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B C Chung

Publications and source records attributed to B C Chung.

103 records · Page 6Linked to original sources
PubMed1986

Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta.

Conversion of cholesterol to pregnenolone is mediated by P450scc [cholesterol, reduced-adrenal-ferrodoxin: oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.67]. RNA from several human adrenal samples was translated in vitro and immunoprecipitated with anti-bovine P450scc, indicating that P450scc mRNA represents about 0.5% of human adrenal mRNA in normal, hypertrophied, and malignant adrenals. A 1626-base-pair human adrenal P450scc cDNA was cloned in bacteriophage lambda gt10. Primer extension data indicated P450scc mRNA is about 1850 bases long and that all adrenal P450scc mRNA has the same 5' end. A full-length clone containing 1821 bases was obtained from a human testis cDNA library to yield the complete sequence. The encoded human preP450scc contains 521 amino acids with a molecular weight of 60189.65. The testis and adrenal sequences were identical; the human cDNA and amino acid sequences are 82% and 72% homologous, respectively, with the bovine sequences. P450scc cDNA was used to probe DNA from a panel of mouse-human somatic cell hybrids, showing that the single human P450scc gene lies on chromosome 15. The human P450scc gene is expressed in the placenta in early and midgestation; primary cultures of placental tissue indicate P450scc mRNA accumulates in response to cyclic AMP.

Amino Acid Sequence↗
PubMed1986

Study of cholesterol side-chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes.

Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.

Adrenal Hyperplasia, Congenital↗
PubMed1986

Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human granulosa cells.

Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to 32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10(-6) M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin-specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with 32P-labeled human P450c17 cDNA (P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450c17 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.

Aldehyde-Lyases↗
PubMed1986

Assignment of the gene for adrenal P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase) to human chromosome 10.

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20 lyase activities. We identified several human P450c17 cDNA clones in a human adrenal cDNA library we constructed in lambda gt10. A short clone containing the 3'-terminal 650 bases of the full-length sequence was used to examine Southern blots of DNA from normal persons and from a panel of mouse/human somatic cell hybrid lines. The pattern of hybridization of this cDNA to normal human DNA cut with 8 restriction endonucleases suggests the human genome has two (or more) P450c17 genes. The pattern of hybridization to the somatic cell hybrid cell lines, each containing a limited, known number of human chromosomes, indicates the human adrenal P450c17 gene lies on chromosome 10. The chromosomal locations of the other P450c17 genes could not be determined.

Adrenal Glands↗
PubMed1985

Cloning and characterization of the bovine gene for steroid 21-hydroxylase (P-450c21).

Steroid 21-hydroxylase activity is required for the synthesis of both cortisol and aldosterone. Using two manually synthesized oligonucleotide probes, we screened a bovine genomic DNA library and identified a phage, lambda E11, carrying the gene for the steroid 21-hydroxylase, P-450c21. The identity of lambda E11 was initially proven by initiating dideoxy sequencing from the two oligonucleotides directly on the full-length, uncleaved phage template. Hybridization of total bovine genomic DNA to lambda E11 restriction fragments indicates the presence of repetitive sequences in or near the P-450c21 gene. Northern blots indicate that the mature mRNA exists in two principal forms of about 2.2 and 2.4 kb in length. Southern blots indicate there are two copies of the gene in the bovine genome. Sequence analysis of a 1141-bp Eco RI fragment of the gene shows three complete exons and a portion of a fourth exon, correctly determining the amino acid sequence of 148 amino acids of this important enzyme. This cloned 1141-bp fragment cross-hybridizes to human genomic DNA, indicating it should be a useful probe for studying the human P-450c21 gene in patients having impaired 21-hydroxylase activity.

Amino Acid Sequence↗
PubMed1984

Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA.

P450scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450scc indicates P450scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the PstI site of pBR322 by dC X dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.

Adrenal Glands↗
PubMed1981

A clinical trial with cefoperazone in pneumonia and pyelonephritis.

