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)

Cytoskeleton not only controls cell morphology but also regulates cell growth, migration, differentiation, and gene expression, events which are fundamental to embryogenesis, carcinogenesis, and wound healing. We have recently reported that reorganization of cytoskeleton induces expression of mRNA for transforming growth factor-beta 1 (TGF-beta 1), collagenase, and tissue inhibitor of metalloproteinase-I (TIMP-I) in dermal fibroblasts. In this report we have examined the role of gene transcription in this induction. As judged by nuclear run-on assay, trypsin, EGTA (ethylene glycol-bis (beta-aminoethyl ether) N, N, N', N', tetra-acetic acid), or cytochalasin C (Chs) increased the rate of transcription of the TGF-beta 1 gene by 2.0, 2.7, and 1.6 fold, respectively, and of the collagenase gene by 5.3, 6.2, and 3.3 fold. The rate of transcription of the TIMP-I gene was increased by trypsin (4.3 fold) or EGTA (3.8 fold) but unaffected by Chs. Cytochalasin induced an increase in the rate of transcription of procollagen I (alpha 1), procollagen I (alpha 2), and fibronectin genes by 1.4, 1.5, and 1.9 fold respectively, while trypsinization or EGTA treatment had no or little effects on these gene. Since transcription of the TGF-beta 1 gene is believed to be largely governed by the activating protein 1 (AP1) complex, we also examined the expression of mRNA for c-fos and c-jun protoon-coproteins. Trypsinization induced rapid (within 30 min) and transient expression of c-fos mRNA. A 2.4 fold increase in c-jun mRNA was apparent after 4 hr and persisted for at least 24 hr. Actinomycin D (Act D) suppressed the induction of TGF-beta 1 mRNA by Chs but had less effect on the TGF-beta 1 mRNA in trypsinized cells which had been replated for 4 hr, suggesting that the half life of TGF-beta 1 mRNA is reduced in cells with a disassembled cytoskeleton. Simultaneous treatment with Chs and cycloheximide (Cxm) resulted in a superinduction of TGF-beta 1 mRNA by 88 +/- 23% (n = 4, P < 0.05), which was abrogated by preexposure to Act D. In contrast, the induction of collagenase mRNA by Chs was totally blocked by Cxm, indicating that the Cxm-mediated superinduction is selective and that protein synthesis is required for induction of this mRNA. Our results suggest that the activities of genes for proteins involved in the structure (Type I collagen and fibronectin), turnover (collagenase and TIMP-1) and regulation (TGF-beta 1) of extracellular matrix (ECM), are all governed at least in part by the status of the cytoskeleton. Since the cytoskeleton is reorganized during cell division, migration, and differentiation, these results may have implications for the regulation of ECM during such processes as embryogenesis, carcinogenesis, and wound healing.
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PMID:Cytoskeleton regulates expression of genes for transforming growth factor-beta 1 and extracellular matrix proteins in dermal fibroblasts. 925 40

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

Ets-1 is a transcription factor that activates expression of matrix-degrading proteinases such as collagenase and stromelysin. To study the control of ets-1 gene expression in rat vascular smooth muscle cells (VSMC), cells were exposed to factors known to regulate VSMC migration and proliferation. Platelet-derived growth factor-BB (PDGF-BB), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) induced a dose-dependent expression of ets-1 mRNA. These effects were abrogated by inhibition of protein kinase C (PKC) by H-7 or chronic PMA treatment. Ets-1 mRNA was superinduced by PDGF-BB and ET-1 in the presence of cycloheximide. The chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester and the depletion of endoplasmic reticulum intracellular Ca2+ concentration ([Ca2+]i) by thapsigargin inhibited PDGF-BB- and ET-1-induced ets-1 mRNA, whereas ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid had no effect. However, [Ca2+]i release alone was not sufficient to increase ets-1 mRNA. Forskolin blocked ET-1-, PDGF-BB-, and PMA-induced ets-1 mRNA, as well as inositol phosphate formation, consistent with an effect through impairment of PKC activation. Inhibitors of ets-1 gene expression, such as H-7 and herbimycin A, inhibited the ET-1 induction of collagenase I mRNA. We propose that ets-1 may be an important element in the orchestration of matrix proteinase expression and of vascular remodeling after arterial injury.
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PMID:Ets-1 is an early response gene activated by ET-1 and PDGF-BB in vascular smooth muscle cells. 948 38

