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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Development of suitable methods for the quantification of the proliferative response of airway smooth muscle (ASM) cells in culture will assist the investigation of the cellular mechanisms underlying the hyperplasia and hypertrophy of ASM as seen in asthmatic airways. In this study, two rapid and simple colorimetric assays have been modified to enable the growth of human bronchial and rabbit tracheal smooth muscle in culture to be assessed. One method depends upon the reduction by living cells of the tetrazolium salt MTT to form a blue formazan product, whereas the other relies on rapid binding of the dye Coomassie brilliant blue to protein at acidic pH. Experiments demonstrated the validity of both assays in quantifying the proliferative response of cultured human and rabbit ASM cells. The increase in optical density observed for each assay correlated directly, throughout the duration of culture, with the increase in cell number determined by hemocytometry in human and rabbit ASM cells proliferating in response to fetal calf serum (1.25 to 10%). This relationship held also for rabbit tracheal ASM cells proliferating in response to the heterodimer of platelet-derived growth factor (1 to 50 ng/ml). Application of these methods to adherent proliferating cultures of human and rabbit ASM cells demonstrated their adaptability to the generation of growth curves in response to serum and to a defined growth factor. These methods allow both total cellular protein and proliferation to be estimated in human and rabbit ASM cells in culture, using assays that are rapid, reproducible, inexpensive, and easy to perform while negating the use of radioisotopes. It is intended that these additional methods should be useful in delineating some of the mechanisms that might contribute to the proliferative response of these cells--particularly since there has been a resurgence in interest in culturing smooth muscle cells derived from the airways.
Am J Respir Cell Mol Biol 1992 Dec
PMID:Quantifying proliferation of cultured human and rabbit airway smooth muscle cells in response to serum and platelet-derived growth factor. 144 5

Human keratinocytes are the most appropriate target cells for evaluating mechanisms of skin cytotoxicity and pharmacology of chemical agents. After having formed a confluent stratified epithelium with proliferating basal cells and differentiated cell layers, human keratinocytes were harvested after enzymatic detachment as stable, three-dimensional cell aggregates or used as adherent multilayers in microtiter plates to study the local cytotoxicity of different toxic compounds. The advantage of this test is that it uses the adequate target cells and that it evaluates both the ability of the test chemical to penetrate several cellular layers as well as the ability to interfere with cellular function. The end points are cell viability and cell metabolism, which are determined by neutral red uptake and MTT reduction, respectively. For the 13 chemicals evaluated in this study we found good correlation (r = .819) between the potency rankings of keratinocyte NR 50 values and in vivo irritancy data. There was also good agreement (r = .945) between ranking of these chemicals according to midpoint toxicity of both the 3T3 assay and the keratinocyte assay. This test system might be at the present stage a supplementation of the current test battery, which shall replace in vivo irritation tests like the Draize test.
Mol Toxicol
PMID:Multilayered keratinocyte culture used for in vitro toxicology. 247 74

Until recently the alcohol dehydrogenase of Drosophila melanogaster was thought to act only in the first step of primary alcohol oxidation, producing an aldehyde. Instead, acetic acid is the main product of a two-step process. A rapid procedure was developed for the isolation and purification of two allozymes. The thermostability of the purified enzymes was found to be very different, t 1/2 at 35 degrees C, being 45 min and 130 min for ADH-F and ADH-71k respectively. The kinetic parameters of ethanol oxidation by the two purified allozymes were determined within physiological substrate and coenzyme ranges. The use of artificial electron acceptors has a notable influence on the ethanol oxidation: the apparent Michaelis constants increase; the oxidation rate with ADH-71k increases, whereas it decreases with ADH-F. Purified ADH is shown to be able to catalyze the oxidation of acetaldehyde solely in the presence of NAD+, and PMS and MTT as artificial electron acceptors. From the kinetic data the relative in vivo oxidation rates of ethanol by both ADH allozymes were calculated. ADH-F turned out to be somewhat less effective (30%-40%) than ADH-71k. The physiological consequences of these differences are discussed.
Mol Gen Genet 1985
PMID:Dual function of the alcohol dehydrogenase of Drosophila melanogaster: ethanol and acetaldehyde oxidation by two allozymes ADH-71k and ADH-F. 315 99

