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Microsomes
Kinds of Microsomes Selected AbstractsGenotoxicity of nitrosulfonic acids, nitrobenzoic acids, and nitrobenzylalcohols, pollutants commonly found in ground water near ammunition facilitiesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2006Tamara Grummt Abstract 2-Amino-4,6-dinitrobenzoic acid (2-A-4,6-DNBA), 4-amino-2,6-dinitrobenzoic acid (4-A-2,6-DNBA), 2,4,6-trinitrobenzoic acid (2,4,6-TNBA), 2-amino-4, 6-dinitrobenzylalcohol (2-A-4,6-DNBAlc), 4-amino-2,6-dinitrobenzylalcohol (4-A-2,6-DNBAlc), 2,4-dinitrotoluol-5-sulfonic acid (2,4-DNT-5-SA), 2,4-dinitrotoluol-3-sulfonic acid (2,4-DNT-3-SA), and 2, 4-dinitrobenzoic acid (2,4-DNBA) are derivatives of nitro-explosives that have been detected in groundwater close to munitions facilities. In the present study, the genotoxicity of these compounds was evaluated in Salmonella/microsome assays (in strains TA100 and TA98, with and without S9 and in TA98NR without S9), in chromosomal aberration (CA) tests with Chinese hamster fibroblasts (V79), and in micronucleus (MN) assays with human hepatoma (HepG2) cells. All compounds except the sulfonic acids were positive in the bacterial mutagenicity tests, with 2,4,6-TNBA producing the strongest response (8023 revertants/,mol in TA98 without S9 activation). 2-A-4,6-DNBA was a direct acting mutagen in TA98, but negative in TA100. The other positive compounds were ,1,3 orders of magnitude less mutagenic than 2,4,6-TNBA in TA98 and in TA100; relatively strong effects (,50,400 revertants/,mol) were produced by the benzylacohols in the two indicator strains. With the exception of 2,4-DNBA, the mutagenic responses were lower in the nitroreductase-deficient strain TA98NR than in the parental strain. 2,4-DNBA produced a marginally positive response in the V79-cell CA assay; the other substances were devoid of activity. Only the benzoic acids were tested for MN induction in HepG2 cells, and all produced positive responses. As in the bacterial assays, the strongest effect was seen with 2,4,6-TNBA (significant induction at ,1.9 ,M). 4-A-2,6-DNBA was positive at 432 ,M; the weakest effect was observed with 2,4,-DNBA (positive at ,920 ,M). The differences in the sensitivity of the indicator cells to these agents can be explained by differences in the activities of enzymes involved in the activation of the compounds. The strong responses produced by some of the compounds in the human-derived cells suggest that environmental exposure to these breakdown products of nitro-explosives may pose a cancer risk in man. Environ. Mol. Mutagen., 2006. © 2005 Wiley-Liss, Inc. [source] Application of toxicity identification evaluation to sediment in a highly contaminated water reservoir in southeastern BrazilENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2006Rosalina P. A. Araújo Abstract Rasgão Reservoir, located close to the Metropolitan region of São Paulo, Brazil, has been analyzed previously, and its sediment was found to be highly toxic, with high levels of metals and polycyclic aromatic hydrocarbons and a complete absence of benthic life. Polychlorinated biphenyls also were present, as was mutagenic activity, detected with the Salmonella/microsome assay. Because of the extremely complex mixture of contaminants in these sediments, a toxicity identification evaluation was performed on the pore water and elutriate using Ceriodaphnia dubia and Vibrio fischeri. Toxicity characterization, identification, and confirmation procedures were performed in one representative sample of the reservoir, and the results indicated that ammonia was the main cause of the toxicity detected with C. dubia in both sediment pore water and elutriate. Chemical analysis corroborated this observation by revealing un-ionized ammonia concentrations as high as 5.14 mg/L in pore water and 2.06 mg/L in elutriate. These high ammonia levels masked possible toxicity caused by other classes of compounds. The toxicity detected with V. fischeri decreased with the time of sample storage and was related to the organic fraction of the pore water and the elutriate, in which compounds such as benzothiazole and nonylphenol were detected. [source] The use of toxicity bioassays to monitor the recovery of oiled wetland sedimentsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2003Danica C. Mueller Abstract Six toxicity assays were compared to determine their efficacy in assessing toxicity dynamics during a wetland bioremediation study. The toxicity bioassays used were the Microtox® 100% elutriate test, Microtox Solid Phase Test (SPT), amphipod assay, P450 reporter gene system, Toxi-ChromoPadÔ test and a Salmonella/microsome assay. Oiled sediments were analyzed for toxicity in the petroleum biostimulation experiment conducted along the San Jacinto River, near Houston (TX, USA). The bioassays were evaluated for their ability to measure acute toxicity, chronic toxicity, and the mutagenic potential of amended oiled plots as compared to oiled and unoiled control plots. Amendments were diammonium phosphate alone or in combination with potassium nitrate, which served as an alternate electron acceptor. With exception of the Toxi-ChromoPad and Salmonella tests, the bioassays exhibited a significant increase in toxicity after oil application. Microtox bioassays detected significant sediment toxicity up to 29 d after oil and amendment application. The Microtox solid phase test results correlated strongly with gas chromatographymass spectrometry analyses of total target saturate and aromatic hydrocarbons. The amphipod assay detected initial toxicity with a decline to day 70, followed by a significant increase in toxicity on day 140 in plots receiving nutrient amendments, which may be in response to excessive nutrient application. Low levels of enzyme induction were observed with the P450 reporter gene system assay in all oiled sediments throughout the study, suggesting low but persistent levels of polycyclic aromatic hydrocarbons. Of the six tests, the two