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)

Previous studies on ultrastructural changes that occur in cultured human fibroblasts during their in vitro life-span indicate that "senescent" cells characteristically possess structurally altered mitochondria, highly lobed nuclei, and an abundance of secondary lysosomes when compared to early passage cells. In the present study, we demonstrate that improper preparative methods can induce altered mitochondrial morphology in preparations of both IMR-90 and HF730A fibroblasts, regardless of passage level. We also show that nuclei of both living and fixed IMR-90 fibroblasts are ovoid in shape, not lobulate, in well-spread cells, regardless of either the passage level or the proliferative capacity of the cell. Fibroblasts contain lobulated nuclei only when they have not spread completely on the culture substrate. Lobulations can be induced at any passage level by collagenase/trypsin or trypsin/EDTA treatment prior to fixation, but not by cytochalasin B treatment or by cold temperatures. We conclude that any treatment that affects cytoskeleton-membrane-culture substrate interactions will induce this aberrant nuclear morphology, but that this is not indicative of "senescence" and does not relate to proliferative decline.
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PMID:Changes in nuclear shape and mitochondrial structure do not accompany the loss of division potential in human fibroblasts in vitro. 732 28

Euro-Ficoll and bovine serum albumin (BSA) are two of the most commonly used density gradient media for the purification of pancreatic islets. Euro-Ficoll is based upon Euro-Collins, a cold storage medium, and must, therefore, be used at 4 degrees C. The ionic composition of BSA, however, is likely to contribute to hypothermic cellular swelling, and this may influence the efficiency of islet purification using this medium at 4 degrees C. Experience in this laboratory also suggested that batch-to-batch variation in islet purity using BSA was related to differences in BSA osmolality. The aim of this study was to assess the effect of gradient medium temperature and osmolality on the purification of human and porcine islets using BSA. Pancreata were collagenase-digested, and islets were purified on continuous linear density gradients of BSA. The distribution of insulin and amylase in each gradient was assayed, and used to calculate the median density of islets and exocrine tissue, and the efficiency of islet purification (% amylase contamination at a fixed insulin yield), using: 1) gradient osmolalities of 300, 400, and 500 mOsm/kg H2O (seven porcine pancreata), and 2) gradients at 4 degrees C and at 22 degrees C (eight human and seven porcine pancreata). Increase in density gradient osmolality produced increases in porcine exocrine tissue density which exceeded changes in islet density, resulting in improved islet purity, maximal at a BSA osmolality of 400 mOsm/kg H2O. For human pancreata there was no significant change in pancreatic tissue densities nor islet purity with temperature.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pancreatic islet purification using bovine serum albumin: the importance of density gradient temperature and osmolality. 751 74

The effect of surface roughness on osteoblast proliferation, differentiation, and protein synthesis was examined. Human osteoblast-like cells (MG63) were cultured on titanium (Ti) disks that had been prepared by one of five different treatment regimens. All disks were pretreated with hydrofluroic acid-nitric acid and washed (PT). PT disks were also: washed, and then electropolished (EP); fine sandblasted, etched with HCl and H2SO4, and washed (FA); coarse sandblasted, etched with HCl and H2SO4, and washed (CA); or Ti plasma-sprayed (TPS). Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by brightfield and darkfield microscopy, cold field emission scanning electron microscopy, and laser confocal microscopy, while chemical composition was mapped using energy dispersion X-ray analysis and elemental distribution determined using Auger electron spectroscopy. The effect of surface roughness on the cells was evaluated by measuring cell number, [3H]thymidine incorporation into DNA, alkaline phosphatase specific activity, [3H]uridine incorporation into RNA, [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagenase-digestible protein (NCP), and [35S]sulfate incorporation into proteoglycan. Based on surface analysis, the five different Ti surfaces were ranked in order of smoothest to roughest: EP, PT, FA, CA, and TPS. A TiO2 layer was found on all surfaces that ranged in thickness from 100 A in the smoothest group to 300 A in the roughest. When compared to confluent cultures of cells on plastic, the number of cells was reduced on the TPS surfaces and increased on the EP surfaces, while the number of cells on the other surfaces was equivalent to plastic. [3H]Thymidine incorporation was inversely related to surface roughness. Alkaline phosphatase specific activity in isolated cells was found to decrease with increasing surface roughness, except for those cells cultured on CA. In contrast, enzyme activity in the cell layer was only decreased in cultures grown on FA- and TPS-treated surfaces. A direct correlation between surface roughness and RNA and CDP production was found. Surface roughness had no apparent effect on NCP production. Proteoglycan synthesis by the cells was inhibited on all the surfaces studied, with the largest inhibition observed in the CA and EP groups. These results demonstrate that surface roughness alters osteoblast proliferation, differentiation, and matrix production in vitro. The results also suggest that implant surface roughness may play a role in determining phenotypic expression of cells in vivo.
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PMID:Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63). 754 45

