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)

A method is described for the preservation and subsequent recovery of hepatocytes obtained by collagenase perfusion of cynomolgus monkey (Macaca fascicularis) livers. The fresh cells are suspended in fetal bovine serum containing 10% dimethylsulfoxide and, using a microprocessor-controlled, liquid nitrogen freezing chamber and a specific cooling protocol, processed in such a way that they can be stored in liquid nitrogen for several months and still restored to active culture. When the cryopreserved cells were established in culture, they were found to actively synthesize and secrete both albumin and apolipoprotein A-I. That, taken together with morphologic evidence, was viewed as indication that the cells recovered in culture were in fact hepatocytes and not some other cell type from the monkey liver. The availability of this procedure for storing hepatocytes should contribute significantly to the efficient use of nonhuman primates as models with which to study hepatic metabolism.
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PMID:Cryopreservation of cynomolgus monkey (Macaca fascicularis) hepatocytes for subsequent culture and protein synthesis studies. 231 97

Proteolytic destruction of pig aortal valves was studied at various steps of their preparation to implantation (native tissue, the tissue treated with terrylytine as well as with terrylytine and glutaric aldehyde) using pig pancreatic elastase and bacterial collagenase. The rate of the tissue destruction was estimated by means of monitoring an increase in content of protein and amino nitrogen in the hydrolysates. The tissue treated with terrylytine and glutaricaldehyde was 10-40-fold more resistant to proteolysis as compared with native heart valve tissue. Inadequate stabilizing effect of glutaric aldehyde on elastin, as compared with that on collagen, was found, when proteolysis of native and modified with glutaric aldehyde elastin from bovine cervical ligament and of calf skin collagen was studied using elastase and collagenase, respectively.
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PMID:[Resistance of heart valve bioprosthesis to the proteolytic effect of collagenase and elastase]. 256 Aug 68

The viability and drug responses of cryopreserved adult Onchocerca have been examined in vitro. Male worms were cryopreserved in liquid nitrogen (-196 degrees C) using ethanediol as a cryoprotectant in a 2-step incubation procedure. After thawing, 85-90% of O. gutturosa males were normally motile. These motile worms were evaluated for viability using 4 measurements (long-term motility/survival in culture; [U-14C]adenine uptake and leakage; glucose utilization; MTT-formazan colorimetry) and were no different from unfrozen controls. Subsequent experiments demonstrated that the motility responses of cryopreserved worms exposed to the antifilarial drugs ivermectin, CGP 6140 and levamisole were virtually identical to unfrozen controls. Some success was also obtained with this technique in cryopreserving O. volvulus males, with 2 thawed specimens surviving in culture for 93 and 106 d respectively. Following collagenase isolation, female worms were cryopreserved in medium +10% serum without protectant at -79 degrees C. A batch of 8 female O. gutturosa were all motile when thawed 14 d later, with a mean survival time (based on 5 specimens) of 71 d (range 60-90). However, a batch of worms transferred from -79 degrees C to -196 degrees C were badly damaged when thawed. Female O. volvulus were cryopreserved at -79 degrees C in Guatemala and sent by air freight on solid CO2 to the UK. Most specimens were active when thawed. Survival of motile specimens ranged from 7 to 272 d in culture. It is concluded that these techniques are of practical value for the storage and transportation of adult Onchocerca.
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PMID:Onchocerca gutturosa and O. volvulus: studies on the viability and drug responses of cryopreserved adult worms in vitro. 261 29