Microtox tests and the amphipod test showed the most potential in evaluating petroleum toxicity in wetland sediments. [source] Mutagenic and antimutagenic potential of the medicinal plants M. laevigata and C. xanthocarpaPHYTOTHERAPY RESEARCH, Issue 3 2003J. B. F. Fernandes Abstract Aqueous extracts of medicinal plants (Mikania laevigata and Campomanesia xanthocarpa) were screened for the presence of mutagenic activity in the Salmonella/microsome assay. The extracts of Campomanesia xanthocarpa showed frameshift (TA97a strain) signs of mutagenic activity without exogenous metabolism (S9 fraction). The infusions of Mikania laevigata, negative for mutagenic activity, showed high percentages of inhibition of mutagenesis induced by mutagens 2AF (2-amino,uorene), in the presence of exogenous metabolism (S9 fraction), for frameshift (TA98) and base pair substitution (TA100) lesions. In addition, these inhibitions were observed against mutagen SAZ (sodium azide) in assays with the TA100 strain, without exogenous metabolism (S9 fraction). A synergistic effect was also observed in frameshift mutagenic events, with direct action in the presence of 4NQO (4-oxide-1-nitroquinoline) and a tendency to a low percentage of action enhancement, in the presence of the 2AF mutagen. The variable responses observed in the extract assays show the potentials for interaction of the different active principles in genetic material. Copyright © 2003 John Wiley & Sons, Ltd. [source] Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite In Vivo, and acute toxicity in miceJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2009Silvia Romão Abstract The mesoionic derivative 4-phenyl-5-[4-nitrocinnamoyl]-1,3,4-thiadiazolyl-2-phenylamine chloride (MI-D) has antitumoral and anti-inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI-D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI-D in high-performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI-D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI-D showed the presence of the metabolite product. The kinetic parameters Km (19.5 ± 4.5 ,M) and Vmax [1.5 ± 0.4 units of fluorescence/(100 ,g of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI-D and indicating that the reaction follows Michaelis-Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD-50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD-50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI-D as a future chemotherapeutic drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:394,405, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20303 [source] Prediction of human pharmacokinetics , renal metabolic and excretion clearanceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2007Urban Fagerholm The kidneys have the capability to both excrete and metabolise drugs. An understanding of mechanisms that determine these processes is required for the prediction of pharmacokinetics, exposures, doses and interactions of candidate drugs. This is particularly important for compounds predicted to have low or negligible non-renal clearance (CL). Clinically significant interactions in drug transport occur mostly in the kidneys. The main objective was to evaluate methods for prediction of excretion and metabolic renal CL (CLR) in humans. CLR is difficult to predict because of the involvement of bi-directional passive and active tubular transport, differences in uptake capacity, pH and residence time on luminal and blood sides of tubular cells, and limited knowledge about regional tubular residence time, permeability (Pe) and metabolic capacity. Allometry provides poor predictions of excretion CLR because of species differences in unbound fraction, urine pH and active transport. The correlation between fraction excreted unchanged in urine (fe) in humans and animals is also poor, except for compounds with high passive Pe (extensive/complete tubular reabsorption; zero/negligible fe) and/or high non-renal CL. Physiologically based in-vitro/in-vivo methods could potentially be useful for predicting CLR. Filtration could easily be predicted. Prediction of tubular secretion CL requires an in-vitro transport model and establishment of an in-vitro/in-vivo relationship, and does not appear to have been attempted. The relationship between passive Pe and tubular fraction reabsorbed (freabs) for compounds with and without apparent secretion has recently been established and useful equations and limits for prediction were developed. The suggestion that reabsorption has a lipophilicity cut-off does not seem to hold. Instead, compounds with passive Pe that is less than or equal to that of atenolol are expected to have negligible passive freabs. Compounds with passive Pe that is equal to or higher than that of carbamazepine are expected to have complete freabs. For compounds with intermediate Pe the relationship is irregular and freabs is difficult to predict. Tubular cells are comparably impermeable (for passive diffusion), and show regional differences in enzymatic and transporter activities. This limits the usefulness of microsome data and makes microsome-based predictions of metabolic CLR questionable. Renal concentrations and activities of CYP450s are comparably low, suggesting that CYP450 substrates have negligible metabolic CLR. The metabolic CLR of high-Pe UDP-glucuronyltransferase substrates could contribute to the total CL. [source] Prediction of human pharmacokinetics , improving microsome-based predictions of hepatic metabolic clearanceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2007Urban Fagerholm Physiologically based methods generally perform poorly in predicting in-vivo hepatic CL (CLH) from intrinsic clearance (CLint) in microsomes in-vitro and unbound fraction in blood (fu,bl). Various strategies to improve the predictability have been developed, and inclusion of an empirical scaling factor (SF) seems to give the best results. This investigation was undertaken to evaluate this methodology and to find ways to improve it further. The work was based