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 cDNA of a T-cluster binding protein (TCBP) has been cloned using the Southwestern method. The cDNA sequence of TCBP reveals that it has 78% homology to that of nucleolysin, a factor involved in apoptosis in cytolytic lymphocyte target cells. In particular, the 0.8kb sequences of the 5'-half of both cDNAs were identical. However, nucleolysin has a lysosome-targeting motif at the carboxy terminus, while TCBP has a hydrophobic sequence instead. Southern blot experiments have revealed that both cDNA sequences existed in the same YAC clone derived from chromosome 10. This strongly suggests that the TCBP cDNA is an alternatively spliced product of the nucleolysin/TCBP gene. The TCBP mRNA is ubiquitously expressed, not only in megakaryocytic cells but also in other hematopoietic cells. However, when HEL cells were induced to differentiate to megakaryocytes by DMSO, TCBP mRNA was reduced, while PF4 gene expression increased simultaneously. Gel mobility shift analysis demonstrated that recombinant TCBP specifically bound to the T-cluster and the proximal T-rich region of the PF4 promoter. Co-transfection experiments showed that TCBP reduced the gene expression from the PF4 promoter. On the other hand, TCBP did not affect expression from the PF4 promoter in which the T-cluster and the T-rich region had been removed. These results indicate that TCBP may participate in the regulation of PF4 gene expression by binding to the T-cluster and the T-rich sequence.
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PMID:An alternative form of nucleolysin binds to a T-cluster DNA in the silencer element of platelet factor 4 gene. 920 9

The provision of an immediate supply of isolated porcine hepatocytes for artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions. By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatment facilities. Additionally, cryopreservation provides an adequate period for quality control testing to be completed prior to use of any specific cell lot. We are reporting a dual approach, namely the preservation of porcine hepatocytes, at 4 degrees C and at -196 degrees C in liquid nitrogen (LN2). Using a combination of cryoprotectant agents with Chee's modified Eagle's culture media (CEM), collagenase isolated hepatocytes stored at 4 degrees C for 24 h maintained 80% of the initial diazepam metabolism measured in freshly isolated cells and nearly 100% of initial function was preserved in hepatocytes stored up to 6 mo at -196 degrees C. University of Wisconsin solution (UW) was also tested and while adequate for 4 degrees C storage, it certainly did not match the performance of the CEM formulations for preservation of metabolic function of cells stored in liquid nitrogen. Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN2 frozen cells. Other findings in these studies underlined the importance of the gradual introduction of DMSO in the prefreezing process, the period of osmotic equilibration, and the rapid postthaw withdrawal of this agent to minimize cytotoxic effects at these critical stages. Our freezing methodology provides the foundation for further technological developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices.
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PMID:Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems. 933 95

The biotransformation pathway of tamoxifen and alpha-hydroxytamoxifen to DNA-binding species was investigated in rat hepatocytes in vitro. Rat hepatocytes were isolated by in situ collagenase perfusion and then maintained in sulphate-free Dulbecco's modified Eagle's medium. Magnesium sulphate was added to the medium to give concentrations of 0-10 microM, prior to treatment for 18 h with solvent vehicle (DMSO), tamoxifen (10 microM), alpha-hydroxytamoxifen (1 microM) or benzo[a]pyrene (BaP) (10 and 50 microM). DNA was isolated and analysed by 32P-post-labelling. For tamoxifen and alpha-hydroxytamoxifen, the level of DNA adduct formation was directly proportional to the concentration of sulphate in the medium. Between 0 and 10 microM MgSO4, the DNA adduct level increased 10-fold with both compounds. Rat hepatocytes were also maintained in normal Dulbecco's modified Eagle's medium and pretreated with dehydroisoandrosterone-3-sulphate (DHEAS, a sulphotransferase inhibitor) at concentrations ranging from 0-1 mM, prior to treatment with solvent vehicle (DMSO), tamoxifen (10 microM), alpha-hydroxytamoxifen (1 microM) or BaP (50 microM). For tamoxifen and alpha-hydroxytamoxifen the level of DNA adducts was reduced to approximately one-fifth by the addition of DHEAS (0.1 mM). BaP-DNA adduct formation, which proceeds by a pathway that does not require sulphation, was not significantly affected by sulphate concentration or by addition of DHEAS, which demonstrates that the general metabolic capacity and viability of the hepatocytes were not compromised. It is concluded that the activation of tamoxifen in rat liver cells to DNA binding products proceeds predominantly through hydroxylation followed by sulphate ester formation at the alpha-position of the ethyl side chain.
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PMID:The metabolic activation of tamoxifen and alpha-hydroxytamoxifen to DNA-binding species in rat hepatocytes proceeds via sulphation. 963 75

