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 cytochrome P-450IIE1 (CYP2E1) isozyme activates several toxins and procarcinogens. Recent studies employing immunohistochemical and immuno-analysis techniques have shown that this isozyme is predominantly localized in the pericentral zone of the liver acinus. Experiments were conducted to evaluate whether microsomes isolated from the pericentral region of the liver display elevated catalytic activity towards effective substrates for CYP2E1 such as dimethylnitrosamine (DMN) as compared with periportal microsomes. Rats were treated with pyrazole to induce CYP2E1 and hepatocytes prepared from periportal or pericentral zones of the livers by the digitonin-collagenase procedure. Microsomes isolated from these hepatocytes had similar total P-450 contents; however, the microsomes from the pericentral hepatocytes displayed an increased DMSO binding spectrum suggesting an increased content of CYP2E1. Low Km DMN demethylase activity (but not high Km activity) as well as the oxidation of aniline and p-nitrophenol were 2- to 3-fold higher in pericentral compared to periportal microsomes. The oxidation of DMN by both microsomal preparations, as well as the increased rates obtained with the pericentral microsomes, was sensitive to inhibition by carbon monoxide as well as to other CYP2E1 substrates such as ethanol, pyrazole, or 4-methylpyrazole. Anti-CYP2E1 IgG inhibited the oxidation of DMN by both microsomal preparations 75% to 85% and prevented most of the increase found with the pericentral microsomes. Oxidation of aniline and p-nitrophenol was elevated in pericentral hepatocytes compared with periportal hepatocytes to the same extent as in the isolated microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Increased oxidation of dimethylnitrosamine in pericentral microsomes after pyrazole induction of cytochrome P-4502E1. 168 70

The in vitro succinate dehydrogenase inhibition (SDI) test was adapted to be used with microtiter plates and this microtiter SDI (mSDI) test was evaluated for clinical use of chemosensitivity testing, as compared to findings with the SDI test. The optimal conditions of the mSDI test were determined: (1) 2-5 x 10(4) cells/well; (2) enzymatic disaggregation of solid tumors with the use of a mixture of 0.2% pronase, 0.25% collagenase, 0.1% DNase for 20 min at 37 degrees C; (3) addition of 10 mM sodium succinate in the colorimetric reaction; and (4) use of dimethyl sulfoxide (DMSO) as a solvent for extraction of formazan product. Good correlations were observed between the mSDI and the SDI tests when S-180 cells (r = 0.890-0.996) or 16 human fresh tumor cells (r = 0.731-0.999) were exposed to six anti-cancer drugs (carboquone, adriamycin, mitomycin C, aclacinomycin A, cisplatin, 5-fluorouracil). Thus, the mSDI test facilitates testing of a large number of drugs with minimal amounts of specimens, and is expected to replace the SDI test for chemosensitivity testing of clinical tumor cells.
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PMID:The microtiter SDI test is more advantageous than the SDI test for assessing the chemosensitivity of human tumor cells. 195 59

Relation between processes of proliferation and synthesis of the embryonal serum protein alpha-fetoprotein (AFP), the influence on these processes of polyelectrolyte dextran sulfate (DS) and dimethyl-sulfoxide (DMSO) has been studied in the monolayer culture of mouse hepatocytes. In control cultures the correlations between the time of appearance and the level of DNA and AFP synthesis were observed. DS and DMSO were found to inhibit both processes. Cell proliferation could be reestablished by addition of epidermal growth factor. In case of the influence of DMSO, it wasn't followed by the induction of AFP synthesis. This the processes of DNA and AFP synthesis in monolayer cultures of mouse hepatocytes can be separated. The elongated incubation of hepatocytes with collagenase during their obtaining, abolished the effects of DS. This shows that surface components of hepatocytes, lost upon enzyme degradation, may be involved in the mechanism of DS effect.
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PMID:[Synthesis of DNA and alpha-fetoprotein in the monolayer culture of mouse hepatocytes]. 245 78