on a diverse data set taken from Ito and Houston (2005). Another objective was to evaluate whether rationalization of CLH predictions can be made by replacing blood/plasma-concentration ratio (Cbl/Cpl) measurements with SFs. There were apparently no or weak correlations between prediction errors and lipophilicity, permeability (compounds with low permeability missing in the data set) and main metabolizing CYP450s. The use of CLint class (high/low) and drug class (acid/base/neutral) SFs (the CD-SF method) gives improved and reasonable predictions: 1.3-fold median error (an accurate prediction has a 1-fold error), 76% within 2-fold-error, and a median absolute rank ordering error of 2 for CLH (n = 29). This approach is better than the method with a single SF. Mean (P < 0.05) and median errors, fraction within certain error ranges, higher percentage with most accurate predictions, and ranking were all better, and 76% of predictions were more accurate with this new method. Results are particularly good for bases, which generally have higher CLH and the potential to be incorrectly selected/rejected as candidate drugs. Reasonable predictions of fu,bl can be made from plasma fu (fu,pl) and empirical blood cell binding SFs (B-SFs; 1 for low fu,pl acids; 0.62 for other substances). Mean and median fu,bl prediction errors are negligible. The use of the CD-SF method with predicted fu,bl (the BCD-SF method) also gives improved and reasonable results (1.4-fold median error; 66% within 2-fold-error; median absolute rank ordering error = 1). This new empirical approach seems sufficiently good for use during the early screening; it gives reasonable estimates of CLH and good ranking, which allows replacement of Cbl/Cpl measurements by a simple equation. [source] Toxico-kinetics, recovery efficiency and microsomal changes following administration of deltamethrin to black Bengal goatsPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 3 2001Sanis Juliet Abstract A study of the toxico-kinetics, recovery percentage from different substrates, cytotoxicity and role of cytochrome P450 and b5 of liver microsome in the metabolism of deltamethrin were carried out in female black Bengal goat. The ALD50 value of deltamethrin in goat by intravenous route lies between 0.2 and 0.6,mg,kg,1. Intravenous disposition kinetics using a dose of 0.2,mg,kg,1 showed that the maximum blood concentration of deltamethrin was recorded at 0.5,min, followed by rapid decline, and a minimum concentration was detected at 6,min after administration. The following values were obtained,:,Vdarea 0.148 (±,0.02) litre,kg,1; t1/2 (,) 0.22 (±,0.02),min; t1/2 (,) 2.17 (±,0.37),min; Kel 1.05 (±,0.24) min,1; AUC 4.30(±,0.45),µg min,ml,1; ClB 0.05 (±,0.006) litre,kg,1 min,1; T,B 1.93 (±,0.58); fc 0.40(±,0.05). After 10,min, liver retained the maximum residue, and heart, adrenal gland, kidney, spleen, fat and brain also held the insecticide; liver, fat, heart and spleen retained residue after 30,min, and bone, liver and fat retained residue after 60,min of intravenous administration. Oral absorption of deltamethrin was poor and inconsistent, and approximately 65% of administered dose was recovered from faeces and gastrointestinal contents. The excretion of deltamethrin through urine was meagre, and only 0.01 and 0.013% of the administered dose was recovered after 3 and 5 days of oral administration respectively. All the tissues retained the residue after 3 days; while fat, rumen, reticulum, omasum, abomasum, large and small intestine and bone retained the residue after 5 days of oral administration; and the percentage recoveries were 1.73 and 0.027 respectively. Deltamethrin reduced the level of cytochrome P450 content of liver microsomal pellet of goat after 5 days of oral administration. Histopathological examination of liver, kidney, heart, spleen brain and lung sections of treated goats did not reveal any pathological changes. © 2001 Society of Chemical Industry [source] Establishment of a PF2D-MS/MS platform for rapid profiling and semiquantitative analysis of membrane protein biomarkersPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2008Hyoung-Joo Lee Abstract Current proteome profiling techniques have identified relatively few mammalian membrane proteins despite their numerous important functions. To establish a standard throughput-potential profiling platform for membrane proteins, Triton X-100-solubilized rat liver microsomal proteins were separated on a 2-D separation system (2-D liquid phase fractionation (PF2D)) in two different pH ranges (4.0,8.5 and 7.0,10.5). This system produced 182 proteins with more than two transmembrane domain (TMD), including 16,TMDs with high confidence. Comparative 2-D liquid maps with high resolution and reproducibility have been constructed for liver microsome from the phenobarbital (PB) treated rats. PF2D was also found to be useful for the semiquantification of some representative cytochrome P450 family proteins (e.g., cytochrome P450 2B2) that were induced by PB treatment compared with untreated controls. Thus, the combination of both high-detection capacity and rapid preliminary semiquantification in a PF2D platform could become a standard system for the routine analysis of membrane proteins. [source] Determination of triclosan metabolites by using in-source fragmentation from high-performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2010Jian-lin Wu Triclosan is a widely used broad-spectrum antibacterial agent that acts by specifically inhibiting enoyl,acyl carrier protein reductase. An in vitro metabolic study of triclosan was performed by using Sprague-Dawley (SD) rat liver S9 and microsome, while the invivo metabolism was investigated on SD rats. Twelve metabolites were identified by using in-source fragmentation from high-performance liquid chromatography/negative atmospheric pressure chemical ionization ion trap mass spectrometry (HPLC/APCI-ITMS) analysis. Compared to electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) that gave little