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Methods in Molecular Biology (Clifton,... 2018Microsomes are vesicles derived from the endoplasmic reticulum (ER) when cells are broken down in the lab. These microsomes are a valuable tool to study a variety of ER...
Microsomes are vesicles derived from the endoplasmic reticulum (ER) when cells are broken down in the lab. These microsomes are a valuable tool to study a variety of ER functions such as protein and lipid synthesis in vitro.Here we describe a protocol to isolate ER-derived microsomes Arabidopsis thaliana seedlings and exemplify the use of these purified microsomes in enzyme assays with the auxin precursors tryptophan (Trp) or indole-3-pyruvic acid (IPyA) to quantify auxin synthetic capacity in microsomal and cytosolic fractions.
Topics: Arabidopsis; Arabidopsis Proteins; Cell Fractionation; Endoplasmic Reticulum; Microsomes
PubMed: 29043673
DOI: 10.1007/978-1-4939-7389-7_9 -
Drug Metabolism and Disposition: the... Nov 1999Twenty-nine drugs of disparate structures and physicochemical properties were used in an examination of the capability of human liver microsomal lability data ("in vitro...
Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes.
Twenty-nine drugs of disparate structures and physicochemical properties were used in an examination of the capability of human liver microsomal lability data ("in vitro T(1/2)" approach) to be useful in the prediction of human clearance. Additionally, the potential importance of nonspecific binding to microsomes in the in vitro incubation milieu for the accurate prediction of human clearance was investigated. The compounds examined demonstrated a wide range of microsomal metabolic labilities with scaled intrinsic clearance values ranging from less than 0.5 ml/min/kg to 189 ml/min/kg. Microsomal binding was determined at microsomal protein concentrations used in the lability incubations. For the 29 compounds studied, unbound fractions in microsomes ranged from 0.11 to 1.0. Generally, basic compounds demonstrated the greatest extent of binding and neutral and acidic compounds the least extent of binding. In the projection of human clearance values, basic and neutral compounds were well predicted when all binding considerations (blood and microsome) were disregarded, however, including both binding considerations also yielded reasonable predictions. Including only blood binding yielded very poor projections of human clearance for these two types of compounds. However, for acidic compounds, disregarding all binding considerations yielded poor predictions of human clearance. It was generally most difficult to accurately predict clearance for this class of compounds; however the accuracy was best when all binding considerations were included. Overall, inclusion of both blood and microsome binding values gave the best agreement between in vivo clearance values and clearance values projected from in vitro intrinsic clearance data.
Topics: Atmospheric Pressure; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Half-Life; Humans; Mass Spectrometry; Microsomes, Liver; Pharmacokinetics
PubMed: 10534321
DOI: No ID Found -
Environmental and Molecular Mutagenesis Jul 2018The Salmonella/microsome assay (Ames test) is the most widely used mutagenicity test for the evaluation of pure chemicals and environmental samples. There are several...
The Salmonella/microsome assay (Ames test) is the most widely used mutagenicity test for the evaluation of pure chemicals and environmental samples. There are several versions of protocols available in the literature, including those that reduce the amount of sample needed for testing with liquid and agar media. The microsuspension version of the Salmonella/microsome assay is more sensitive than the standard protocol. It is performed using 5-times concentrated bacteria and less sample and S9 mixture, but still uses conventional Petri dishes (90 × 15 mm). It has been extensively used for environmental sample testing, including in effect-directed analysis (EDA). The objective of this study was to miniaturize the microsuspension assay using 12-well microplates instead of the conventional plates. For validation of this miniaturization, thirteen mutagenic compounds were tested using three Salmonella strains that were selected based on their different spontaneous reversion frequencies (low, medium, and high). The conditions of the miniaturized procedure were made as similar as possible to the microsuspension protocol, using the same testing design, metabolic activation, and data interpretation, and the tests were conducted in parallel. The miniaturized plate assay (MPA) and microsuspension procedures provided similar sensitivities although MPA is less laborious and require less sample and reagents, thereby reducing overall costs. We conclude that the MPA is a promising tool and can be particularly suitable for environmental studies such as EDA or monitoring programs. Environ. Mol. Mutagen. 59:488-501, 2018. © 2018 Wiley Periodicals, Inc.
