Biomedical subjects

Elizabeth A Shephard

Publications and source records attributed to Elizabeth A Shephard.

11 recordsLinked to original sources

PubMed2006

Mutation, polymorphism and perspectives for the future of human flavin-containing monooxygenase 3.

Flavin-containing monooxygenases (FMOs) catalyze NADPH-dependent monooxygenation of soft-nucleophilic nitrogen, sulfur, and phosphorous atoms contained within various drugs, pesticides, and xenobiotics. Flavin-containing monooxygenase 3 (FMO3) is responsible for the majority of FMO-mediated xenobiotic metabolism in the adult human liver. Mutations in the FMO3 gene can result in defective trimethylamine (TMA) N-oxygenation, which gives rise to the disorder known as trimethylaminuria (TMAU) or "fish-odour syndrome". To date 18 mutations of FMO3 gene have been reported that cause TMAU, and polymorphic variants of the gene have also been identified. Interindividual variability in the expression of FMO3 may affect drug and foreign chemical metabolism in the liver and other tissues. It is important therefore to study how base sequence variation of the FMO3 gene might affect the ability of individuals and different ethnic population groups to deal with the variety of environmental chemicals and pharmaceutical products that are substrates for FMO3.

Adult↗

PubMed2006

Expression of recombinant flavin-containing monooxygenases in a baculovirus/insect cell system.

The baculovirus/insect cell heterologous expression system provides an important tool for investigating the catalytic activity of individual drug-metabolizing enzymes toward a particular substrate. In this chapter we describe a baculovirus/insect cell system that we have used for the expression of human and mouse flavin-containing monooxygenases. Methods are described for the generation of recombinant baculoviral DNAs, via both site-specific transposition in Escherichia coli and site-specific recombination in vitro; adaptation of Spodopterafrugiperda (Sf) 9 cells to shaking culture and to serum-free medium; cryopreservation and transfection of Sf9 cells; amplification of baculovirus and determination of viral titer; analysis of baculoviral DNA; and expression and analysis of recombinant proteins.

Animals↗

PubMed2006

Transfection of primary cultures of rat hepatocytes.

Five different transfection reagents-calcium phosphate, TransFast Transfection Reagent, Superfect Transfection Reagent, Effectene Transfection Reagent, and Tfx-20--were compared for their ability to effectively transfect primary cultures of male rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and then cultured on Matrigel-coated plates for 24 h before transfection. The cells were transfected with either pGL3-Control or pGL3-Basic plasmids. The efficiency of transfection of each reagent was monitored using the dual luciferase reporter gene assay system. Superfect Transfection Reagent, Effectene Transfection Reagent and Tfx-20 were the most effective for the transfection of primary hepatocytes and gave comparable transfection efficiencies. Calcium phosphate was found to be the least effective transfection reagent and gave the most variable transfection results. Tfx-20 gave the least variable transfection results when different hepatocyte preparations were compared.

Animals↗

PubMed2006

Determination of cellular localization of expression of flavin-containing monooxygenase genes in mouse tissues by in situ hybridization.

Methods are described for the cellular localization of expression of flavin-containing monooxygenase (FMO) genes in various mouse tissues by in situ hybridization. These include the production of digoxigenin (DIG)-labeled antisense and sense RNA probes by transcription from FMO cDNA templates, the preparation of paraffin wax-embedded and cryostat tissue sections, the hybridization of RNA probes to tissue sections, and the specific detection of hybridized probes using an antibody to DIG.

Animals↗

PubMed2006

Deletion of genes from the mouse genome using Cre/loxP technology.

The steps required to delete genes from the mouse genome are illustrated by showing how a cluster of three flavin-containing monooxygenase (Fmo) genes (Fmol, Fmo2, and Fmo4) were deleted from mouse chromosome 1. Such large deletions are accomplished using loxP/Cre recombinase technology. Genomic clones corresponding to the genes to be deleted are first isolated, and then appropriate genomic fragments are cloned into vectors containing a loxP site. This produces targeting vectors, which are electroporated into mouse embryonic stem (ES) cells to allow a homologous recombination event to take place between the mouse genomic fragment, present within the vector, and the homologous sequences in the ES cell genome. Screening of ES cells for recombinants in which loxP sites have been inserted on either side of the gene cluster to be deleted is described. Recombination by Cre recombinase to produce ES cell lines carrying the deletion on chromosome 1 is also described.

Animals↗

PubMed2006

Characterization of targeted mouse embryonic stem cell chromosomes: karyotyping and fluorescence in situ hybridization of metaphase spreads.

The manipulation of genes in mouse embryonic stem (ES) cells can result in chromosome abnormalities. This chapter describes methods for karyotyping of the manipulated ES cell line before injection into blastocysts and the use of fluorescence in situ hybridization to confirm the deletion of a targeted gene. The method is illustrated by describing how an ES cell line targeted for the deletion of Fmo genes was characterized.

Animals↗

PubMed2006

Microinjection of targeted embryonic stem cells and establishment of knockout mouse lines for Fmo genes.

Methods are described for the injection of mouse embryonic stem cells, in which Fmo genes have been targeted to disrupt gene function, into 3.5-d-old blastocysts and the implantation of these into foster mothers. Successful injection and implantation of blastocysts will produce mice of mixed coat color (the chimera). Also described are methods to establish the success of blastocyst injection and implantation of germ-line transmission of the knockout (KO) mutation. Breeding strategies to produce congenic and isogenic KO mouse lines are outlined. Simple methods for the isolation of tail DNA, the tagging of mice, and record keeping of the line are also given.

