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E A Shephard

Publications and source records attributed to E A Shephard.

At least 55 records · Page 3Linked to original sources
PubMed1989

Cloning, expression and chromosomal localization of a member of the human cytochrome P450IIC gene sub-family.

We have isolated and sequenced a cDNA clone (pB8) that codes for a novel member of the cytochrome P450IIC sub-family of man. Analysis, by Southern blot hybridization, of DNA isolated from a panel of nine independent human-rodent somatic cell hybrids demonstrated that the corresponding gene (CYP2C) is located on human chromosome 10. Northern blot hybridization of RNA isolated from human livers revealed a 10-fold inter-individual variation in the expression of the gene.

Base Sequence↗
PubMed1989

Isolation of a human cytochrome P-450 reductase cDNA clone and localization of the corresponding gene to chromosome 7q11.2.

We have isolated and sequenced cDNA clones that code for rat and human NADPH-dependent cytochrome P-450 reductase. The cDNA coding for the human protein was used to analyse, by Southern blot hybridization, DNA isolated from a panel of 8 independent human-rodent somatic cell hybrids. The results indicate that cytochrome P-450 reductase is encoded by a single gene (POR) located on human chromosome 7(pter-q22). Analysis of human metaphase chromosomes by hybridization in situ confirmed the results and refined the localization to 7q11.2. Northern blot hybridization revealed that in human liver the expression of the gene varies by less than 3-fold between different individuals.

Animals↗
PubMed1988

The effect of terpenoid compounds on cytochrome P-450 levels in rat liver.

We have investigated the ability of camphor, menthol, pinene, limonene and myrcene to induce in rats members of a cytochrome P-450 sub-family termed PB P-450. These proteins have recently been designated as members of the P450IIB sub-family. None of these naturally occurring terpenoids significantly changed the total content of cytochromes P-450 or cytochrome b5. Radioimmunoassay results showed that PB P-450 was induced 6-fold by camphor and to a lesser extent by menthol and pinene. The induction was confirmed by Western blotting. It was shown by nucleic acid hybridization that induction of PB P-450 by terpenoids was mediated by an increase in the amount of the corresponding mRNA. Analysis of the denaturation of mRNA-cDNA hybrids demonstrated that the mRNA induced by the terpenoids was encoded by a member of the P450IIB sub-family. None of the terpenoids had an effect on the amount of mRNA coding for P450IA2 (a cytochrome P-450 inducible by beta-naphthoflavone and isosafrole). The results indicate that cytochromes P-450 induced by a synthetic compound, phenobarbital, may have originally evolved in response to terpenoid compounds normally present in the environment.

Animals↗
PubMed1988

The major phenobarbital-inducible cytochrome P-450 gene subfamily (P450IIB) mapped to the long arm of human chromosome 19.

We have recently isolated a cloned cDNA that codes for a human orthologue of the major phenobarbital-inducible cytochrome P450IIB subfamily of rodents. The cloned human cDNA was used to analyse, by Southern blot hybridization, DNA extracted from a panel of 9 independent human-rodent somatic cell hybrids. The results indicate that all members of the P450IIB gene subfamily of man are located on chromosome 19. Evidence from hybrids containing different regions of human chromosome 19 localizes this cytochrome P-450 gene subfamily further to the long arm of chromosome 19 in the region cen-q13.3. We propose the designation CYP2B for this locus.

Animals↗
PubMed1988

Cloning and chromosomal mapping of human cytochrome b5 reductase (DIA1).

We have isolated a cDNA clone that codes for human cytochrome b5 reductase. The cDNA was used to analyse, by Southern-blot hybridization, DNA isolated from a panel of 11 independent human-rodent somatic cell hybrids. The results indicate that cytochrome b5 reductase is encoded by a single gene located on human chromosome 22.

Amino Acid Sequence↗
PubMed1986

Regional localization of a human cytochrome P-450 (CYP1) to chromosome 19q13.1-13.3.

A phenobarbitone inducible cytochrome P-450 gene family (CYP1) has recently been localized to chromosome 19q13.1-qter. We have used a human liver cDNA probe in in situ hybridization experiments to metaphase chromosomes from two balanced translocation carriers, 46,XX,t(11;19) (q13;q13.1) and 46,XX,t(7;19) (q31.3;q13.3) to obtain a more precise localization. The results suggest a regional assignment for CYP1 to chromosome 19q13.1-13.3.