Topics: Environmental Pollutants; Equipment Design; Microsomes; Miniaturization; Mutagenicity Tests; Mutagens; Salmonella typhimurium; Sample Size
PubMed: 29668047
DOI: 10.1002/em.22195 -
Current Drug Metabolism 2018Over the past two decades, saccharolactone has been routinely used in in vitro microsomal incubations, and sometimes in incubations with recombinant Uridine... (Review)
Review
BACKGROUND
Over the past two decades, saccharolactone has been routinely used in in vitro microsomal incubations, and sometimes in incubations with recombinant Uridine diphosphoglucuronosyl transferases (UGT) while investigating glucuronidation reactions. The addition of saccharolactone is aimed at completely inhibiting β-glucuronidases that may be present in the microsomes, in the anticipation of accurate identification and quantification of the formed glucuronide metabolites. Recent research has demonstrated that saccharolatone may not serve the intended objective, and may even lead to inhibition of certain UGTs.
OBJECTIVE
This report investigates the historic evidence in the practice of saccharolactone addition in relation to β- glucuronidases and UGTs. The chemical nature and inhibition potency of saccharolactone are explored in an attempt to unravel the myth in its application. Finally, the collective evidence is discussed in an effort to provide guidance to drug metabolism scientists on the utilization of saccharolactone.
CONCLUSION
In-depth evaluation of the experimental evidence in the literature points toward a weak rationale for general in vitro application of saccharolactone. Furthermore, inhibition of recombinant or microsomal UGTs by saccharolactone may be model dependent. Overall, the integrated data suggests that saccharolactone should not be utilized in in vitro microsomal incubations with the objective of inhibiting β-glucuronidases.
Topics: Glucaric Acid; Glucuronosyltransferase; Humans; Microsomes
PubMed: 29298647
DOI: 10.2174/1389200219666171229232007 -
Cold Spring Harbor Protocols Sep 2014When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough... (Review)
Review
When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.
Topics: Animals; Endoplasmic Reticulum; Humans; Microsomes; Protein Transport; Subcellular Fractions
PubMed: 25183824
DOI: 10.1101/pdb.top074567 -
Nihon Rinsho. Japanese Journal of... Nov 1999
Review
Topics: Adolescent; Autoantibodies; Autoimmune Diseases; Child; Child, Preschool; Female; Humans; Infant; Kidney; Male; Microsomes; Microsomes, Liver
PubMed: 10635886
DOI: No ID Found -
Biochemical Pharmacology Aug 1976
Topics: Animals; DNA; Dimethylnitrosamine; In Vitro Techniques; Male; Methylation; Microsomes, Liver; NADP; Nitrosamines; Proteins; Rats
PubMed: 9095
DOI: 10.1016/0006-2952(76)90203-3 -
Methods in Molecular Biology (Clifton,... 2009Proteomic profiling of subcellular compartments has many advantages over traditional proteomic approaches using whole cell lysates as it allows for detailed proteome...
Proteomic profiling of subcellular compartments has many advantages over traditional proteomic approaches using whole cell lysates as it allows for detailed proteome analysis of a specific organelle and corresponding functional characteristics. The microsome is a critical, membranous compartment involved in the synthesis, sorting, and secretion of proteins as well as other metabolic functions. This chapter will describe detailed methods for the isolation of microsomal organelles including the ER, Golgi, and prechylomicron transport vesicle (PCTV), a recently identified vesicular system involved in intestinal lipoprotein assembly and secretion. Particular focus is given to the isolation of microsomes from primary hepatocytes and enterocytes freshly isolated from rodent liver and intestinal tissue, and their proteomic profiling using a combination of two-dimensional gel electrophoresis and mass spectrometry.
Topics: Animals; Cell Fractionation; Cricetinae; Electrophoresis, Gel, Two-Dimensional; Enterocytes; Hepatocytes; Humans; Isoelectric Focusing; Male; Mass Spectrometry; Mesocricetus; Microsomes; Proteins; Proteome; Proteomics
PubMed: 19381589
DOI: 10.1007/978-1-59745-281-6_17 -
Carcinogenesis Oct 1992Microsome-mediated metabolism of [3H]4-aminobiphenyl (ABP) and binding of [3H]N-hydroxy-4-aminobiphenyl (N-OH-ABP) to nucleic acids by dog hepatic and bladder microsomes...