To elucidate the relatively short retention of crosslinked poly(1-vinyl-2-pyrrolidinone) hydrogels in the eye when used as potential vitreous substitutes, a 14C-labeled hydrogel was produced and subjected to both in vitro biodegradation assays and in vivo experiments. The polymer was synthesized by the free-radical copolymerization of 99% 1-vinyl-2-pyrrolidinone with 1% 14C-methyl methacrylate in the presence of ethylene glycol dimethacrylate (0.1%) as crosslinking agent. The in vitro protocol for assessing the biodegradation included the incubation of hydrogel with hydrolases (trypsin or collagenase), followed by examination of changes in its physical characteristics and by monitoring its residual radioactivity, as well as by detection of possible degradation products. Within the maximum duration of experiments (4 weeks), none of the procedures indicated biodegradation of polymer. The hydrogel was also injected into the vitreous humor of rabbits and followed up to 4 weeks. Residual radioactivity measurements of the vitreous contents indicated that 50% of the polymer was removed by the end of this period. Histopathologic examination revealed cell infiltrates of the mononuclear phagocyte system in both vitreous and retinal tissue. A possible phagocyte-mediated mechanism for the dissipation of hydrogel is discussed.
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PMID:Biodegradation in vitro and retention in the rabbit eye of crosslinked poly(1-vinyl-2-pyrrolidinone) hydrogel as a vitreous substitute. 949 28

Calcium signaling mechanisms were examined in vessel segments and dispersed single smooth muscle cells (SMC) of interlobular arteries and afferent arterioles (< 50 microns diameter) from the rat kidney. These resistance vessels were isolated from rat kidneys, using an iron oxide-sieving technique with subsequent collagenase digestion. Individual cells were identified by their characteristic oval appearance and positive staining for smooth muscle-specific alpha-actin and heavy chain myosin SM-1 and SM-2. Cytosolic calcium concentration ([Ca2+]i) was measured using fura 2 ratiometric fluorescence at 340 and 380 nm wavelength with a microscope-based photometer. Angiotensin II (ANG II) and arginine vasopressin (AVP), at concentrations of 10(-10)-10(-6) M, produced dose-dependent increases in [Ca2+]i; maximum increases were 221 +/- 49 nM for ANG II and 237 +/- 49 nM for AVP. The temporal response patterns for both agonists were characterized by a square-shaped, immediate step increase in [Ca2+]i to a near maximum level that was maintained through the recording period of 150-200 s. Responses of individual dispersed SMC and short vessel segments were similar. Losartan antagonized the action of ANG II, indicating mediation by AT1 receptors on preglomerular arteriolar SMC. The V1-selective antagonist [d(CH2)5Tyr(Me)2Tyr(NH2)9]AVP completely inhibited AVP-induced [Ca2+]i changes. The importance of calcium entry in hormone-induced changes in [Ca2+]i was demonstrated by the finding that neither ANG II nor AVP elicited a [Ca2+]i response in media rendered nominally calcium free by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Calcium entry occurred primarily through L-type, voltage-gated calcium channels as the dihydropyridine, nifedipine, completely prevented or reversed [Ca2+]i changes normally elicited by either hormone. Our results provide new information about the similarity of calcium signaling in single SMC and short segments freshly isolated from renal interlobular arteries and afferent arterioles. The observations indicate that AT1 and V1 receptors are coupled to signal transduction pathways leading to rapid changes in [Ca2+]i. Calcium mobilization appears to play a minor to nonexistent role under the experimental conditions. The predominant mechanism involves calcium entry through dihydropyridine-sensitive, voltage-gated calcium channels in single SMC from these resistance vessels.
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PMID:ANG II and vasopressin stimulate calcium entry in dispersed smooth muscle cells of preglomerular arterioles. 953 Feb 66

Our previous studies have shown that the degree of damage to a liposome corresponds to the variability of the animal species from which the serum comes, and that a complement activating factor (CAF) plays an important role in inducing the activation of the complement system, ultimately leading to the lysis of the liposomes. In this study, our attention focused on the characterization of the bovine serum factor (bCAF) that is involved in complement-mediated immune lysis of the liposome. The active fraction containing CAF partially purified with PEG and ammonium sulfate results in marked activation of the complement system via the alternative pathway when interacted with CAF-depleted serum, whereas the active fraction or CAF-depleted serum alone does not activate the complement. The interaction between lipopolysaccharide (LPS), heparin, zymosan or their mixture in place of CAF and CAF-depleted serum does not result in any significant activation of the complement system. Results from pretreatment with rabbit anti-bovine IgM IgG and rabbit anti-bovine IgG IgG indicate that activation of the complement system is not attributable to the antibody which is generally involved in activation of complement via the classical pathway. The results have further been proven by pretreatment with Concanavalin A (Con A) sepharose and protein G sepharose ruling out the possibility of antibody-mediated activation of complement. Our studies on collagenase and trypsin digestion demonstrate that the relative activity of CAF does not diminish with increase in collagenase concentration, and decreases with increase in trypsin concentration, strongly indicating that CAF does not have a collagen-like domain in its structure. The relative activity of CAF is dramatically inhibited after reduction with 2-mercaptoethanol (2-ME), clearly demonstrating that CAF is sensitive to reduction with 2-ME and confirming a sulhydryl-dependent protein. The optimal activity of CAF is observed in the range of 35-45 degrees C and its half-life at 37 degrees C is about 105 h. Furthermore, the relative activity of CAF increases and gradually approaches a plateau level with the increase of Mg2+ concentration. Obviously, complement activation induced by CAF depends on adequate Mg2+ concentration, confirming that this dependence is characteristic of the alternative pathway.
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PMID:Characterization of bovine serum factor triggering the lysis of liposomes via complement activation. 958 79