15 cases of pneumonia and 15 cases of pyelonephritis were included in a clinical trial of parenteral cefoperazone. The organisms isolated from patients with bacterial pneumonia were: Staphylococcus aureus (6), Klebsiella pneumoniae (5), Diplococcus pneumoniae (3), Pseudomonas species (2), and 6 others. Amongst those with pyelonephritis, Escherichia coli (12), Enterobacter (1), and 3 other pathogens, were isolated. All were sensitive to cefoperazone. The efficacy of cefoperazone in patients with pneumonia was: good in 11 (73%), fair in 3, and poor in 1. In pyelonephritis, 12 (80%) responded well and in the other 3 response was fair. There were no significant side effects or hypersensitivity reactions.

Adult↗
PubMedDate not supplied

Cell-type specificity of human CYP11A1 TATA box.

Expression of the CYP11A1 (SCC) genes, which encode the enzyme important for the first step of steroid biosynthesis, occurs in the adrenal gland and gonads, and is stimulated by cAMP. Transfection of serial deletions of the SCC promoter, which drives reporter gene expression, showed that a minimal promoter containing only the TATA box could direct cAMP-dependent transcription. Transcription factor SF1, which binds to a site next to the TATA box, can stimulate basal transcription but not cAMP response, either in adrenal cell lines or in COS-1 co-transfected with the SF1 expression plasmid. These data lead to the conclusion that the minimal promoter containing only the TATA box can drive cell type-specific, cAMP-dependent transcription. Additional experiments replacing the TATA sequence of SCC with other TATA sequences suggested that the TATA sequence itself is important for this cAMP-dependent transcription.

Animals↗
PubMedDate not supplied

Analysis of anabolic steroids using GC/MS with selected ion monitoring.

This study describes the use of gas chromatography/mass spectrometry with selected ion monitoring to screen 18 anabolic steroids banned by the International Olympic Committee. These anabolic steroids are analyzed in two fractions depending on their excretion pattern: nonconjugated (free) or conjugated fraction. The wet procedure of extracting steroids from urine consists of an initial isolation of lipophilic compounds on a column packed with Amberlite XAD-2 resin, followed by enzymatic hydrolysis with beta-glucuronidase from Escherichia coli. After extraction, the hydrolyzed steroids are derivatized to the corresponding trimethylsilyl ethers. The derivatized steroids are analyzed by gas chromatography/mass spectrometry with selected ion monitoring of their characteristic ions. It takes 12 and 26 min to run GC/MS and edit the raw data for nonconjugated and conjugated fractions respectively.

Anabolic Agents↗
PubMedDate not supplied

Structure and expression of the CYP21 (P450c21, steroid 21-hydroxylase) gene with respect to its deficiency.

Steroid 21-hydroxylase (P450c21) deficiency is the major cause of a common genetic disease, congenital adrenal hyperplasia, with the symptoms of virilization due to steroid imbalance. We have devised a fast diagnostic method to detect common mutations in the c21B gene by a two-step gene amplification procedure coupled to restriction digestion. This procedure does not require isotopes and is suitable for routine use in a hospital setting. In addition, we have developed a procedure for the production of active P450c21 in E. coli. We tested many different vector and bacterial strain combinations to find out the best condition for P450c21 expression. The bacteria harboring the P450c21 expression plasmid were grown in a rich media supplemented with trace metals, heme biosynthesis precursor delta-levulinic acid, and induced with IPTG at 20 degrees C for 48 h. We found that low growth temperature and long induction time were important for abundant synthesis of P450c21 in E. coli.

Adrenal Hyperplasia, Congenital↗
PubMedDate not supplied

Cloning of zebrafish cDNA for 3beta-hydroxysteroid dehydrogenase and P450scc.

P450scc and 3beta-HSD cDNA were isolated from a zebrafish lambda gt10 cDNA library using trout SCC and 3beta-HSD cDNA as the probes. The zebrafish SCC cDNA encodes a protein of 509 amino acids, which shares a 78% similarities with the trout SCC and 58% with the human SCC. As for 3beta-HSD, two forms of cDNA were isolated, termed HSD 5 and HSD 17, which may have resulted from alternative splicing. HSD 5 and HSD 17 encode proteins of 374 and 341 amino acids respectively. Both share 77% amino acid similarities with trout 3beta-HSD and 53% similarities with the mouse 3beta-HSD. Zebrafish has been increasingly used as a genetic model system to study organ development and to investigate human diseases. The cloning and the characterization of zebrafish P450scc and 3beta-HSD should facilitate study of steroidogenesis and human disease associated with steroid imbalance.

3-Hydroxysteroid Dehydrogenases↗