fragmentation for triclosan and its metabolites, the in-source fragmentation under APCI provided intensive fragmentations for the structural identifications. The invitro metabolic rate of triclosan was quantitatively determined by using HPLC/ESI-ITMS with the monitoring of the selected triclosan molecular ion. The metabolism results indicated that glucuronidation and sulfonation were the major pathways of phase II metabolism and the hydroxylated products were the major phase I metabolites. Moreover, glucose, mercapturic acid and cysteine conjugates of triclosan were also observed in the urine samples of rats orally administrated with triclosan. Copyright © 2010 John Wiley & Sons, Ltd. [source] Evaluation of the metabolism of propranolol by linear ion trap technology in mouse, rat, dog, monkey, and human cryopreserved hepatocytesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2009Todd M. Baughman Propranolol is a widely used quality control and validation compound for liver microsome and hepatocyte metabolism studies. A multitude of literature reports describing the identification of propranolol metabolites exists today. However, no literature reports currently exist showing hepatocyte metabolism across the five species commonly used during pre-clinical drug discovery, namely mouse, rat, dog, monkey, and human. Herein, we present full metabolic profiles of propranolol in mouse, rat, dog, monkey and human hepatocytes. As expected, extensive phase I and phase II metabolism was observed across all five species and species-specific metabolites were detected in monkey and dog hepatocytes. Of particular interest was the detection of an N -hydroxylamine glucuronide metabolite in monkey and dog hepatocytes. Copyright © 2009 John Wiley & Sons, Ltd. [source] P71 Metabolism of delta-3-Carene by human cytochrom 450 enzymesCONTACT DERMATITIS, Issue 3 2004Mike Duisken Occupational exposure to monoterpenes occurs in saw mills, particle-board plants, carpentry shops and other types of wood-treating industries. The bicyclic monoterpene delta-3-Carene, one of the components of turpentine, may irritate the skin and muceous membranes and prolonged exposure may result in allergic contact dermatitis or chronic lung function impairment. The effects of low concentrations of delta-3-Carene on alveolar macrophages in vitro were examined and a dose-dependent relationship between the cell viability and the delta-3-Carene concentration was found. Little is known about the metabolism of delta-3-Carene in mammalians. In order to determine the toxic potential of this monoterpene we studied the human metabolism of delta-3-Carene in vitro. Therefore we used pooled human liver S9 and human liver microsomal cytochrome P450 enzymes. By using GC-MS analysis we found one main metabolite produced at high rates. The structure was identified by its mass spectra. The mass fragmentation indicated hydroxylation in allyl position. After synthesis of the assumed product in a four step reaction, it was characterized as delta-3-Carene-10-ol. There was a clear correlation between the concentration of the metabolite production, incubation time and enzyme concentration, respectively. Kinetic analysis showed that Km and Vmax values for the oxidation of delta-3-Carene by human liver microsomes were 0.39 ,M and 0.2 nmol/min/nmol P450. It is the first time that delta-3-Carene-10-ol is described as human metabolite of delta-3-Carene. [source] No relationship between enzyme activity and structure of nucleotide binding site in sarcoplasmic reticulum Ca2+ -ATPase from short-term stimulated rat muscleACTA PHYSIOLOGICA, Issue 4 2009T. Mishima Abstract Aim:, We examined whether structural alterations to the adenine nucleotide binding site (ANBS) within sarcoplasmic (endo) reticulum Ca2+ -ATPase (SERCA) would account for contraction-induced changes in the catalytic activity of the enzyme as assessed in vitro. Methods:, Repetitive contractions were induced in rat gastrocnemius by electrical nerve stimulation. Measurements of sarcoplasmic reticulum properties were performed on control and stimulated muscles immediately after or at 30 min after the cessation of 5-min stimulation. In order to examine the properties at the ANBS, the binding capacity of SERCA to fluorescence isothiocyanate (FITC), a competitive inhibitor at the ANBS, was analysed in microsomes. Results:, Short-term electrical stimulation evoked a 23.9% and 32.6% decrease (P < 0.05) in SERCA activity and in the FITC binding capacity, respectively, in the superficial region of the muscle. Whereas SERCA activity reverted to normal levels during 30-min recovery, a restoration of the FITC binding capacity did not occur. Conclusion:, The discordant changes between the enzyme activity and the FITC binding suggest that, at least during recovery after exercise, changes in SERCA activity may not correlate closely with structural alterations to the ANBS within the enzyme. [source] Subcellular compartmentalization of aromatase is sexually dimorphic in the adult zebra finch brainDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2007Kevin N. Rohmann Abstract The vertebrate brain is a source of estrogen (E) via the expression of aromatase (E-synthase). In the zebra finch (Taeniopygia guttata), despite documented dimorphisms in E-action, no differences are detectable in circulating E, or the neural levels of aromatase transcription, activity, or somal protein expression. Studies of aromatase expression at the light- and electron-microscope levels reveal greater numbers of fibers and presynaptic boutons in adult males relative to females. We assayed aromatase activity and content in synaptosomes and microsomes from the anterior [containing lMAN and Area X (males)] and posterior telencephalon (containing HVC and RA) of adult birds. In contrast to non-song birds and mammals, both cell fractions contain abundant aromatase measurable in terms of activity (enzyme assays) and content (Western blots) with minimal enrichment in microsomes. From brain homogenates