Synthetic activity of collagen types was examined in cultured arterial smooth muscle cells during modulation from synthetic to contractile phenotype by treatment with dimethyl sulfoxide (DMSO). Smooth muscle cells of rabbit thoracic aorta cultured with a 1% supplement of DMSO for 8 days (DMSO group) predominantly exhibited cellular features of the contractile type with abundant microfilaments and a distinct basement membrane. Cultured cells in the DMSO group or in controls during stationary or subconfluent phase were labeled with [3H]proline for 24 h, and the samples including the cell layer and medium were analyzed. The incorporation of proline into bacterial collagenase-digestible fractions was increased slightly in the DMSO group. Type analysis of the collagenous protein by SDS-PAGE and subsequent fluorography disclosed a markedly increased ratio of type IV/I collagen and a slightly increased type V/I collagen ratio, as compared with those of controls. A decrease of type III collagen production in DMSO-treated cells probably due to their lower cell density was also recognized. From these biochemical and morphological observations, it is suggested that increased synthesis of minor collagen types, particularly type IV collagen, is closely associated with smooth muscle phenotypic expression following DMSO treatment. Similar cellular events may occur in smooth muscle cells migrating into the intima during the process of arteriosclerosis in vivo.
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PMID:Altered synthesis of collagen types in cultured arterial smooth muscle cells during phenotypic modulation by dimethyl sulfoxide. 265 78

Isolated and cultured human hepatocytes provide a useful model for studies of the liver cell function in man. In vitro studies using human hepatocytes are scarce, due to the limited availability and the lack of suitable methods for storage. In this study, we report the effect of deep freezing storage on the viability, fine structures and albumin synthesis of human adult hepatocytes in classical culture conditions. Hepatocytes were isolated using collagenase perfusion (9 isolations). The cell yield was 4-37 X 10(8) with a viability of 60-87%. Cryopreservation was performed in medium containing 10% DMSO and 20% fetal calf serum using a Cryoson BV-4 programmable freezer (0 degree C for 5 min, followed by a freezing rate of 1.5 degrees C/min for 20 min and 7 degrees C/min for 10 min). The cells were stored for 25-275 days in the liquid nitrogen vapor phase (-150 degrees C). Within 16 h about 80% of viable cells from freshly isolated hepatocytes whereas after cryopreservation, 55% of viable cells as determined by Trypan Blue exclusion before the cryopreservation attached to plastic and survived. Electron microscopy showed well developed tight junctions, structures similar to bile canaliculi. Cell polarity was evident. However, 'bleb' formation, more lipid droplets and lysosomes were found in cryopreserved hepatocytes during a short period after thawing. At the 3rd week, cells detached and died. These changes were associated with increased secretion of lactate dehydrogenase, whereas the albumin secretion dropped (from 10 to 4 micrograms/micrograms DNA), regardless of whether hepatocytes were cultured from fresh preparations or after cryopreservation. These findings suggest the cryopreservation is a useful technique to preserve hepatocytes for in vitro studies. Nevertheless, an improved method is necessary to increase the efficiency of cell seeding after cryopreservation.
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PMID:Cryopreservation of adult human hepatocytes. The influence of deep freezing storage on the viability, cell seeding, survival, fine structures and albumin synthesis in primary cultures. 374 87

Pancreatic islet cells of rats obtained by the collagenase digestion method were preserved from 7 to 10 days at -80 degrees C in a deep freezer or at -196 degrees C in liquid nitrogen with Dulbecco modified Eagle's medium, supplemented with 10% or 20% DMSO (dimethyl sulfoxide); thawing was performed in a 37 degree C water bath. Cryopreserved islets were morphologically almost intact, and possessed approximately 50% of the insulin secretion activity of the control groups. About 300 pancreatic islets preserved at -196 degrees C with 20% DMSO were transplanted into the portal veins of streptozotocin-induced diabetic rats. Rats recovered from the diabetic state, and the normalized condition was maintained up to 20 weeks, although 4 weeks were needed before blood and urine glucose reached normal levels after transplantation. Intact B cells were found in the transplanted islet cell masses in the liver of the recipients, but B cells of the recipient's pancreases (streptozotocin-treated rats) showed a marked decrease, as well as degenerative changes.
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PMID:Transplantation of cryopreserved pancreatic islets into the portal vein. 678 60

We studied the effect of 95% DMSO on dermal/epidermal healing and microbiol flora in partial-thickness wounds. Wounds of 0.3 mm were made in the skin of Yorkshire pigs with a keratome and treated daily with either 95% DMSO, water, or they were left untreated. Wounds were excised on Days 2-7 and the dermis was separated from the epidermis. The dermis was assayed for collagen biosynthesis (by measuring the production of [14C]hydroxyproline (HP) and amount of radioactive peptides released after collagenase digestion) and absolute HP (by spectrophotometric analysis). The epidermis was evaluated macroscopically for resurfacing. Aerobic bacteria from unwounded and wounded skin were identified and quantitated. There were no significant differences between treatment groups in HP incorporation or absolute collagen content from Days 2-6 after wounding. HP incorporation in the total protein fractions and in the collagenase digestible fractions were analogous. Collagen biosynthesis was similar in both unwounded, untreated, and unwounded DMSO-treated skin. Epidermal healing did not differ between treatment groups. There were no differences in the number or types of bacteria in wounds between treatment groups. These results indicate that topical DMSO is neither beneficial nor harmful in the healing of superficial wounds.
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PMID:Dimethyl sulfoxide (DMSO) does not affect epidermal wound healing. 684 37