Cryopreservation is the only available technique for long-term storage of pancreatic islets. The freezing/thawing protocol may cause considerable loss of viable islet tissue and impair its function in vivo. The aim of this study was to investigate glucose and insulin levels after transplantation of fresh and cryo/thawed rat islets. Rat pancreatic islets were isolated following intraductal collagenase injection and Ficoll gradient purification. After isolation, islets were cultured for 24 h and then either transplanted or frozen after stepwise addition of DMSO according to Rajotte et al. and stored in liquid nitrogen. After rapid thawing islets were stepwise transferred into RPMI medium and cultured for another 24 h. The recipients were athymic mice with streptozotocine-induced diabetes. Two hundred fresh (n=13) or cryo/thawed (n=15) islets were transplanted beneath the renal capsule. Glucose levels were measured for 14 days and blood samples for insulin determination were obtained 15 min after i.p. glucagon (10 mg/kg) administration on day 14. Glucose levels were normalized (<9 mmol/l) in all recipients within 3 days since transplantation. On day 14, mean fasting values+/-SE in fresh and cryo/thawed islet groups were 4.0+/-0.6 and 4.4+/-0.4 mmol/l, respectively (P>0.05). Fasting insulin levels were higher in the cryo/thaw than in the fresh islet group (1.67+/-0.33 vs 0.57+/-0.13 ng/ml; P<0.01). Post-glucagon levels did not differ significantly (1.45+/-0.24 vs 0.86+/-0.24 ng/ml; P=0.06). While glucagon significantly increased insulin levels (P<0.01) in the fresh islet group, no change in insulin levels was observed (P>0.05) in the cryo/thaw group. Immunohistochemical staining demonstrated fragmentation of viable islet tissue which was more apparent in the cryo/thaw group. We conclude that in a short-term study cryo/thawed rat islets produce higher insulin levels than fresh islets transplanted into nude mice. This may be due to better islet survival or loss of feed-back regulation.
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PMID:Increased glucagon-stimulated insulin secretion of cryopreserved rat islets transplanted into nude mice. 993 Sep 40

Future improvements in the recovery and function of pancreatic islets following cryopreservation will require a more precise quantification of the stresses that occur at each stage of the cryopreservation protocol. Changes in solution osmolality during the addition and dilution of cryoprotectants and during freezing and thawing induce changes in islet volume that may exceed tolerable limits. The aim of this study was to determine the range of solution osmolalities that results in significant changes in islet function. Islets were isolated from canine pancreases by collagenase digestion and Euro-Ficoll purification. Following 12-h culture at 37 degrees C, islets were counted and dispensed into multiwell plate inserts. Islet function was assessed in each well immediately before and 24 h following a 10-min osmotic challenge with hypo- or hyperosmotic solutions of PBS (0, 75, 150, 300, 600, 1200, or 2300 mOsm/kg) at 22 degrees C. Canine islets reached their osmotic equilibrium within 10 min. Duplicate wells were used for each osmolality treatment for each of six donors (n = 12). No significant differences in basal or glucose-stimulated insulin secretion were found between wells prior to the osmotic challenge (3.35 +/- 0.45 and 20.98 +/- 3.36 microIU/IE/h, respectively). Following the osmotic challenge and 24-h in vitro tissue culture, a significant increase in basal secretion was observed for islets exposed to 0 and 75 mOsm/kg solutions and a significant decrease for islets exposed to 2300 mOsm/kg solution. Islets exposed to 0 and 2300 mOsm/kg solutions showed significant decreases in the stimulated insulin secretion when compared to controls. Solution osmolalities of 150-1200 mOsm/kg appear to be tolerated by canine islets with no significant deviations in insulin secretion. The corresponding tolerable volume range was 152.6 +/- 6.8% to 60 +/- 5.1% of the isotonic islet volume. The minimum critical volume was used in a theoretical analysis of the islet volumes that would result from equilibrium freezing in dimethyl sulfoxide (DMSO). The calculations show that 1.5 mol/l DMSO is sufficient to prevent damage to islets due to excessive shrinkage. Further refinement of cryoprotectant addition and dilution protocols, and cooling and warming protocols for canine islets, are now possible.
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PMID:Osmotic tolerance limits of canine pancreatic islets. 1044 40