The relationship between the enhanced responses of gluconeogenesis to norepinephrine (NE) and glucagon and its zonal distribution was studied in liver lobules of cold-exposed rats by examination of preparations enriched for periportal hepatocytes (PP-H) and for perivenous hepatocytes (PV-H) by the digitonin-collagenase perfusion technique. In the control group, gluconeogenesis from lactate (10 mM) plus pyruvate (1 mM) was higher in PP-H than in PV-H. NE (100 nM) and glucagon (100 nM) increased the rate of gluconeogenesis by 80 and 70%, respectively, in both PP-H and PV-H. Gluconeogenesis in PP-H was unchanged by cold exposure. The rate in PV-H increased to the rate in PP-H at 5 days after cold exposure, and then the rate returned to the control value at 20 days. The gluconeogenic response to the alpha-adrenergic action of NE in both PP-H and PV-H doubled after 5 days. The response to glucagon tripled in PP-H and was cut in half in PV-H after 20 days. Phorbol 12-myristate 13-acetate (PMA; 1 microM), A-23187 (100 nM), and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; 1 mM) increased the rate of gluconeogenesis by 200, 100, and 80%, respectively, in both PP-H and PV-H from the control group. The responses to PMA and A-23187 were unchanged by exposure to cold. The response to DBcAMP was doubled in PP-H and was cut in half in PV-H after 20 days of cold exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cold acclimation induces zonal heterogeneity in gluconeogenic responses to glucagon in rat liver lobule. 761 95

Large numbers of human hepatocytes were obtained from split and whole livers by using an adaptive form of the collagenase perfusion technique employed in rodent and human biopsies. In order to guarantee a homogenous distribution of the perfusate within the whole specimen, major hepatic veins were cannulated with large bore catheters. This technique allowed for the isolation of human hepatocytes on a large scale (up to 18.5 x 10(9) in one case) from normal and diseased liver specimens. The yield of isolated normal viable hepatocytes is inversely proportional to the donor age. In addition, it was noted that a short time between declared death and organ harvest (cross clamp time) results in higher viability of hepatocytes. In contrast, the time of cold organ preservation did not correlate with the viability or the yield of isolated hepatocytes. We conclude that the technique presented here allows isolation of large numbers of human hepatocytes from specimens unsuitable for transplantation but very valuable for biomedical research.
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PMID:A new technique for isolating and culturing human hepatocytes from whole or split livers not used for transplantation. 782 76

In recent years, tumor-infiltrating lymphocytes (TILs) have been reported to be effective for tumors in experimental and clinical research. In order to increase the therapeutical effect, we modified some steps of Rosenberg's approach: a. cold digestion with collagenase at 4 degrees C for 24 hours; b. sedimentation instead of centrifugation; c. elimination of tumor cells before the cultivation procedure. Compared with the original approach, the proliferation, activity and cytotoxicity of TILs obtained by the modified procedure were much improved. TILs' expansion-fold was greater than that with the original approach. Cytotoxicity against tumor cells was more potent. Increased TILs' subsets were CD3 and CD8 cells. Meanwhile, we took tumor cells from tumor tissues to test their in vitro chemosensitivities to different drugs in order to select highly sensitive antitumor drugs for treatment of cases with advanced tumors. According to the design of using highly active TILs and highly sensitive drugs (H & H therapy), preliminary clinical results of 50 cases showed higher response rates than those in treatment with TIL/IL2, LAK/IL2 and TIL+IL2+CTX. Less toxic side effects were observed in 14 patients.
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PMID:A new experimental and clinical approach of combining usage of highly active tumor-infiltrating lymphocytes and highly sensitive antitumor drugs for the advanced malignant tumor. 786 84

We have retrospectively evaluated islet isolation records collected from 230 consecutive adult pancreases with 146 pancreases fulfilling all criteria for our evaluation. Fifty-six pancreases procured by our local organ procurement team (33 before in situ vascular flush and 23 after in situ vascular flush with University of Wisconsin [UW] solution) were compared with 90 pancreases received from distant centers that were shipped in UW solution after in situ vascular flushing. Cold storage of 3-26 hr preceded islet isolation using collagenase digestion and Ficoll purification. Recoveries of islets were assessed by duplicate counts of dithizone-stained aliquots before and after purification. Isolations were considered successful if > 100,000 viable islets (islet equivalents to 150 microns) were recovered after purification. Islet function was assessed by in vitro glucose-stimulated perifusion and islets were considered viable if the stimulation index (glucose stimulated over basal insulin secretion) was > 2. Eighty-three percent of the isolations from locally procured, UW-flushed pancreases were successful, as compared with 86% for pancreases that were stored for 3 to 8 hr, 73% for 8 to 16 hr of storage, and 38% for isolations following > 16 hr of cold storage. Increasing the duration of cold storage prior to islet isolation was associated with an increased proportion of failed isolations and decreased measures of islet viability. The actual numbers of islets per gram of pancreas before and after purification were significantly reduced if the hypothermic cold storage was > 16 hr. In vitro viability of isolated islets during glucose-stimulated perifusion showed a higher percentage of viable islets from pancreases with shorter durations of cold storage. For pancreases with > 16 hr of cold storage prior to islet isolation, islet viability was significantly reduced. We conclude that, with the current methods available to recover and store cadaver donor pancreases and the methods currently used to isolate human islets, yields of purified islets decline significantly from human pancreases that have been subjected to cold storage of > 16-18 hr.
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PMID:Human pancreas preservation prior to islet isolation. Cold ischemic tolerance. 788 93