Animals↗

PubMed2004

Cell-, tissue-, sex- and developmental stage-specific expression of mouse flavin-containing monooxygenases (Fmos).

The cell-, tissue-, sex- and developmental stage-specific expression profiles of five members of the flavin-containing monooxygenase (FMO) family, FMO1, 2, 3, 4 and 5, were investigated in 129/SV mice, using isoform-specific antisense RNA probes. In situ hybridization localized FMO1 and 5 mRNAs to the perivenous, and FMO 2, 3 and 4 mRNAs to the periportal, regions of the liver. In kidney, each FMO mRNA is localized to the distal and proximal tubules and collecting ducts; FMO1 mRNA is present also in the glomerulus. In lung, FMO1 and 3 mRNAs are expressed in the terminal bronchiole, and FMO1 mRNA also in the alveoli. FMO1 mRNA is present in neurons of the cerebrum and in the choroid plexus. RNase protection assays showed that the most abundant isoform in newborn liver, lung, kidney and brain, and in adult lung and kidney is FMO1, but in adult liver FMO5 is present in greatest amounts. In liver, lung and kidney, expression of Fmo1, 3 and 5 peaks at 3 or 5 weeks of age, but in the brain, Fmo1 expression is greatest in newborns. In the kidney, FMO5 mRNA abundance is fourfold greater in males than in females, at all stages of development. Our results demonstrate that Fmo1, 2, 3, 4 and 5 exhibit distinct cell-, tissue-, sex- and developmental stage-specific patterns of expression.

Animals↗

PubMed2004

Organization and evolution of the flavin-containing monooxygenase genes of human and mouse: identification of novel gene and pseudogene clusters.

OBJECTIVES: To date, six flavin-containing monooxygenase (FMO) genes have been identified in humans, FMOs 1, 2, 3, 4 and 6, which are located within a cluster on chromosome 1, and FMO5, which is located outside the cluster. The objectives were to review and update current knowledge of the structure and expression profiles of these genes and of their mouse counterparts and to determine, via a bioinformatics approach, whether other FMO genes are present in the human and mouse genomes. RESULTS AND CONCLUSIONS: We have identified, for the first time, a mouse Fmo6 gene. In addition, we describe a novel human FMO gene cluster on chromosome 1, located 4 Mb telomeric of the original cluster. The novel cluster contains five genes, all of which exhibit characteristics of pseudogenes. We propose the names FMO 7P, 8P, 9P, 10P and 11P for these genes. We also describe a novel mouse gene cluster, located approximately 3.5 Mb distal of the original gene cluster on Chromosome 1. The novel mouse cluster contains three genes, all of which contain full-length open-reading frames and possess no obvious features characteristic of pseudogenes. One of the genes is apparently a functional orthologue of human FMO9P. We propose the names Fmo9, 12 and 13 for the novel mouse genes. Orthologues of these genes are also present in rat. Sequence comparisons and phylogenetic analyses indicate that the novel human and mouse gene clusters arose, not from duplications of the known gene cluster, but via a series of independent gene duplication events. The mammalian FMO gene family is thus more complex than previously realised.

Amino Acid Sequence↗

PubMed2003

Trimethylaminuria and a human FMO3 mutation database.

Trimethylaminuria (TMAuria), or fish-odor syndrome, is due to defective flavin-containing monooxygenase 3 (FMO3). In the liver, this protein catalyzes the NADPH-dependent oxidative metabolism of odorous trimethylamine (TMA), derived in the gut from dietary sources, to nonodorous trimethylamine N-oxide (TMA N-oxide). Affected individuals are unable to carry out this reaction and consequently exude a fishy body odor, due to the secretion of TMA in their breath and sweat and its excretion in their urine. This leads to a variety of psychosocial problems, including disruption of schooling, clinical depression, and attempted suicide. Twelve missense, three nonsense, and one gross deletion mutation are known to cause TMAuria. FMO3 is also a drug-metabolizing enzyme and compromised activity is expected to have implications for the efficacy of drug treatment and the possibility of adverse drug reactions both in TMAuric patients and in the general population. To date eight polymorphic variants, not associated with TMAuria, have been reported. A human FMO3 mutation database was created using MuStar, a locus-specific database system for maintaining data about allelic variants and distributing these via the World Wide Web. The database currently contains 24 entries and is accessible on the World Wide Web via the URL http://human-fmo3.biochem.ucl.ac.uk/Human_FMO3. Additional entries can be submitted via the curator of the database or via a web-based form.

Alleles↗

PubMed2002

Genetic polymorphisms of flavin-containing monooxygenase (FMO).

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU). The TMAU patient excretes large amounts of TMA in urine and sweat. A more recent ethnically related polymorphism in expression of the major FMO in lung, FMO2, has been described. All Caucasians and Asians genotyped to date are homozygous for a CAG --> TAG amber mutation resulting in a premature stop codon and a nonfunctional protein truncated at AA 472 (wildtype FMO2 is 535 AA). This allele has been designated hFMO2*2A. Twenty-six percent of individuals of African descent and 5% of Hispanics genotyped to date carry at least one allele coding for full-length FMO2 (hFMO2*1 allele). Preliminary evidence indicates that FMO2.1 is very active toward the S-oxygenation of low MW thioureas, including the lung toxicant ethylene thiourea. Polymorphic expression of functional FMO2 in the individuals of African and Hispanic descent may markedly influence drug metabolism and/or xenobiotic toxicity in the lung.

Animals↗