Established renal epithelial cell lines of human, pig, and dog origin (293, LLC-PK1, MDCK) were examined in terms of nephrotoxicity and ability to biotransform cyclosporine A (CsA). All three cell lines exhibited a comparable concentration dependent cytotoxicity to CsA treatment. Alterations in cell function included a decreased transport of lysine, an inhibition of growth, and an activation of lysosomal and mitochondrial activity as indicated by the increased uptake of neutral red (NR) and increased reduction of the tetrazolium dye MTT at 1-6 microM CsA. Increased leakage of lactic dehydrogenase and activities of gamma-glutamyl transpeptidase (GGT) and N-acetyl-beta-D-hexosaminidase were observed at 48 h and 12 microM CsA. A discrimination between CsA and the less nephrotoxic cyclosporine-(CsH) was shown for DNA synthesis and NR uptake. The contribution of extrarenal parameters on kidney cell function was studied by the addition of medium from hepatocytes exposed to CsA to the kidney cell lines. A more potent inhibition of DNA synthesis and enhanced reduction of MTT resulted than by addition of equimolar CsA directly to the kidney cells. These data indicate that hepatocyte constituents present in the medium due to CsA treatment affect kidney cell function; additionally, the presence of CsA metabolites may contribute to the CsA-induced nephrotoxicity. The vascular nephrotoxicity induced by CsA, an increased deposition of platelets in the renal arterioles, was mimicked by cocultures of endothelial cell monolayers and platelets. CsA increased the aggregability and adherence of platelets to the endothelial cell monolayers, whereas CsH had no effect.
Mol Toxicol
PMID:Cyclosporine A nephrotoxicity studied by the combined application of kidney cell lines, hepatocytes, and endothelial-platelet cocultures. 350 90

Different methods of measuring cytotoxicity have been investigated in order to establish a test system for assessment of the toxicity of water samples. Ideally this should be highly sensitive and rapid to perform. Four variations of the neutral red uptake test have been compared with the MTT test and ATP determination. Chinese hamster V79/4 cells were used as the test system with 10 model toxins. All tests gave essentially similar results, with linear regression analysis producing correlation coefficients in excess of .93. No single test was most sensitive to all 10 compounds. The neutral red uptake and MTT tests could be performed in a single working day (approximately 8 h) if test compound and cells were plated out simultaneously. These tests are preferable to ATP determination, which is a complex and lengthy procedure, requiring expensive reagents.
Mol Toxicol
PMID:Development of cytotoxicity tests for assessment of the toxicity of water samples taken from the environment. 350 94

A major improvement in dental restoratives is possible through the development of biomaterials that do not shrink upon polymerization, hence, avoid leakage and subsequent breakdown. Polymers containing spiroorthocarbonates (SOCs) show promise in this respect, but their toxicology in copolymerized materials has not been explored. In this study, the in vitro toxicology of these materials in homopolymer form and in two trial non-shrinking epoxy co-polymers was evaluated for cytotoxicity and mutagenicity. Cytotoxicity was determined by the MTT test to measure the lethality effect on mouse L929 cells. Mutagenicity was evaluated using the Ames-Salmonella Test. For comparison, commercial composite and adhesive materials as well as several other materials of current interest in dentistry were also evaluated. Epoxy resin samples containing 5% of either T/T SOC or Dp SOC reduced the cytotoxicity (TC50) from approximately 400 to 800 micrograms/200 microliters. The epoxy-spiro copolymers had more favorable TC50 values than the commercial product Super-Bond. They showed TC50 values on the order of 35% greater than Super-Bond and 45% less than Scotchbond 2, the latter two being materials currently used in the clinic. These two comparatives demonstrated dose response curves with lower doses at maximum cell kill values than the spiro materials. The epoxy formulations all showed weak mutagenesis, but this is attributed to the epoxy formulation and not the SOCs. Although considerable toxicology is yet be conducted, these in vitro results suggest that biocompatible copolymer formulations for spiroorthocarbonates are a developmental reality.
Res Commun Mol Pathol Pharmacol 1994 Dec
PMID:The effect of spiroorthocarbonate volume modifier co-monomers on the in vitro toxicology of trial non-shrinking dental epoxy co-polymers. 771 11