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

Previous studies in rodent and canine animal models have suggested a detrimental impact on islet recovery and function when pancreas excision is preceded by in situ vascular flushing with cold preservation solutions. We studied the efficacy of islet isolation from 19 consecutive cadaver pancreases procured alternately by initial pancreatectomy before in situ flush (group 1, our standard procurement technique, n = 9) or pancreas removal following in situ vascular flushing with cold University of Wisconsin solution (group 2, n = 10). Once procured, pancreases were weighed, the main pancreatic duct was cannulated, and 150 ml of collagenase solution was injected. The pancreases were transported to the isolation laboratory and processed within 2 hr. Islets were counted and sized after dithizone staining, and the islet equivalents were calculated. Aliquots of isolated islets were cryopreserved using established techniques. Islet function of both freshly isolated and frozen-thawed islets was assessed using a glucose-stimulated perifusion system. Significantly more pancreas was harvested after University of Wisconsin flush (90.6 +/- 6.9 g for group 1 versus 66.7 +/- 4 for group 2, P < 0.05). The quantity of islets per gram of processed pancreas released during enzymatic digestion from each of the experimental groups did not differ significantly (4.5 +/- 0.6 x 10(3) islet equivalents per gram for primary pancreatectomy versus 4.0 +/- 0.4 x 10(3) University of Wisconsin flush). Similarly, following Ficoll purification, the overall yields of islets did not differ significantly. Total islet yield in the primary pancreatectomy group was 181 +/- 25 x 10(3) islet equivalents (2.7 +/- 0.3 x 10(3) IE/g) versus 217 +/- 41 x 10(3) for the University of Wisconsin flush group (2.9 +/- 0.8 x 10(3) islet equivalents/g; P not significant). No differences were observed in in vitro viability. Perifusion stimulation indexes (peak/basal insulin release) were 5.9 +/- 1.3 for group 1 and 7.1 +/- 1.5 for group 2. These results conflict with published results in animal models and indicate that large numbers of viable islets can be recovered from cadaver pancreas utilizing either procurement technique. The decreased operating time, simplicity, and safety favor the use of total pancreatectomy after limited in situ vascular flushing as the method of choice for pancreas procurement for subsequent islet isolation.
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PMID:Cadaver pancreas recovery technique. Impact on islet recovery and in vitro function. 797 19

One of the major steps toward successful islet transplantation for the treatment of type diabetes is to obtain islets of sufficient number and viability. Using a standardized method of isolating islets, the goal of this study was to analyze the factors influencing the outcome of islet isolation. A total of 104 cadaveric human pancreata were processed for islets by the same team. Data from the islet-processing charts were reviewed retrospectively. The two endpoints were the recovery of islets, viable after 2 days of culture (group V = viable, group NV = nonviable) and the islet yield. Viable islets were recovered in 61% of cases (n = 63). Minimal blood glucose recorded during hospitalization was very significantly lower in group V (124 +/- 5 vs. 148 +/- 9, P = 0.01). Lack of significant medical history in the donor was associated with better viability as compared with various donor predispositions (chi-2 4.21, P = 0.04). Cold ischemia time (8.1 +/- 0.5 hr in group V vs. 9.8 +/- 0.9 hr in group NV, P = 0.07) and collagenase lot (5 lots tested, chi-2 13.1, P = 0.01) also affected the recovery of viable islets. Hospital time was shorter in group V (65.3 +/- 6.8 vs. 80.9 +/- 17.9 hr, P = 0.35). Multivariate logistic regression analyses of viable islet recovery identified minimal blood glucose (P = 0.03) and collagenase lot (P = 0.06) as the most significant risk factors. However, the best multivariate predictive model--which includes blood glucose, collagenase lot, donor age and surgical procurement team--correctly predicted 66.2% of cases only. Multivariate analysis of final islet yield designed hospitalization length, cardiorespiratory arrest, surgical procurement team, and collagenase lot as the best predictors. These data obtained in a large series of pancreata emphasized several donor and technical factors that should target the attention of islet transplant researchers in order to improve islet yield and viability.
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PMID:Human islet isolation in 104 consecutive cases. Factors affecting isolation success. 801 87


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