ABSTRACT
In mammals, the final 11 beta-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450(11 beta), together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11 beta-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450(11 beta) whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH-adrenodoxin-reductase-like activity. Adrenodoxin and P-450(11 beta) behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450(11 beta). The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.
Subject(s)
Cortodoxone/metabolism , Hydrocortisone/metabolism , Mitochondria/enzymology , Saccharomyces cerevisiae/enzymology , Steroid 11-beta-Hydroxylase/metabolism , Adrenodoxin/genetics , Adrenodoxin/metabolism , Androstenedione/metabolism , Base Sequence , Cloning, Molecular , Corticosterone/metabolism , DNA Primers , Desoxycorticosterone/metabolism , Electron Transport Complex IV/metabolism , Escherichia coli/genetics , Ferredoxin-NADP Reductase/metabolism , Gene Expression , Hydroxylation , Molecular Sequence Data , NADP/metabolism , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Steroid 11-beta-Hydroxylase/geneticsABSTRACT
In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient 'recombination in vivo' technique. Two sets of vectors were developed. The first set, called 'expression vectors', contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a 'transfer vector' which is compatible with the second set of E. coli-yeast shuttle vectors. This set of 'recombination vectors' contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 microns replicon. Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis.
Subject(s)
Cloning, Molecular/methods , Genetic Vectors/genetics , Plasmids/genetics , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Centromere , DNA, Single-Stranded , Drug Resistance, Microbial/genetics , Escherichia coli/genetics , Genetic Markers , Molecular Sequence DataABSTRACT
A high-pressure liquid chromatographic method for the determination of diuron and its metabolites in human urine and blood is presented. The synthesis of different metabolites and of a suitable internal standard is described and the structure of the compounds is determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The method is applied to an overdose case.
Subject(s)
Diuron/metabolism , Adult , Chromatography, High Pressure Liquid , Diuron/poisoning , Female , Gas Chromatography-Mass Spectrometry , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , Reference Standards , Spectrophotometry, InfraredABSTRACT
A sensitive and selective method for the determination of cotinine in plasma and urine is presented. Quantitation is effected by capillary gas chromatography-mass spectrometry after liquid--liquid extraction of 0.25-1 ml of biological specimens with a trideuterated cotinine internal standard. The procedure is linear and has acceptable precision over the range of concentrations encountered in pharmacokinetic studies of nicotine or cotinine. The suitability of the assay is shown by a number of plasma concentration--time curves after a single oral or intravenous administration of cotinine to a human volunteer and after multiple-dose intravenous administration of nicotine.
Subject(s)
Cotinine/analysis , Pyrrolidinones/analysis , Body Fluids/analysis , Cotinine/blood , Cotinine/urine , Gas Chromatography-Mass Spectrometry , Humans , Smoking , Time FactorsABSTRACT
The increase in the concentration of lactic and succinic acid during storage was measured in an attempt to determine the age of eggs. The increase in succinic acid alone is not sufficient to indicate egg age. Although lactic acid concentrations increase more rapidly, the levels are still very low, the increase is not linear, and concentrations in fresh eggs vary widely. On the other hand, refractive indices showed a nearly linear correlation with age and very little variation between eggs. These measurements are reproducible and easy to perform and, with further study, should provide an alternative to the AOAC method.