Microsome-mediated metabolism of [3H]4-aminobiphenyl (ABP) and binding of [3H]N-hydroxy-4-aminobiphenyl (N-OH-ABP) to nucleic acids by dog hepatic and bladder microsomes were investigated. HPLC analysis of the ethyl acetate extracts of hepatic microsomal incubates of [3H]ABP in the presence of 4-acetylaminobiphenyl (AABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), or acetyl coenzyme A (AcCoA) as acetyl donors showed the formation of [3H]AABP, suggesting that microsomes catalyze N-acetylation of ABP involving transacetylation. Dog hepatic microsomes also catalyzed the binding of [3H]N-OH-ABP to RNA in the presence of AABP, N-OH-AABP or AcCoA, and the binding was blocked by paraoxon, an inhibitor of microsomal deacetylases. Binding of [3H]N-OH-ABP to DNA was catalyzed also by dog hepatic microsomes, and the extent of binding was 266, 156 and 135 pmol/mg DNA for AABP, N-OH-AABP and AcCoA as acetyl donors respectively. HPLC analyses of the DNA hydrolysates showed that the major adduct formed was N-(deoxyguanosine-8-yl)-4-aminobiphenyl, based on mobility of the adduct in comparison with the synthetic standard. The acetyl adduct N-(deoxyguanosine-8-yl)-4-acetylaminobiphenyl was not detected in the DNA hydrolysates. Adduct profiles obtained from 32P-postlabeling of DNA samples from the microsome-mediated binding of [3H]N-OH-ABP showed similarities to the profile obtained previously from the chemical interaction of N-OH-ABP with DNA under acidic conditions, suggesting that the microsome-mediated binding of N-OH-ABP may proceed via formation of aryl nitrenium ions as the ultimate electrophilic species. Microsomes from dog bladder also catalyzed the binding of [3H]N-OH-ABP to RNA and DNA in the presence of AABP, N-OH-AABP or AcCoA as acetyl donors, though the levels of binding were less than those observed with hepatic microsomes. The prevalence of these acetyl transferases in the target organs for ABP and AABP carcinogenesis raises the possibility that metabolic activation of the proximate metabolite N-OH-ABP could occur directly in these tissues and these reactions could play a critical role in the initiation of cancers.
Topics: Acetylation; Aminobiphenyl Compounds; Animals; Carcinogens; DNA; Dogs; Liver; Microsomes; Microsomes, Liver; Nucleic Acids; RNA, Transfer; Urinary Bladder
PubMed: 1423829
DOI: 10.1093/carcin/13.10.1705 -
Mutation Research Sep 1989Butylated hydroxyanisole (BHA) was found to induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence of Aroclor-induced rat-liver S9. The...
Butylated hydroxyanisole (BHA) was found to induce chromosome aberrations in Chinese hamster ovary (CHO) cells in the presence of Aroclor-induced rat-liver S9. The effects were more marked when washed microsomes were employed and chromosome damage was considerably reduced in the presence of catalase, suggesting that hydrogen peroxide was involved. Stimulation of H2O2 production by BHA in S9 or microsome incubation mixtures was demonstrated using the catalase-mediated production of formaldehyde from methanol. One of the major microsomal metabolites of BHA, tert.-butyl hydroquinone (t-BHQ), which autoxidises in solution producing H2O2 also induced extensive catalase-sensitive chromosome damage in the absence of metabolic activation. These observations suggest that extracellular generation of reactive oxygen species may be implicated in the mechanism of BHA clastogenicity in vitro. However, chromosome damage was not completely abolished by catalase and the end product of t-BHQ oxidation, tert.-butyl quinone, was also weakly clastogenic, suggesting that intracellular effects of quinone metabolites may also be involved in the clastogenicity of BHA.
Topics: Animals; Biotransformation; Butylated Hydroxyanisole; Catalase; Cell Line; Cell Survival; Chromosome Aberrations; Cricetinae; Cricetulus; Hydrogen Peroxide; Hydroquinones; Lethal Dose 50; Male; Microsomes; Mutagenicity Tests; Rats; Rats, Inbred Strains
PubMed: 2770757
DOI: 10.1016/0027-5107(89)90203-0