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C B Kasper

Publications and source records attributed to C B Kasper.

At least 55 records · Page 3Linked to original sources
PubMed1983

Genetic polymorphisms for a phenobarbital-inducible cytochrome P-450 map to the Coh locus in mice.

Southern blot analysis suggests that multiple sequences homologous to a phenobarbital-inducible cytochrome P-450 cDNA are present in the rat and mouse genomes. A cDNA (pP-450b-5) to a major phenobarbital-inducible cytochrome P-450 mRNA species in the rat detected 6 polymorphic DNA fragments when hybridized to DNA from C57BL/6J and DBA/2J mice restricted with endonucleases EcoRI, BamHI, and PvuII. Using the BXD recombinant inbred strains, five of these polymorphisms were mapped to the Coh (coumarin hydroxylase) locus on chromosome 7 of the mouse. The Coh locus has previously been shown to code for a phenobarbital-inducible enzyme, believed to be a cytochrome P-450, which catalyzes the conversion of coumarin to 7-hydroxycoumarin (umbelliferone). The DNA polymorphisms appear to reflect changes in either cytochrome P-450 genes or pseudogenes that are very closely linked to the gene responsible for differential coumarin hydroxylase in mice or it may represent a change(s) in the Coh gene itself. The region of the Coh locus on chromosome 7 may be the site of a cluster of cytochrome P-450 genes.

Animals↗
PubMed1983

Cloning of DNA complementary to cytochrome P-450 induced by pregnenolone-16 alpha-carbonitrile. Characterization of its mRNA, gene, and induction response.

The major form of cytochrome P-450 (P-450PCN) was isolated from rats administered pregnenolone-16 alpha-carbonitrile (PCN). Messenger RNA coding for P-450PCN was enriched by polysome immunoadsorption and utilized to construct a library of cDNA clones in pBR322. P-450PCN clones were isolated from this library by differential colony hybridization using [32P]cDNA probes transcribed from PCN-induced and PCN-induced P-450PCN immunoenriched poly(A) RNA. The P-450PCN clone with the largest cDNA insert (pP450PCN-10) was verified to contain sequences complementary to P-450PCN mRNA by hybrid selection-translation. pP450PCN-10 was composed of approximately 1900 base pairs and had a restriction map that overlapped at least 3 other cDNA clones selected by differential colony hybridization. Denaturing-agarose gel electrophoresis and nitrocellulose blot-hybridization using nick-translated 32P-labeled pP450PCN-10 indicated that pP450PCN mRNA is 2500 +/- 150 nucleotides in length; pP450PCN-10, therefore, represents approximately 76% of its corresponding mRNA sequence. Southern blot analysis of rat DNA using pP450PCN revealed that approximately 50 to 60 kilobases of DNA reacted with the PCN probe, suggesting the P-450PCN gene is either a very large gene or other genomic segments exist that react with the probe, such as pseudogenes or related P-450 genes that share homology. The mechanism of P-450PCN induction was examined by isolating poly(A) RNA at various times after steroid administration and quantitating for P-450PCN mRNA using pP450PCN-10 as a hybridization probe. PCN administration produced a rapid elevation of P-450PCN mRNA which reached maximal levels (7-fold above control) 12 h after administration. In contrast, cytochrome P-450b mRNA, which is readily induced by phenobarbital, was only slightly elevated (approximately 2-fold) after PCN administration.

Animals↗
PubMed1983

Transcriptional regulation of rat liver epoxide hydratase, NADPH-Cytochrome P-450 oxidoreductase, and cytochrome P-450b genes by phenobarbital.

The relative rates of transcription of epoxide hydratase, NADPH-cytochrome P-450 oxidoreductase, and cytochrome P-450b genes were determined in purified hepatocyte nuclei isolated at different times after phenobarbital administration. The rates of transcription of the epoxide hydratase and oxidoreductase genes were increased 4- and 9-fold, respectively, above control levels 1 h after administration of the drug. Transcription rates for the oxidoreductase gene slowly decreased to control values at 24 h while transcription of the epoxide hydratase gene rapidly declined to approximately 2-fold above control values at 2.5 h and remained at this level up to 24 h after administration. Interestingly, transcription of the cytochrome P-450b gene followed a markedly different induction time course than the epoxide hydratase and oxidoreductase genes. Specifically, cytochrome P-450b gene transcription increased 23-fold above control values 6 to 8 h after phenobarbital administration and then slowly declined to 14-fold at 24 h. The increased levels of intranuclear pre-mRNA and cytoplasmic mRNA for each enzyme correlated well with transcriptional activity. In contrast to these results, the rate of transcription of the serum albumin gene was not affected, and levels of albumin mRNA actually decreased after administration of phenobarbital. The rapid increase in the rates of transcription and the appearance of elevated levels of nuclear pre-mRNAs and cytoplasmic mRNAs unequivocally demonstrates that phenobarbital elevates levels of these drug-metabolizing enzymes primarily by augmenting the rate of transcription of their respective genes.

Animals↗
PubMed1983

Cloning and characterization of the rat NADPH-cytochrome P-450 oxidoreductase gene.