Chromosome Mapping↗
PubMed1985

Induction of cytochrome P-450 by phenobarbital is mediated at the level of transcription.

We have previously shown that the 43-fold induction by phenobarbital of the major phenobarbital-inducible cytochrome P-450 of rat liver microsomal membranes (PB P-450) is mediated by a 20-fold increase in the amount of its mRNA in the cytoplasm. Here we demonstrate that the induction of the mRNA can be almost entirely accounted for by an increase in the rate of transcription of genes coding for PB P-450, and involves little or no change in the rates of processing, transport or degradation of the mRNA. Phenobarbital treatment resulted in no amplification or rearrangement of PB P-450 genes.

Animals↗
PubMed1985

Isolation and sequence of a human cytochrome P-450 cDNA clone.

A previously reported cDNA clone [pP450(1)] coding for a phenobarbital-inducible cytochrome P-450 variant of rat liver microsomal membranes, designated P-450e(U.C.), was used as a specific hybridization probe to screen a human liver cDNA library. Restriction mapping showed that two of the colonies isolated contained plasmids coding for overlapping regions of the same cDNA sequence. The clone [pHP450(1)] having the longer cDNA insert (1.25 kilobase pairs) was sequenced. The homology between the rat and human cDNAs is 62% in their coding regions but is only random (24%) in the 3'-noncoding nucleotides. The amino acid sequence deduced from the human cDNA is 50% identical to that of P-450e(U.C.). The homology increases to 72% if conservative changes in amino acid residues are permitted. The hydropathy profile of the polypeptide encoded by pHP450(1) is almost identical to that of P-450e(U.C.). Regions known to be highly conserved in cytochrome P-450 isozymes isolated from rat, rabbit, and mouse were found to be conserved in the amino acid sequence derived from pHP450(1). Analysis by Southern blotting indicated that the human cytochrome P-450 encoded by pHP450(1) is part of a multigene family.

Amino Acid Sequence↗
PubMed1985

A cytochrome P-450 gene family mapped to human chromosome 19.

We have recently isolated a cloned cDNA coding for a cytochrome P-450 of human liver microsomal membranes, which corresponds to a major phenobarbital-inducible cytochrome P-450 of rat liver. This human cytochrome P-450 is encoded by a member of a multigene family. DNA extracted from a panel of 12 independent human-rodent somatic cell hybrids was analysed by Southern blot hybridization with the cloned cDNA. The results indicate that all components of this cytochrome P-450 gene family are located on chromosome 19. Evidence from hybrids derived from an individual carrying a balanced translocation suggests a regional localization of 19p13.2----qter. Analysis of human metaphase chromosomes by in situ hybridization localizes this cytochrome P-450 gene family further to the long arm of chromosome 19 in the region q13.1----qter. We propose the designation P450PB for this locus.

Animals↗
PubMed1984

Hormonal control of carbonic anhydrase III.

Using radioimmunoassay, the concentration of carbonic anhydrase III (CA III) in the livers of adult male rats was found to be approximately 30 times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver CA III concentrations that could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomized female rats induced about a 5-fold increase in liver CA III concentration. Immunoprecipitational analysis of the products of liver mRNA translation in vitro with antiserum specific for CA III showed that hormonal control of the levels of CA III in rat liver is mediated by changes in the amount of translatable CA III mRNA. Marked changes in liver CA III concentrations were also observed in developing and aging male rats. Different control mechanisms appear to operate in mouse and man.

Aging↗
PubMed1983

Sexual differentiation of rat liver carbonic anhydrase III.

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.

Age Factors↗
PubMed1983

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique.

We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.

Animals↗
PubMed1983

Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.

Animals↗
PubMed1983

Androgen-linked control of rat liver carbonic anhydrase III.

The concentration of carbonic anhydrase III (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.

Aging↗
PubMed1983

A rapid one-step purification of NADPH-cytochrome c (P-450) reductase from rat liver microsomes.

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.

Animals↗