Cryopreservation allows accumulation of the necessary islet transplantable mass as well as adequate time for tissue typing and infectious disease screening. Cryopreservation protocols may be optimized by modeling the osmotically induced volume excursions that occur during the addition and removal of cryoprotective agents (CPAs). To that end, three transport parameters were measured at 22 degrees C in canine and human islets isolated by collagenase digestion and euroficoll purification: (i) the apparent hydraulic conductivity (Lp), (ii) the permeability coefficient of the CPA (Ps), and (iii) the associated reflection coefficient (sigma). The parameters were determined by volumetric analysis of islets upon abrupt exposure to 1, 2, and 3 M dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), and propylene glycol (PG). The parameters were calculated using the Kedem-Katchalsky theory to describe islet volume excursion kinetics (assuming islets to be single equivalent osmotic units with the same volume and surface area of the actual islet) and a three-parameter curve fit was performed using the Marquardt-Levenberg method. It was determined that the permeability characteristics of pancreatic islets are species specific, and based upon the measured parameters, the highest Ps values for canine islets were observed following exposure to 2 M EG, and the highest Ps values for human islets were observed following exposure to 2 M PG. The permeability parameters were analyzed adjusting for islet radius using ANCOVA procedures to acquire least square means. For canine islets exposed to 2 M EG these values were determined to be 0.936 microm/min/atm, 2.47 microm/s, and 0.90 (for Lp, Ps, and phi, respectively) and for human islets exposed to 2 M PG the values were determined to be 1.56 microm/min/atm, 3.48 microm/s, and 0.85 (for Lp, Ps, and sigma, respectively). These parameters were used in a model to calculate osmotically induced islet volumetric response upon addition/dilution of the optimum CPAs, taking into consideration critical volume excursion limits at which irreversible damage occurs.
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PMID:Water and cryoprotectant permeability characteristics of isolated human and canine pancreatic islets. 1058 Mar 49

The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and beta-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22 degrees C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40 degrees C before storage in liquid nitrogen (-196 degrees C). Frozen islets were rapidly thawed (200 degrees C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcryopreservation tissue culture was 81.5 +/- 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 +/- 1.5 (mean +/- SEM) for the frozen-thawed islets, compared with 12.4 +/- 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single beta-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single beta-cells in real time. Secretion from single beta-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed beta-cells with an average of 7.0 +/- 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 +/- 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, beta-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that beta-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.
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PMID:Secretion from islets and single islet cells following cryopreservation. 1070 97

