMI for canine islet isolation and subsequent purification in either a conventional hyperosmotic density gradient of dextran in Hanks, or a novel normosmotic density gradient of Percoll in UWS. The isolation solution did not affect islet yield before purification (51% of the native islet mass). Loss of amylase (30%) and swelling of the acinar cells were observed in RPMI. In contrast, no loss of amylase and slight shrinkage of the acinar cells were observed in the UWS. Cell swelling affected the density separation and viability of the cells. Dextran density separation resulted in a 15% purity and 41% recovery of the islets isolated in RPMI, as compared to a 93% purity and 52% recovery of islets isolated in UWS. Percoll density separation improved the purity (99%) and recovery (74%) of islets isolated in UWS. Islets isolated in UWS demonstrated a superior basal and glucose stimulated insulin release during perifusion. Electron microscopy demonstrated a well-preserved islet ultrastructure after isolation in both solutions--except for slightly swollen mitochondria after isolation in RPMI. Autotransplantation of islets in pancreatectomised dogs was successful both after isolation in UWS and RPMI. We conclude that prevention of cell swelling during isolation and purification in the UWS resulted in an improved yield of viable and consistent virtually pure islets. Prevention of cell swelling during islet isolation should facilitate the analysis and control of other factors affecting outcome in man.
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PMID:Cell preservation in University of Wisconsin solution during isolation of canine islets of Langerhans. 792 36


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