of identical concentration, aromatase activity was higher in the synaptosomal relative to the microsomal fraction, in males relative to females, and in the posterior compared to anterior telencephalon. These effects were driven by high levels of synaptosomal aromatase in the male posterior telencephalon. These data suggest that males possess more aromatase per presynaptic bouton, or a greater number of aromatase-containing presynaptic boutons than females in the posterior telencephalon. Further, the present report reveals synaptic aromatization as a considerable source of E in the zebra finch brain, and supports the idea that telencephalic synapses in and around the adult male song production nuclei may be exposed to higher levels of E compared to the female brain. © 2006 Wiley Periodicals, Inc. J Neurobiol 67: 1,9, 2007 [source] Pharmacodynamics and pharmacokinetics of YM128, a GPIIb/IIIa antagonist prodrugDRUG DEVELOPMENT RESEARCH, Issue 3 2002Ken-ichi Suzuki Abstract We examined the biochemical properties of YM-57029 ({4-[4-(4-Carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino}acetic acid monohydrochloride trihydrate) and the pharmacodynamics and pharmacokinetics of its prodrug, YM128 (Ethyl (Z)-(4-{4-[4-(N2 -hydroxycarbamimidoyl)phenyl]-3-oxopiperazin-1-yl}piperidino)acetate), an orally-active glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist. YM-57029 strongly inhibited aggregation of human platelets induced by various agonists, with IC50 values ranging from 3.6 to 51 nM. YM-57029 specifically inhibited fibrinogen binding to purified GPIIb/IIIa about 1,000-fold more potently than Arg-Gly-Asp-Ser (RGDS). Moreover, YM-57029 effectively inhibited an Arg-Gly-Asp (RGD) peptide binding to platelets, suggesting that YM-57029 competed with the RGD sequence of ligand. YM-57029 or YM128 dose-dependently inhibited ex vivo platelet aggregation after iv bolus injection or oral administration to beagle dogs and cynomolgus monkeys. However, YM128 exerted more potent and prolonged inhibitory effects on platelet aggregation than YM-57029 after oral administration to cynomolgus monkeys. Furthermore, YM-57029 prolonged template bleeding time at a dose that inhibited ex vivo platelet aggregation during cumulative iv infusion to cynomolgus monkeys. Metabolic and pharmacokinetic studies showed that YM128 effectively converted into YM-57029 in liver microsomes from humans as well as dogs and monkeys, and that bioavailabilities of YM128 in dogs and monkeys were 32.3 and 22.2%, respectively. These results suggest that YM128, a prodrug of YM-57029, may be a valuable GPIIb/IIIa antagonist with good bioavailability in humans. Drug Dev. Res. 55:149,161, 2002. © 2002 Wiley-Liss, Inc. [source] Comparison of DNA-Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes SensorsELECTROANALYSIS, Issue 9 2009Sadagopan Krishnan Abstract Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy) as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500,nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response. [source] Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samplesELECTROPHORESIS, Issue 8 2006Minoo Afshar Abstract A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N -despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid,liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH,2.0 running buffer composed of 100,mM sodium phosphate, 19% methanol, and 0.6% highly sulfated ,-CD. For each compound, the S -enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30,min. Enantiomer levels between 25 and 1000,ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12,ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N -dealkylation and hydroxylation rates. [source] Interaction of bisphenol a with human UDP-glucuronosyltransferase 1A6 enzymeENVIRONMENTAL TOXICOLOGY, Issue 3 2008Nobumitsu Hanioka Abstract The effects of bisphenol A (BPA) on UDP-glucuronosyltransferase 1A6 (UGT1A6) activities in microsomes from human livers and yeast cells expressing human UGT1A6 (humUGT1A6) were investigated. Serotonin (5-HT) and 4-methylumbelliferone (4-MU) were used as the substrates for UGT1A6. BPA dose-dependently inhibited 5-HT and 4-MU glucuronidation activities in both enzyme sources. The IC50 values of BPA for 5-HT and 4-MU glucuronidation activities were 156 and 163 ,M for liver microsomes, and 84.6 and 80.3 ,M for yeast cell microsomes expressing humUGT1A6, respectively. The inhibitory pattern of BPA for 5-HT and 4-MU glucuronidation activities in human liver microsomes exhibited a mixture of competitive and noncompetitive components, with Ki values of 84.9 and 72.3 ,M, respectively. In yeast cell microsomes expressing humUGT1A6, 5-HT glucuronidation activities were noncompetitively inhibited by BPA (Ki value, 65.5 ,M), whereas the inhibition of 4-MU glucuronidation activities by BPA exhibited the mixed type (Ki value, 42.5 ,M). These results suggest that BPA interacts with human UGT1A6 enzyme, and that the interaction may contribute to the toxicity, such as hormone disruption and reproductive effects, of BPA. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Interaction of tributyltin with hepatic cytochrome P450 and uridine diphosphate-glucuronosyl transferase systems of fish: In vitro studiesENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2004Yolanda Morcillo Abstract Hepatic microsomes of red mullet (Mullus barbatus) and flounder (Platichthys flesus) were preincubated in the presence of a concentration range of the antifouling agent tributyltin (TBT) chloride, and the interactions of TBT with cytochrome P450 and uridine diphosphate,glucuronyl transferase systems were investigated. The enzyme systems were examined in terms of cytochrome P4501A (CYP1A)-catalyzed 7-ethoxyresorufin O -deethylase (EROD) activity and benzo[a]pyrene (BaP) metabolism and in terms of glucuronidation of testosterone and 17,-estradiol, respectively. Ethoxyresorufin O -deethylase and BaP hydroxylase (BPH) activities of both fish species were progressively inhibited by