A rat liver DNA genomic library was prepared using the lambda phage cloning vector Charon 28. Recombinant phage were screened with a cDNA clone (pOR-7) containing sequences complementary to mRNA coding for NADPH-cytochrome P-450 oxidoreductase. This cDNA clone contains the poly(A) addition site and 60% of the mRNA sequence (Gonzalez, F. J., and Kasper, C. B. (1982) J. Biol. Chem. 257, 5962-5968). Four positive phage were identifed and plaque-purified, and their DNA was isolated and subjected to restriction endonuclease mapping. All phage DNA inserts, which ranged from 11 to 16 kilobases, contained several overlapping restriction fragments. The clone with the largest insert (lambda OR-2) was found to contain restriction fragments identical with those of rat DNA when both were subjected to Southern blotting with nick-translated pOR-7 DNA; this finding established the presence in lambda OR-2 of the 3' end (poly(A) addition site) of the oxidoreductase gene. When [32P]cDNA synthesized from enriched oxidoreductase poly(A) RNA was utilized as a probe, additional fragments were identified. The fragment most distal to the 3'-specific fragment was assumed to contain the 5' cap site and was subcloned into pBR322 for further analysis. Restriction mapping and Southern blot analysis further localized the 5' end of the gene to an AvaII fragment of 540 base pairs (bp). Hybridization of this fragment with oxidoreductase mRNA-enriched poly(A) RNA resulted in the arrest of translation of oxidoreductase; this confirmed that it contained an exon region of the oxidoreductase gene. S1 nuclease mapping and DNA sequencing identified to within +/- 1 bp the 5' cap site of the gene which corresponds to an A start. DNA sequencing of the 5'p flanking region revealed no "TATA box" in the vicinity of -25 to -30 bp of the cap site. R-loop analysis of lambda OR-2 revealed the presence of a minimum of seven introns in the 6000-bp oxidoreductase gene and eight exons with a total length of approximately 2600 bp.

Animals↗
PubMed1982

Mixed-function amine oxidase of the rat hepatocyte nuclear envelope. Demonstration and effects of phenobarbital and 3-methylcholanthrene.

Mixed-function amine oxidase (EC 1.14.13.8) has been demonstrated in highly purified rat hepatocyte nuclear envelope . The enzyme was present in the nuclear envelope at a level 20 percent of that observed in microsomes. Induction studies indicated that nuclear envelope amine oxidase as well as its microsomal counterpart were refractory to the effects of phenobarbital and 3-methylcholanthrene. Phenobarbital administration increased the specific activity of the microsomal N, N-dimethylaniline N-demethylase and benzo[a]pyrene hydroxylase by 600 and 190 percent, respectively, but decreased the specific activity of the nuclear enzymes by 30-50 percent. In contrast, 3-methylcholanthrene increased the specific activity of benzo[a]pyrene hydroxylase in nuclear envelope and microsomes by 42- and 11-fold, respectively. The hydrocarbon also increased the microsomal and nuclear N, N-dimethylaniline N-demethylase by 40 and 60 percent, respectively, but the specific activity of microsomal and nuclear aniline 4-hydroxylase was decreased by 50 percent. Demonstration of amine oxidase in rat hepatocyte nuclear envelope implicates this enzyme in the toxicity and carcinogenicity of certain drugs and chemicals.

Animals↗
PubMed1981

Cloning of epoxide hydratase complementary DNA.

Tightly membrane-bound polysomes were isolated from livers of rats administered trans-stilbene oxide. Epoxide hydratase mRNA was enriched from these polysomes using immunochemical techniques and oligo(dT)-cellulose chromatography. This resulted in an increase in message concentration over that found in noninduced membrane-bound cDNA, synthesized from enriched mRNA, was inserted into the ampicillin resistance gene of pBR322 using oligo(dG)-oligo(dC) tailing. Clones containing sequences complementary to epoxide hydratase mRNA were selected by differential colony hybridization using [32P]cDNA synthesized from immunoenriched mRNA and [32P]cDNA synthesized from nonenriched mRNA. Plasmids from four clones, which only annealed with the enriched probe, were isolated and shown to specifically hybridize with epoxide hydratase mRNA by hybrid selection-translation. A composite restriction endonuclease map of the plasmid inserts was constructed which spanned 1310 base pairs and represented approximately 80% of the message sequence. The 3'-5' orientation of this map relative to the epoxide hydratase mRNA was also determined.

Cloning, Molecular↗
PubMed1980

Pyrophosphate:protein phosphotransferase: a membrane-bound enzyme of endoplasmic reticulum.

Recently, we demonstrated that highly purified rat liver microsomal membrane was capable of selectively phosphorylating two intrinsic membrane polypeptides (M(r) 145,000 and M(r) 130,000) and that the course of the reaction was kinetically divided into two distinct stages [Lam, K. S. & Kasper, C. B. (1980) J. Biol. Chem. 255, 259-266]. Evidence was also presented that strongly suggested that a phosphoryl donor other than ATP was involved in the second stage of phosphorylation. In the present study, we demonstrate that incubation of microsomal membrane with [gamma-(32)P]ATP produces a prominent (32)P-labeled compound detectable by thin-layer chromatography on polyethyleneimine-impregnated cellulose. DEAE-cellulose fractionation of detergent-solubilized microsomal membrane generated a protein fraction that could convert in excess of 90% of the [gamma-(32)P]ATP into this newly (32)P-labeled unknown compound (I approximately P) without the formation of significant levels of (32)P(i). When [alpha-(32)P]ATP was used, I approximately P was unlabeled. Enzymically synthesized I approximately P was purified and determined to be pyrophosphate by using (31)P NMR spectroscopy. [(32)P]Pyrophosphate, synthesized chemically or enzymically, was capable of selectively phosphorylating the M(r) 145,000 and M(r) 130,000 polypeptides. Time course studies utilizing pyrophosphate as the phosphate source showed only one phase of phosphorylation that was strongly inhibited by micromolar levels of ATP as well as by NaF (5 mM). These studies further establish that pyrophosphate is the phosphoryl donor involved in the second stage of phosphorylation.

Animals↗