Immunosuppressive factor (ISFnp) which inhibits proliferation and viability of thymoma EL-4 cells was isolated from mouse liver. The testing procedure based on the biotransformation of MTT-tetrazolium by mitochondrial enzymes of viable cells allowed us to purify this factor as individual peak of protein, that allowed to obtain polyclonal rabbit antibodies to this factor. By the methods of double immunodiffusion, gel-filtration and SDS-PAGE with subsequent immunoblotting we shown that this factor specifically localized in liver and consists two subunits of 40 and 42 kDa which form dimers with apparent M(r) about 70-80 kDa. This factor induced olygonucleosomal DNA cleavage in EL-4 cells in vitro similar to dexamethasone-induced DNA-degradation in thymocytes. This cleavage preceded to lysis of EL-4 cells assessed by 51Cr-release, that strongly suggested an involvement of apoptosis in cell death mechanism. ISFnp strongly inhibited blast-transformation and proliferation in MLC-responses to mutant MHC class 2 molecule. This effect was not due to deletion of allo-reactive clones, because removing of this factor from MLC cultures treated with one for 4 days resulted in blast-transformation without any reduction of the number of viable cells as well as their capacity for secondary responses to the same antigen as compared with control cultures.
Mol Biol (Mosk)
PMID:[Isolation, study of activity, and characteristics of an immunosuppressive liver factor causing apoptosis of EL-4 thymoma cells in vitro]. 772 57

Severe chronic asthma is associated with structural changes in the airway wall including airway smooth muscle (ASM) hyperplasia. We have used cultured ASM cells isolated from rabbit trachealis as a model with which to investigate possible mechanisms of accelerated ASM growth to mitogenic stimuli. To elucidate the role that protein kinase C (PKC)- and protein tyrosine kinase (PTK)-dependent pathways play in the control of ASM mitogenesis, we have investigated the effect of reportedly selective inhibitors of PKC (3-[1-[3-(amidinothio)propyl]-3-indolyl]-4-(1-methyl-3-indolyl)-1H - pyrrole-2,5-dionemethanesulfonate [Ro31-8220] and 3-[1-(aminopropyl)indolyl]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dione acetate [Ro31-7549]) and PTK (alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide [ST638]) on partially purified PKC, fetal calf serum (FCS)-stimulated protein phosphotyrosine content and on FCS-induced proliferation. Anion-exchange chromatography of lysed ASM cells resolved two peaks of Ca(2+)-activated, phospholipid-dependent PKC activity and one peak of Ca(2+)- and phospholipid-independent PKC activity. The selective PKC inhibitors, Ro31-8220 and Ro31-7549, abolished the main peak of PKC activity and the Ca(2+)- and phospholipid-independent peak that co-eluted with the main peak. The inhibition was dependent on the concentration of ATP in the reaction cocktail (IC50: 10 microM ATP: Ro31-8220 0.026 microM, Ro31-7549 0.073 microM; 100 microM ATP: Ro31-8220 0.065 microM, Ro31-7549 0.271 microM), consistent with these compounds inhibiting PKC at the ATP-binding site. Ro31-8220 was more potent (2- to 3-fold) than Ro31-7549. Concentrations of each inhibitor that produced maximal inhibition of the pooled kinase activity also abolished the second peak of Ca(2+)-dependent activity. The PTK inhibitor, ST638, had no effect on the kinase activity associated with any of the Ca(2+)-dependent or -independent peaks that eluted from the column. ST638, however, maximally inhibited FCS-stimulated PTK activity (IC50 25 microM). FCS-stimulated PTK was also inhibited by Ro31-8220 (IC50 0.15 microM), but only by 60%, revealing an Ro31-8220-insensitive component to the response. The ability of each protein kinase inhibitor to inhibit proliferation was also studied using four independent indices of ASM cell growth and division: 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) dye conversion, Coomassie blue protein determination, hemacytometer cell counts, and DNA synthesis. Ro31-8220 and Ro31-7549 produced concentration-dependent inhibition of FCS-stimulated proliferation of growth-arrested ASM cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Am J Respir Cell Mol Biol 1995 Feb
PMID:Inhibition of fetal calf serum-stimulated proliferation of rabbit cultured tracheal smooth muscle cells by selective inhibitors of protein kinase C and protein tyrosine kinase. 786 14