increasing concentrations of TBT, and the effects were more pronounced for EROD than for BPH (maximal inhibition at 100 ,M TBT for EROD and 250,500 ,M TBT for BPH). Hydroxylated metabolites of BaP (3-hydroxy-, 7,8-dihydrodiol, and 9,10-dihydrodiol), representing 95% of the total metabolites formed, were reduced up to 75 % in the presence of 100 to 500 ,M TBT, whereas the formation of other metabolites was less affected. This may alter BaP toxicity and carcinogenicity. Overall, the results were consistent with a specific inhibitory effect of TBT on CYP1A in the two fish species. Additionally, the conjugation of testosterone was significantly inhibited (20%) at low TBT doses (5 ,M), with no effect on the glucuronidation of estradiol. [source] Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2003Hongxia Li Abstract Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccu-mulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho -Cl substituents (CB-15 [4,4,-Cl2] and CB-77 [3,3,,4,4,-Cl4]), vicinal meta and para -H atoms (CB-52 [2,2,,5,5,-Cl4], and ,101 [2,2,,4,5,5,-Cl5]) or with both characteristics in combination with either only one ortho -Cl (CB-26 [2,3,,5-Cl3], CB-31 [2,4,,5-Cl3]) or two ortho -Cl substituents (CB-44 [2,2,,3,5,-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations ,1.0 ,M because these inhibitors became decreasingly selective at higher concentrations. At <1.0 ,M, ellipticine (CYP1A1/2 inhibitor) selectively inhibited CB-15, ,26, ,31, and ,77 metabolism, with no significant inhibition of CB-44, ,52, and ,101 metabolism. Inhibition of CB-52 and ,101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 ,M and maximized at about 100% at 10 ,M. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, ,31, and ,44 metabolism relative to CB-15, ,77, and ,52 at concentrations ,1.0 ,M. Major testosterone metabolites formed in vitro were 2,-(CYP3A), 6,- (CYP3A, CYP1A), and 16,- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory animals. Inhibition of 2,- and 6,-hydroxytestosterone formation at ellipticine and ketoconazole concentrations ,1.0,M suggested that both inhibitors were good substrates of CYP3A-like enzymes in grey seal. Chloramphenicol (model for CYP2B) is apparently not a good inhibitor of CYP1A and CYP3A activities in grey seal because the chemical did not inhibit any metabolic route of testosterone at concentrations from 0.1 to 10 ,M. Our findings demonstrated that at least CYP1A- and CYP3A-like enzymes in the liver of grey seals are capable of metabolizing PCBs with ortho - meta and/or meta - para vicinal hydrogens. A CYP2B form might also be involved, but this could not be proven by the results of our experiments. Defining the profiles of CYP enzymes that are responsible for PCB biotransformation is necessary to fully understand the bioaccumulation, toxicokinetics, and risk of PCB exposure in seals and other free-ranging marine mammals. [source] Vitamins A1 and A2 in hepatic tissue and subcellular fractions in mink feeding on fish-based diets and exposed to Aroclor 1242ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2002Anne Käkelä Abstract Two-month-old female mink were fed diets based on either Baltic herring (Clupea harengus membras) or freshwater smelt (Osmerus eperlanus) for 21 weeks. A portion of the smelt-fed mink were exposed orally to polychlorinated biphenyls (PCBs), Aroclor 1242 (1 mg/d). Retinol (vitamin A1), 3,4-didehydroretinol (vitamin A2), and their different fatty acyl esters were studied in hepatic tissue, microsomes, and cytosol by argentated reversed-phase high-performance liquid chromatography. As a result of Aroclor exposure, concentrations of the fatty acyl esters of vitamins A1 and A2 were about one-tenth and those of unesterified A2 one-fourth those of the control levels. In the fatty acyl esters, percentages of stearates (A1 -18:0 and A2 -18:0) increased at the expense of the other fatty acyl esters. The Aroclor exposure decreased concentrations of alcoholic and esterified forms of the A2 analog more than those of the corresponding A1 analog. In microsomes, Aroclor decreased the alcoholic and esterified vitamin analogs to the same extent (to 9,17%). In the cytosol compared to the control, the concentrations of the vitamin esters fell below 10%, but the alcoholic analogs remained at 30 to 40%. Despite equal dietary supply, in mink fed on Baltic herring, the hepatic levels of vitamin A1 were only about one-third of the values found in the smelt-fed mink. The organochlorines also altered hepatic lipid composition and impaired breeding and kit growth. In the kits of the females fed on Baltic herring, blood hemoglobin was decreased. [source] Biotransformation of n -hexadecane by cell suspension cultures of Cinchona robusta and Dioscorea compositaENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 12 2001Carolina Vega-Jarquin Abstract This manuscript evaluates the phytotoxicity and biotransformation of n -hexadecane as well as peroxidase activity and cytochrome P450 concentration in microsomes for cell suspension cultures of Cinchona robusta and Dioscorea composita. Phytotoxicity was evaluated based on viability and growth. Cell cultures were exposed to a 2 and 4% (v/v) dose of n -hexadecane. The biotransformation of n -hexadecane was determined based on labeled recovery in polar, nonpolar, and cell residue fractions after cell culture extraction during exponential cell growth phase and stationary phase. Differences were observed in accumulation of label during cell growth phase and stationary phase for the cells of the two plants. Differences also were observed between phases for label in polar and nonpolar fractions. Thin-layer chromatography determined labeled intermediates and some were identified. The activity of peroxidase and concentration of cytochrome P450 was lower in C. robusta than in controls and greater in D. composita than in controls. In vitro biotransformation was not successful. [source] Immunohistochemical localization of cytochrome P4501A induced by 3,3,,4,4,,5-pentachlorobiphenyl (PCB 126) in multiple organs of northern leopard frogs, Rana pipiensENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2001Yue-Wern Huang Abstract Monoclonal antibody 1 12,3(MA1 12,3)recognizesanepitopeexclusivetocytochromeP450sinsubfamily1A (CYP1A) from all vertebrates tested so far, including one amphibian species. In this study, we first tested the utility of MAb 1,12-3 for detection of presumed CYP1A proteins in hepatic microsomes of northern leopard frogs treated without or with 3,3,,4,4,,5-pentachlorobiphenyl (PCB 126). Statistical analysis showed that ethoxyresorufin- O -deethylase (EROD) activities and CYP1A equivalents in treated groups were significantly increased at doses >2.3 mg/kg compared with the control groups (p < 0.05), and the increases were maintained for at least four weeks. This result confirmed that MAb 1,12-3 can be used for detection of CYP1A in northern leopard frogs and indicated that CYP1A is the primary catalyst for EROD in this species. In a subsequent experiment, sections of organs of PCB 126-treated frogs were immunohistochemically stained with MAb 1,12-3 to identify localization of the CYP1A in different cell types. The CYP1A staining was seen prominently in hepatocytes and epithelium of nephronic duct, while capillaries close to gastric epithelium and submucosal vascular epithelium in both stomach and intestine exhibited moderate to strong staining. The CYP1A was immunodetected in coronary endothelium and the vascular endothelium of lung and gonad. In skin, mild staining was seen in epithelial cells of mucous glands and serous glands and in vascular endothelium, demonstrating induction of CYP1A in the dermal layer. [source] Anthracene photoinduced toxicity to plhc-1 cell line (Poeciliopsis lucida) and the role of lipid peroxidation in toxicityENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 11 2000Jonghoon Choi Abstract Many polycyclic aromatic hydrocarbons (PAHs) are acutely toxic to fish and other aquatic organisms in the presence of solar ultraviolet radiation (SUVR) of environmentally realistic intensities. In the present study, the photoinduced toxicity of a PAH (anthracene; ANT) to topminnow hepatoma cell line (PLHC-1) was assessed. After the toxicity was characterized, the role of lipid peroxidation in PAH photoinduced toxicity was examined by measuring lipid peroxidation products and by assessing the effect of lipid peroxidation antagonist (Trolox) treatment. In cytotoxicity tests using two assays (MTT, neutral red), the SUVR/ANT treatment elicited toxicity to PLHC-1 cells in a concentration- and SUVR (exposure duration and intensity)-dependent pattern. As found in previous organism-level studies, no significant cytotoxicity was observed in the cells exposed either to fluorescent light/ANT or to SUVR only. The SUVR/ANT treatment elicited the lipid peroxidation process and Trolox pretreatment significantly reduced SUVR/ANT-induced cell mortality. Microscopic observation showed that Trolox pretreatment relieved the SUVR/ANT-inflicted damage, such as cell shrinkage and membrane disruption. Together with a recent finding in our lab that increased production of superoxide anion and a lipid peroxidation product (malondialdehyde) was found in SUVR/ANT-treated fish microsomes, the present study suggests that reactive oxygen radical-induced lipid peroxidation is an important factor in PAH photoinduced toxicity to fish. [source] Evidence of oxidative stress in bluegill sunfish (Lepomis macrochirus) liver microsomes simultaneously exposed to solar ultraviolet radiation and anthraceneENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 7 2000Jonghoon Choi Abstract Many polycyclic aromatic hydrocarbons (PAHs) are acutely toxic to fish and other aquatic organisms in the presence of environmentally realistic intensities of solar ultraviolet radiation (SUVR). However, the biochemical mechanism of this toxicity is not well established. In this study, increased levels of both reactive oxygen species production and lipid peroxidation were hypothesized as a toxic mechanism. To test this hypothesis, the production of superoxide anion and of a lipid peroxidation product (malondialdehyde) was measured in bluegill sunfish (Lepomis machrochirus) liver microsomes. These microsomes were exposed to a representative phototoxic PAH (anthracene [ANT]) and to SUVR and normal laboratory fluorescent light (FLU) in four different combinations: FLU + no ANT, FLU + ANT, SUVR + no ANT, and SUVR + ANT. The highest mean levels of both superoxide anion and malondialdehyde production were observed in the SUVR + ANT group, and these levels were significantly different (p < 0.05) from those in all other treatment groups. We conclude that the photoinduced toxicity of ANT, and possibly of other phototoxic PAHs, manifests at least in part through lipid peroxidation after increased production of reactive oxygen species. [source] Dose-dependent stimulation of hepatic retinoic acid hydroxylation/oxidation and glucuronidation in brook trout, Salvelinus fontinalis, after exposure to 3,3,,4,4,-tetrachlorobiphenylENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2000Patrick M. Boyer Abstract Extremely low stores of vitamin A have been reported in fish and birds inhabiting regions contaminated by coplanar polychlorinated biphenyls (PCBs) and other organochlorines, suggesting many possible effects on retinoid biochemical pathways. Metabolic imbalances associated with biologically active retinoids (e.g., retinoic acid) could be associated with teratogenesis, edema, growth inhibition, reproductive impairment, immunosuppression, and susceptibility to cancer. Sexually mature brook trout were injected intraperitoneally with the coplanar PCB 3,3,,4,4,-tetrachlorobiphenyl (TCBP) and again 4 weeks later. At 8 weeks, retinoic acid metabolism was measured in liver microsomes. To our knowledge, retinoic acid conjugation by UDP-glucuronyltransferase is described here for the first time in fish. A substantial rate of glucuronidation was