Luteal cells were isolated from mid-luteal heifer ovaries by collagenase digestion. Cells were cultured with DMEM/Ham's F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. As beta-carotene is almost completely insoluble in all polar solvents, it was added to cultures in either dimethyl sulphoxide (DMSO), tetrahydrofuran (THF) or as high-density lipoprotein (HDL) containing high or low beta-carotene concentrations. Medium was replaced after 24 h, thereafter medium was changed every 48 h. Treatment of cells with DMSO alone or with beta-carotene (5 micromol/l) in DMSO both resulted in significant (P<0.01) stimulation of progesterone production. beta-Carotene (5 micromol/l) in THF did not alter progesterone production but 50 micromol/l beta-carotene in THF resulted in significant inhibition (P<0.02) of progesterone production on days 3 and 7. Cultures were also supplemented with bovine HDL preparations containing equal concentrations of cholesterol (25 microg/ml) but high or low beta-carotene (12.4 or 0.44 microg/mg of cholesterol). Both HDL preparations significantly stimulated progesterone production (P<0. 001) but the high beta-carotene HDL was significantly (P<0.02) more effective than the low beta-carotene HDL. However, when given together with bovine luteinizing hormone (bLH) or dibutyryl cAMP (dbcAMP), the high beta-carotene HDL stimulated progesterone production less than did the low HDL (P<0.01). Uptake and depletion of beta-carotene by luteal cells were also examined in culture. beta-Carotene supplementation increased luteal cell beta-carotene from an initial level of 373 ng per 10(6) cells to 2030 ng per 10(6) cells by day 6. In contrast, the levels in control cells decreased to 14% of starting values during the same period. Cells treated with HDL containing high beta-carotene on day 1 or days 1 and 3 were then incubated with or without bLH or dbcAMP for a further 2 days to investigate the effect of bLH and dbcAMP on depletion of beta-carotene by luteal cells. beta-Carotene depletion in the luteal cells was significantly higher (P<0.05) in LH- and dbcAMP-treated cells than in the control cells in both groups. These results indicate that the use of solvents such as DMSO or THF may have undesirable effects due to alteration of cell membrane permeability. Supplementation with bLH or dbcAMP may increase the metabolism of beta-carotene in luteal cells. bLH or dbcAMP together with high beta-carotene HDL may, when combined with the effect of increased beta-carotene metabolism, give less stimulation than with low beta-carotene HDL.
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PMID:Effects of high density lipoprotein containing high or low beta-carotene concentrations on progesterone production and beta-carotene uptake and depletion by bovine luteal cells. 1092 28

To determine the best and simplest method for cryopreservation of pig hepatocytes, we compared immediate cryopreservation with cryopreservation after short-term culture. Suckling pig hepatocytes were isolated by a modified 2-step in situ collagenase perfusion method, suspended in serum-free medium, and preserved for 10 da by two cryopreservation methods. Serial measurements were made of cell viability, LDH release, synthesis of protein, urea and glucose, glucose-6-phosphatase (G-6-Pase) activity, and diazepam transformation after thawing. These measurements were performed on both groups of cultured hepatocytes, and on freshly isolated hepatocytes, which served as a control. High viability (>95%)of thawed hepatocytes was obtained and maintained in both cryopreservation groups. There were no significant differences in cell viability, protein synthesis, glucose synthesis, G-6-Pase activity, or diazepam transformation between the two cryopreservation groups. In the immediate cryopreservation group, urea synthesis was less than in the group with cryopreservation after short-term culture. Protein synthesis, glucose synthesis, and diazepam transformation were lower in both cryopreserved groups than in the controls. The results showed that a protocol of immediate cryopreservation of hepatocytes in RPMI-1640 medium containing 10% DMSO, hormones, growth factors, and 10% newborn bovine serum, together with rate-controlled freezing and rapid thawing, provides indices of cell viability and function during subsequent serum-free culture that are comparable to hepatocytes cryopreserved after short-term culture, except for lower urea production. This simple procedure can be used in studies of bioartificial liver and hepatocyte transplantation.
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PMID:Cryopreservation of suckling pig hepatocytes. 1168 51

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 units/g liver at 37 degrees C for 20 min) for isolation of intact cells for subsequent comet assay We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation (-85 degrees C for 72 h). To minimize this damage, minced liver (1.0 g) and kidney (0.5 g) tissues were added to 20 ml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO (-85 degrees C for 72 h and 3 weeks), and to a slightly lesser extent with glycerol (72 h), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150units/g liver and 300units/g kidney for 10 min produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at - 85 degrees C for 72 h. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.
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PMID:Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues. 1199 89


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