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 strength, resorption rates, and biocompatibility of collagenous biomaterials are profoundly influenced by the method of cross-linking. The in vitro and in vivo calcification and enzymatic degradation of bovine pericardia (BP) after a series of surface modifications were studied as a function of exposure time. Collagenase degradations of modified BP were monitored by scanning electron microscopy and tensile strength measurements. Bovine pericardium was modified by a combination of different tissue fixatives such as glutaraldehyde (GA), carbodiimide (EDC), diisocyanate (HMDIC), and polyethylene glycol (PEG). GA-PEG-EDC-PEG and GA-PEG-HMDIC-PEG combination treated BP retained maximum stability in collagenase digestion compared to GATBP. In vitro calcification studies and in vivo rat subcutaneous implantations of modified pericardium have shown substantial reduction in the calcification of double cross-linked BP with PEG modification. Further, the biocompatibility aspects of pericardial tissues were established by platelet adhesion and octane contact angle. It seems that cross-links involving amino and carboxyl residues may provide new ways of controlling biodegradation and calcification.
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PMID:Effects of double cross-linking technique on the enzymatic degradation and calcification of bovine pericardia. 1065 44

Pathologic calcification is thought to be the main cause of failure in the present generation tissue valves fabricated from glutaraldehyde pretreated bovine pericardium (BP). The present investigation describes the in vitro calcification and enzymatic degradation of bovine pericardia after hexamethylene diisocyanate (HMDIC) crosslinking and subsequent modification with polyethylene glycol. The enzymatic degradation of these treated surfaces were monitored by scanning electron micrography and tensile strength measurements. Various proteases, such as alpha-chymotrypsin, bromelain, esterase, trypsin and collagenase were investigated for tissue stability. Incubation of these enzymes with crosslinked pericardia had variably reduced their tensile strength. Among these treated surfaces, polyethylene glycol (PEG) grafted BP via isocyanate functionalities had retained maximum strength. The PEG modified tissues had also indicated a substantial reduction in calcification, when compared to other treated tissues. Further, the biocompatibility of various pericardial tissues were established by platelet adhesion and octane contact angle measurements. It is assumed that the PEG modification of pericardium may interfere with the cellular activation of injury (platelets) to reduce tissue associated calcification. In conclusion, it seems the PEG modification of bovine pericardium via HMDIC may provide new ways of controlling tissue biodegradation and calcification. However, more in vivo studies are needed to develop applications.
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PMID:The anticalcification effect of polyethylene glycol-immobilized on hexamethylene diisocyanate treated pericardium. 1170 61

Refinement of techniques to isolate viable hepatocytes began in the late 1960's. It was established that perfusion of the intact liver as opposed to incubation of liver slices or chopped tissue increased the yield of cells. The present study aims to establish a simple, two-step, collagenase digestion method for hepatocyte isolation. A single inbred Fisher rat was used for hepatocyte isolation. The liver was perfused in-situ with perfusion buffer containing ethylene glycol bis N, N1, tetra acetic acid (EGTA), followed by the collagenase buffer. The liver was excised and gently minced. The tissue was resuspended in the collagenase buffer to complete dissociation. The cell suspension obtained was washed, centrifuged and filtered to complete the isolation procedure. The trypan blue exclusion test showed 80-85% cell viability. The isolated cells were transplanted into the splenic parenchyma of syngenic rats. Survival of the transplanted hepatocytes was confirmed by histological examination at the end of 90 days. This two step technique of in-situ liver perfusion gives a high yield of viable hepatocytes which show long term survival after transplantation.
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PMID:Hepatocyte isolation and transplantation in syngenic rats. 1119 86


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