PubMed Health⌕ Search

Biomedical subjects

E A Shephard

Publications and source records attributed to E A Shephard.

At least 37 records · Page 2Linked to original sources
PubMed1994

Interaction of proteins with a cytochrome P450 2B2 gene promoter: identification of two DNA sequences that bind proteins that are enriched or activated in response to phenobarbital.

Cytochromes P450 (CYPs) are of central importance in the metabolism of foreign hydrophobic compounds. Members of the CYP2B subfamily are inducible at the transcriptional level by the barbiturate, phenobarbital. Owing to the lack of a suitable phenobarbital-responsive cell line, very little is known regarding the mechanisms by which phenobarbital induces the expression of these genes. We report the use of gel retardation and DNase I footprinting to investigate the presence of regulatory protein binding sites within a CYP2B2 gene promoter. Two DNA sequences, located between -183 to -199 and -31 to -72, have been identified that bind rat liver nuclear proteins that are enriched or activated in vivo by phenobarbital. Gel retardation competition experiments demonstrated that the two sequences bound different proteins. In vitro transcription competition experiments demonstrated that the sequences and the proteins with which they interact are involved in regulating CYP2B2 gene transcription. These two DNA sequences and their cognate binding proteins may play a role in the induction of CYP2B2 gene expression in response to phenobarbital.

Animals↗
PubMed1993

Maintenance and induction in co-cultured rat hepatocytes of components of the cytochrome P450-mediated mono-oxygenase.

Hepatocytes grown in culture rapidly lose many of the cytochromes P450 (CYP) responsible for metabolizing foreign compounds. Among the proteins most readily lost are members of the CYP2B subfamily. We have investigated, by RNase protection assays, the ability of rat hepatocytes, cultured conventionally or co-cultured with rat liver epithelial cells, to maintain the expression of genes encoding members of the CYP2B subfamily, and the inducibility of this expression by phenobarbital. After 4 days of conventional hepatocyte culture CYP2B mRNAs were undetectable, but remained inducible by phenobarbital. In co-cultured hepatocytes the abundance of the mRNAs remained relatively constant from 4-14 days. After 7 days of co-culture the concentration of the mRNAs was increased 12-15-fold by phenobarbital. RNase protection assays with probes capable of distinguishing between CYP2B1 and 2B2 mRNAs demonstrated that the ratios of the abundance and inducibility of the two mRNAs were the same in co-culture as in vivo. Co-cultured hepatocytes also maintained the expression of genes coding for two other components of the cytochrome P450-mediated mono-oxygenase, namely cytochrome P450 reductase and cytochrome b5.

Animals↗
PubMed1993

Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q.

We have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685-1689) to chromosome 1. We propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and I. R. Phillips, 1991, J. Biol. Chem. 266: 12379-12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261-267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1q23-q25.

Base Sequence↗
PubMed1992

Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family.

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

Amino Acid Sequence↗
PubMed1992

Maintenance of total cytochrome P-450 content in rat hepatocyte culture and the abundance of CYP1A2 and CYP2B1/2 mRNAs.

mRNAs encoding cytochrome P-450s CYP1A2 and CYP2B1/2 have been quantified in rat hepatocytes cultured for periods up to 72 h under several different culture conditions that maintain total cytochrome P-450 content. When hepatocytes were cultured at either 37 or 30 degrees C in Williams E media, both CYP1A2 and CYP2B1/2 mRNAs declined dramatically. However, when cultured at 30 degrees C for 24 h, the decline in these mRNAs was not as great as that observed in cells grown at 37 degrees C. The addition of dimethyl sulphoxide to cells grown at 37 degrees C did not affect the rate of disappearance of the CYP1A2 or CYP2B1/2 mRNAs. These mRNAs also declined rapidly in cells grown in 'P-450 medium' i.e. RPMI 1640 medium without cyst(e)ine but supplemented with 0.1 mM-delta-aminolaevulinic acid. However, the levels of CYP2B1/2 mRNAs were maintained when hepatocytes were cultured in Williams E medium supplemented with 0.5 mM-metyrapone. These conditions did not, however, maintain the levels of CYP1A2 mRNA.

Animals↗
PubMed1992

Localization of cytochrome P-450 gene expression in normal and diseased human liver by in situ hybridization of wax-embedded archival material.

The localization of the expression of several cytochrome P-450 genes in normal and diseased human liver was investigated by in situ hybridization of formalin-fixed, paraffin wax-embedded archival tissue samples with 35S-labeled antisense RNA probes. The results demonstrated that genes coding for members of the cytochrome P-450 3A subfamily (CYP3A) were preferentially expressed in hepatocytes in acinar zone 3 (the centrilobular region), whereas genes coding for CYP1A2, CYP2A, 2B and 2C were expressed uniformly throughout the liver acinus. In cirrhotic livers, CYP2A and 2B genes (and to a lesser extent, CYP3A genes) were highly expressed in isolated hepatocytes located at the junction of parenchyma with fibrous septa. The cause and significance of the position-dependent expression of specific cytochrome P-450 genes in normal and diseased human liver are discussed.

Cytochrome P-450 Enzyme System↗
PubMed1992

Quantification of cytochrome P450 reductase gene expression in human tissues.

We have isolated and sequenced cDNA clones that code for a variant of human cytochrome P450 reductase. An RNase protection assay was used to quantify the corresponding mRNA in adult and fetal tissues. The results demonstrate that, in the samples analyzed, the cytochrome P450 reductase gene displays very little inter-individual variation in its expression in adult liver and is subject to little developmental or tissue-specific regulation.

Aging↗
PubMed1991

Cloning, primary sequence, and chromosomal mapping of a human flavin-containing monooxygenase (FMO1).

cDNA clones that code for a pig and human flavin-containing monooxygenase (FMO) have been isolated. The full-length sequence of the human cDNAs revealed that they encode a polypeptide of 532 amino acid residues containing putative FAD- and NADP-binding sites. The deduced amino acid sequence has 88 and 86% identity, respectively, with the pig and rabbit "hepatic" forms of FMO, but is only 58% similar to the rabbit "pulmonary" FMO, and thus represents the human ortholog of the "hepatic" form of FMO. However, as this FMO is present in low abundance in human adult liver, the general term "hepatic" for this form of the enzyme is misleading, and thus we propose the name FMO1 to describe this human FMO and its mammalian orthologs. Northern blot analysis demonstrated that human FMO1 mRNA is more abundant in fetal than in adult liver, indicating that in man the enzyme is subject to developmental regulation. Southern blot hybridization of human genomic DNA suggests that the protein is encoded by a single gene, which has been designated FMO1 and mapped to chromosome 1.

Amino Acid Sequence↗
PubMed1991

Chromosomal localization of a cytochrome b5 gene to human chromosome 18 and a cytochrome b5 pseudogene to the X chromosome.

We have isolated cDNA clones that code for human cytochrome b5. Owing to the high degree of evolutionary conservation of cytochrome b5 sequences and the existence of human and rodent cytochrome b5 processed pseudogenes, we were unable to map unambiguously the chromosomal localization of the human gene(s) by Southern blot hybridization of DNA from human-rodent somatic cell hybrids. An alternative approach, based on restriction enzyme digestion of PCR-amplified DNA, enabled us to map the human cytochrome b5 gene(s) to chromosome 18 and one of its processed pseudogenes to the X chromosome. We propose the designations CYB5 and CYB5P1 for the gene and pseudogene loci, respectively.

Animals↗