Influenza B virus has been aetiologically linked to Reye Syndrome (RS), but the mechanism(s) by which this pathogen could disrupt liver metabolism and produce the hepatic mitochondrial injury characteristic of the syndrome are unknown. In this study, two mechanisms by which infection of hepatocytes with influenza B virus could disrupt cellular metabolism were investigated. (1) virus-induced increase in pro-oxidant iron with subsequent iron-induced lipid peroxidation (LP) and (2) increased membrane permeability. Hep G2 cells, a well-differentiated continuous human liver cell line derived from a hepatoblastoma, were infected with allantoic-fluid derived influenza B Lee/40 virus (AFDV) at a multiplicity of infection of 10 for 24 h; productive infection was confirmed by both haemagglutination of chick erythrocytes and by plaque assay. Infection of Hep G2 cells preloaded with 59Fe-transferrin resulted in increased release of 59Fe (153 +/- 17% of controls, P < 0.03). However, the iron released did not result in increased LP (assessed by thiobarituric acid reactive substances; TBARS). To confirm that this lack of of increase in TBARS was not due to insensitivity of the cell line to pro-oxidant iron, cells were exposed to 15 microM iron ascorbate for 60 min. Production of TBARS was increased (122 +/- 4% of controls, P < 0.0003). Release of 51Cr from infected cells was also increased (128 +/- 12% of controls, P < 0.05); thus the infected cells exhibited a generalized increase in membrane permeability. However, infection did not depress mitochondrial respiration (as assessed by the formation of MTT-f3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan. To determine if the combination of viral infection and soluble products of activated macrophages would affect mitochondrial respiration, infected hepatocytes were exposed to the supernatant fluid from THP-1 cells which had previously been incubated with lipopolysaccharide at 100 ng ml-1 for 18 h. This supernate did depress the formation of MTT-f (81 +/- 5% of controls, P < 0.03). We conclude that influenza B virus does productively infect Hep G2 cells, and does increase hepatocyte membrane permeability. This effect does not impair mitochondrial respiration directly. However, infection does act in concert with soluble products of activated macrophages to depress hepatic mitochondrial respiration. Whether this interaction can be explained by virus-induced permeability changes and/or other effects of infection deserves further investigation.
Mol Cell Probes 1994 Oct
PMID:Activated THP-1 cells depress mitochondrial respiration in Hep G2 cells infected with influenza B virus. 787 29

In order to address the question whether the virus or immune reactions to the heart induce cardiac damage in the murine model of coxsackievirus B3 induced myocarditis, calcium-resistant cardiac myocytes of a myocarditis susceptible strain of mice were isolated and exposed to myocarditic coxsackievirus B3. The tetrazolium salt MTT was used to visualize the effect of the virus upon cell viability by inverse light microscopy and by an EA test. Coxsackievirus B3 infected isolated adult cardiac myocytes as well as infected cultured cardiac fibroblasts were examined. In vitro the virus killed the myocytes within 16 h of infection whereas the fibroblasts survived the infection. Since coxsackievirus B3 is able to kill cardiac myocytes by itself, immunosuppressive treatment of acute coxsackievirus B3 induced myocarditis may be harmful by eliminating host immune defence mechanisms and, therefore, may lead to an enhanced viral spread and virus induced myocyte necrosis in the heart.
J Mol Cell Cardiol 1994 Jul
PMID:Coxsackievirus B3 infection leads to cell death of cardiac myocytes. 796 59


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