detected in the microsomes from control brook trout, which tended to increase over the dose range of TCBP. Glucuronidation was significantly greater in fish receiving the 10 ,g/g body weight dose level. Metabolism through the cytochrome P450 system was also dose-dependent, resulting in significantly greater production of 4-hydroxyretinoic acid at the 10 ,g/g dose level. In contrast, subsequent oxidation to 4- oxo -retinoic acid was greatest at the 1 ,g/g dose level and did not increase further at higher doses. Liver stores of dehydroretinyl palmitate/oleate were significantly decreased at the 5 and 10 ,g/g dose levels. [source] Antigen-loaded ER microsomes from APC induce potent immune responses against viral infectionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2009Vassiliki Sofra Abstract Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER-enriched microsomal membranes (microsomes) isolated from DC contain more peptide-receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide,MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide-loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide-pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide,MHC complexes armed microsomes from DC can be an important alternative to DC-based vaccines for protection from viral infection. [source] Disruption of the gene encoding 3,-hydroxysterol ,14 -reductase (Tm7sf2) in mice does not impair cholesterol biosynthesisFEBS JOURNAL, Issue 20 2008Anna M. Bennati Tm7sf2 gene encodes 3,-hydroxysterol ,14 -reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on ,14 -unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. The C-terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol ,14 -reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3,-hydroxysterol ,14 -reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3,-hydroxysterol ,14 -reductase activity were detectable in liver microsomes of Tm7sf2(,/,) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2(,/,) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3,-hydroxysterol ,14 -reductase activity determined in liver nuclei showed comparable values in wild-type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2(,/,) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell-cycle progression are downregulated in the liver of Tm7sf2(,/,) mice, whereas genes involved in xenobiotic metabolism are upregulated. [source] CYP7B1-mediated metabolism of dehydroepiandrosterone and 5,-androstane-3,,17,-diol , potential role(s) for estrogen signalingFEBS JOURNAL, Issue 8 2008Hanna Pettersson CYP7B1, a cytochrome P450 enzyme, metabolizes several steroids involved in hormonal signaling including 5,-androstane-3,,17,-diol (3,-Adiol), an estrogen receptor agonist, and dehydroepiandrosterone, a precursor for sex hormones. Previous studies have suggested that CYP7B1-dependent metabolism involving dehydroepiandrosterone or 3,-Adiol may play an important role for estrogen receptor ,-mediated signaling. However, conflicting data are reported regarding the influence of different CYP7B1-related steroids on estrogen receptor , activation. In the present study, we investigated CYP7B1-mediated conversions of dehydroepiandrosterone and 3,-Adiol in porcine microsomes and human kidney cells. As part of these studies, we compared the effects of 3,-Adiol (a CYP7B1 substrate) and 7,-hydroxy-dehydroepiandrosterone (a CYP7B1 product) on estrogen receptor , activation. The data obtained indicated that 3,-Adiol is a more efficient activator, thus lending support to the notion that CYP7B1 catalysis may decrease estrogen receptor , activation. Our data on metabolism indicate that the efficiencies of CYP7B1-mediated hydroxylations of dehydroepiandrosterone and 3,-Adiol are very similar. The enzyme catalyzed both reactions at a similar rate and the Kcat/Km values were in the same order of magnitude. A high dehydroepiandrosterone/3,-Adiol ratio in the incubation mixtures, similar to the ratio of these steroids in many human tissues, strongly suppressed CYP7B1-mediated 3,-Adiol metabolism. As the efficiencies of CYP7B1-mediated hydroxylation of dehydroepiandrosterone and 3,-Adiol are similar, we propose that varying steroid concentrations may be the most important factor determining the rate of CYP7B1-mediated metabolism of dehydroepiandrosterone or 3,-Adiol. Consequently, tissue-specific steroid concentrations may have a strong impact on CYP7B1-dependent catalysis and thus on the levels of different CYP7B1-related steroids that can influence estrogen receptor , signaling. [source] The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum , a quantitative approachFEBS JOURNAL, Issue 19 2007Andreas Weitzmann Traditionally, the canine pancreatic endoplasmic reticulum (ER) has been the workhorse for cell-free studies on protein transport into the mammalian ER. These studies have revealed multiple roles for the major ER-luminal heat shock protein (Hsp) 70, IgG heavy chain-binding protein (BiP), at least one of which also involves the second ER-luminal Hsp70, glucose-regulated protein (Grp) 170. In addition, at least one of these BiP activities depends on Hsp40. Up to now, five Hsp40s and two nucleotide exchange factors, Sil1 and Grp170, have been identified in the ER of different mammalian cell types. Here we quantified the various proteins of this chaperone network in canine pancreatic rough microsomes. We also characterized the various purified proteins with respect to their affinities for BiP and their effect on the ATPase activity of BiP. The results identify Grp170 as the major nucleotide exchange factor for BiP, and the resident ER-membrane proteins ER-resident J-domain protein 1 plus ER-resident J-domain protein 2/Sec63 as prime candidates for cochaperones of BiP in protein transport in the pancreatic ER. Thus, these data represent a comprehensive analysis of the BiP chaperone network that was recently linked to two human inherited diseases, polycystic liver disease and Marinesco,Sjögren syndrome. [source] | |||||||||||||||||||