Fourier transform infrared spectroscopy (FTIR) was used to characterize the organic and mineral phases present during the induction of mineral formation by collagenase-released matrix vesicles (CRMV) during incubation in a synthetic cartilage lymph in vitro. CRMV mineralization, which occurs in the absence of alkaline phosphatase organic phosphate substrates, is characterized by an initial short lag period of limited Ca2+ accumulation, followed by a period of rapid Ca2+ uptake, and finally, by a plateau period during which Ca2+ accumulation continued at a slower rate. FTIR spectra taken at timed intervals during the induction of mineralization revealed the presence of absorptions characteristic of protein, phospholipid, and mineral components in the CRMV. These became progressively more intense with time. To reveal underlying changes occurring during the successive stages of Ca2+ accumulation, FTIR spectra of nascent (or demineralized) CRMV were computer-subtracted from subsequent spectra, nulling on the C-H stretch modes characteristic of the lipid acyl chains. These difference spectra showed little change during early Ca2+ loading, revealing that mineral ions initially accumulated in a form similar to that present in nascent matrix vesicles (MV). During the period of rapid Ca2+ uptake prior to appearance of crystalline mineral, difference spectra revealed subtle changes in the carbonyl and amide nitrogen stretch modes indicative of protein conformational changes. The first definable mineral phase appeared late in the rapid Ca2+ uptake period and was a distinct, crystalline octacalcium phosphate (OCP)-like phase. With time, the OCP-like precursor became more apatitic in character. There was no evidence that any amorphous calcium phosphate phase formed during the MV mineralization sequence. The mature MV mineral phase closely resembled hydroxyapatite formed via an OCP precursor and was similar to other biological apatites that show a substantial incorporation of carbonate.
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PMID:Fourier transform infrared characterization of mineral phases formed during induction of mineralization by collagenase-released matrix vesicles in vitro. 284 33

A range of culture conditions were examined to optimize parasite maintenance. Using male worms in a cell-free system, good results were obtained with medium NCTC 135 + 10% inactivated calf serum (IFCS) in an atmosphere of 95% N2/5% CO2 (median survival time 45 days). Survival was increased to 6-7 months using medium MEM + 10% IFCS + LLCMK2 (monkey kidney) feeder cells in a gas phase of 5% CO2 in air. Worms exposed to collagenase solution (5 mg/ml) were subsequently less motile and survived shorter periods compared to unexposed controls. The drug responses of worms (in vitro) were examined using 13 antiparasitic compounds. Ivermectin and CGP 6140 were among the most active, with the majority of drugs significantly affecting motility levels at a concentration of 5 x 10(-5) M or less. This system may provide useful information on the intrinsic activity of new compounds. A technique was developed for the successful cryopreservation of males in liquid nitrogen using ethanediol as a cryoprotectant in a 2-step incubation procedure, thereby enabling the long-term storage and transportation of worms. In conclusion, the common bovine parasite O. gutturosa provides a practical alternative for research in the absence of O. volvulus.
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PMID:The development of a laboratory model for onchocerciasis using Onchocerca gutturosa: in vitro culture, collagenase effects, drug studies and cryopreservation. 285 98

The tissue-destructive proteinases of B16-BL6 melanoma cells from C57BL/6 mice and subcellular fractions were examined. Cancer cell organelles were isolated following nitrogen cavitation with the use of sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed with the use of radiolabeled proteins and synthetic substrates. Tumor-induced red blood cell lysis was quantitated by measurement of the release of isotope from 59Fe-labeled red blood cells (RBC) cocultivated with melanoma cells; the RBC were from Wistar rats. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact melanoma cells, cell organelles, and cytosol contain proteinases that can degrade collagen and gelatin and lyse normal RBC. Melanoma plasma membranes are highly enriched in collagenase, gelatinase, cysteine proteinase, plasminogen activator, and cytolytic activity. The inhibition of tumor collagenolytic, gelatinolytic, and cytolytic activities by EDTA and 1,10-phenanthroline but not by diisopropyl fluorophosphate and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates that metalloproteinases are the active enzymes in these assays. Minocycline, a synthetic tetracycline with demonstrable inhibitory activity with other mammalian collagenases, also inhibited melanoma collagenolytic and cytolytic activities.
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PMID:Diversity of melanoma plasma membrane proteinases: inhibition of collagenolytic and cytolytic activities by minocycline. 299 28

Large numbers of calcium-tolerant canine cardiocytes can be isolated from the collagenase-perfused canine myocardium. An average yield of 500 million cells provides abundant tissue for the preparation of subcellular fractions. Using nitrogen cavitation, along with extraction by 0.6 M potassium chloride/sucrose buffer, we have been able to prepare, after differential and sucrose gradient centrifugation, membrane fractions that are more than 100-fold enriched in sarcolemmal marker enzymes. This preparation of sarcolemma has the advantage of being essentially free of plasma membranes from endothelial, smooth muscle, and other cell types residing in the myocardium.
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PMID:Characterization of sarcolemma from calcium-tolerant canine cardiocytes. 299 31