The use of autologous cartilage grafts is one of the most common procedures in plastic surgery. Commonly used chemical preservation procedures of the cartilage for a second operation usually lead to the loss of vitality of the graft. In the past few years cryopreservation methods were used in the maintenance of the vitality of the autologous grafts, although these efforts yielded a low vitality of the grafts. It seems that the matrix of the grafts is mainly responsible for the unsuccessful cryopreservation of cartilage. In this work we tried to degrade the matrix of the cartilage grafts by partial enzymatic digestion with collagenase type II and hyaluronidase to facilitate the penetration of dimethylsulfoxide (DMSO). Cell vitality was assessed by trypan dye exclusion. We could demonstrate that it is possible to raise the permeability of the matrix by enzymatic digestion. Cryopreservation of the digested cartilage yielded a vitality of nearly 20%. Our results suggest that pretreatment of cartilage grafts with specific enzymes before preservation enable more successful vital cryopreservation.
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PMID:[Enzyme digestion of autologous cartilage transplants. New possibilities for vital cryopreservation?--Initial results]. 771 Jun 9

Highly purified islets of Langerhans were prepared in the present study from adult pigs by collagenase digestion and density gradient purification. After overnight culture, the tissue was equilibrated with DMSO at 25 degrees C, supercooled to -7.5 degrees C, nucleated, slowly cooled at 0.25 degrees C/min to -40 degrees C, and stored at -130 degrees C. Then, after variable periods of storage, the islets were rapidly thawed at 37 degrees C. Postthaw actual islet and islet equivalent (150-microns sized islets) recovery were 75 +/- 7% and 66 +/- 4%, respectively. The frozen-thawed porcine islets maintained good morphology on histological staining by hematoxylin-eosin and aldehyde-fuchsin. Upon perifusion, basal insulin secretion was 43 +/- 10 and 67 +/- 18 pmol/L from noncryopreserved, control islets, and cryopreserved islets, respectively (P = 0.2). Peak insulin release at 16.7 mmol/L glucose was 85 +/- 28 pmol/L from noncryopreserved islets and 157 +/- 48 pmol/L from the frozen-thawed islets (P = 0.1). When 10 mmol/L theophylline was added to 16.7 mmol/L glucose, the secretion of the hormone peaked to 221 +/- 83 (control islets) and 479 +/- 140 pmol/L (cryopreserved islets, P = 0.1). Total insulin secretion differed significantly for the noncryopreserved and the cryopreserved islets at both 16.7 mmol/L (1412 +/- 306 vs. 3756 +/- 764 pmol/L, respectively, P = 0.007) and 16.7 mmol/L glucose plus 10 mmol/L theophylline (2161 +/- 371 vs. 7505 +/- 2075 pmol/L, respectively, P = 0.011). Normoglycemia was restored within 7 days from implantation in temporarily immunosuppressed (aL3T4 antibody) mice with streptozotocin-induced diabetes by transplanting 1500-2000 cryopreserved porcine islets under the kidney capsule. Mean survival time of frozen-thawed islet xenografts (39 +/- 3 days) was similar to that of noncryopreserved islet xenografts (43 +/- 6 days). This study demonstrates that cryogenic storage is feasible of isolated porcine islets, with the frozen-thawed pancreatic endocrine tissue maintaining morphological integrity and both in vitro and in vivo viability. Further studies are needed to define the effect of cryopreservation on the immunogenic properties of porcine islets.
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PMID:Cryogenic storage of isolated, purified porcine pancreatic islets. 810 68

Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 x 10(6) cells/cm2 in plastic culture dishes precoated with trout skin extract (7.6 micrograms skin protein/cm2) to facilitate cell attachment were maintained at 16 degrees C. Cells were treated with DEX (10(-9) to 10(-7) M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10(-8) to 10(-7) M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10(-8) to 10(-7) M). DEX at 10(-9) M was ineffective. Concomitant addition of 10(-6) M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10(-7) M DEX abolished the DEX effect. RU486 at 10(-8) M was ineffective. Spironolactone (10(-8) to 10(-6) M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10(-6) M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).
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PMID:Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro. 839 84


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