Primary cultures of epithelial cells from human prostate acini proliferate in defined medium. However, the limited availability of human tissue and the lack of knowledge of the conditions required for clonal growth and serial culture of epithelial cells have limited progress in the study of human prostate cell biology. Here we report conditions that permit the proliferation of single epithelial cells from normal, benign hyperplastic, and carcinomatous prostate through three to four serial passages, which represents at least seven to nine cumulative doublings of the cell populations. Primary cultures were prepared from prostatic acini. Monolayers resulting from the outgrowth of epithelial cells from acini were harvested and dissociated into suspensions of single cells which gave rise to discrete colonies in subsequent culture. The requirements for successful serial culture were (a) a low calcium concentration, (b) the presence of a growth factor that is concentrated in bovine neural tissue, (c), detachment of the epithelial cells with collagenase, and (d) harvest of cells before the cell concentration reached 6000 cells/cm2 of culture surface. Suspensions of single cells were successfully stored between subcultures in 10% dimethylsulfoxide with 5% fetal bovine serum and revived after storage for up to 2 months in liquid nitrogen.
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PMID:Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain. 300 May 87

Several dermal fibroblast lines have been established from explants taken from adult rats. The cells have been cultured for 2 yr and possess stable and well-defined growth characteristics through subculture 18. The cells are readily stored in liquid nitrogen with good viability after thawing. Collagenase activity secreted into the culture medium of the cells at different periods of growth has been examined. There is an 88% drop in total enzyme activity present in the medium between 4 and 14 d of culture, when the cells were plated to reach confluence at Day 8 to 10. A more pronounced fall is noted at earlier times when the cells are plated at a higher density. The correlation between DNA content of the cell monolayer and enzyme activity was -0.895, indicating a possible relationship between the growth of cells and collagenase release.
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PMID:Establishment of an adult rat fibroblast cell line for studies of collagenase regulation. 303 61

In most previous studies of cryopreserved isolated pancreatic islets, a slow cooling rate has been employed. We recently observed that faster cooling (5 degrees C/min) resulted in better functional islet preservation than cooling at 0.5 degrees C/min. We found that a culture period after the collagenase isolation of the islets, but prior to freezing, is crucial for the preservation of the islet B cell function. In the present investigation the function of isolated mouse pancreatic islets cooled in Hanks' solution supplemented with 2 M dimethylsulphoxide was compared with that of nonfrozen, cultured islets prepared from the same donors. The islets were cultured in RPMI 1640 + 10% calf serum for 3 days before freezing, and for 3 days after rapid thawing at 37 degrees C. Islets were cooled at rates of 5, 15, or 25 degrees C/min to 70 degrees C and then plunged into liquid nitrogen. All three groups of cryopreserved islets responded with insulin secretion when challenged with high glucose concentrations in batch-type incubations. In further experiments it was found that glucose-stimulated (pro)insulin biosynthesis in islets frozen at 25 degrees C/min was the same as that in the controls. Similar observations were made with respect to glucose-stimulated insulin release in perifusion experiments. However, a 30% reduction in insulin content was observed in the rapidly frozen islets. There was no difference in the replicatory capacity of the islets cells in vitro, as determined by an autoradiographic technique, between control islets and islets cooled at 5 degrees C or 25 degrees C/min. Intrasplenic implantation of 600-800 cryopreserved syngeneic islets into alloxan-diabetic mice led to complete or partial normalization of the hyperglycemia in seven of nine mice. When splenectomy was performed in five animals the serum glucose concentrations increased promptly. We conclude that relatively rapid cooling rates may be useful for cryopreservation of isolated pancreatic islets.
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PMID:Cryopreservation of mouse pancreatic islets. Effects of fast cooling on islet B cell function and on the outcome of islet transplantation. 309 92


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