Biomedical subjects

B C Chung

Publications and source records attributed to B C Chung.

At least 73 records · Page 4Linked to original sources

PubMed1997

Altered hydroxylation of estrogen in patients with postmenopausal osteopenia.

To study the possible contributions of the differences in estrogen metabolism to bone mass in postmenopausal osteopenia, spinal and femoral bone mineral densities (BMD) were measured, and 18 urinary metabolites of estrogen were analyzed by a gas chromatography-mass spectrometry assay system in 59 postmenopausal women (5-10 yr after menopause). The BMD of the spine and femoral neck showed positive correlations with body weight, height, and body mass index as we expected. Compared to nonosteopenic subjects, there were no significant differences in serum estrone (E1) and estradiol (E2) levels in patients with osteopenia. However, the urinary 16 alpha-hydroxyestrone [16 alpha-(OH)E1] level was significantly lower in patients with spinal osteopenia (P < 0.001). Among the 18 urinary metabolites of estrogen, the 16 alpha-(OH)E1 and 16 alpha-(OH)E1/2-hydroxyestrone [2-(OH)E1) ratio showed positive correlations with spinal BMD (P < 0.05), whereas 2-(OH)E2 showed a negative correlation with femoral neck BMD (P < 0.05). The urinary 16 alpha-(OH)E1 level also revealed a positive correlation with the age-matched z score of BMD in the spine (P < 0.05). In multiple stepwise regression analysis, weight, 16 alpha-(OH)E1, interaction between 16 alpha-(OH)E1 and 2-(OH)E2, 2-(OH)E2, and years after menopause were statistically significant for spinal BMD (r2 = 0.4968). For femoral neck BMD and weight, 16 alpha-(OH)E1 and 2-(OH)E2 were the independent determinants (r2 = 0.3369). In conclusion, the activity of estrogen 16 alpha-hydroxylase was decreased and/or the activity of estrogen 2-hydroxylase was enhanced in post-menopausal osteopenia. We speculated that these derangements may serve as contributing factors for the acceleration of bone loss in post-menopausal osteoporosis.

Aged↗

PubMed1997

Metabolic disposition of the new fluoroquinolone antibacterial agent DW116 in rats.

The metabolic disposition of the new fluoroquinolone antibacterial agent DW116 has been studied in Sprague-Dawley rats. The compound was absorbed well and demonstrated excellent oral bioavailability. The plasma kinetic profiles were characterized by monoexponential elimination with an elimination half life of 3-4 hr. The apparent mean total clearance (CL(T)) and the volume of distribution (V(SS)) ranged from 221 +/- 55 to 274 +/- 27 ml/hr/kg and 1.0+/-0.1 to 1.5+/-0.2 l/kg, respectively, and were independent of dose between 4 and 20 mg/kg levels. The renal (CL(R)) clearance was 64.5 ml/hr/kg and the biliary (CL(B)) clearance was 33.8 ml/hr/kg. The combined value accounted for approximately one-half of the total clearance, indicating that the remaining one-half of the administered dose was eliminated via hepatic clearance. The major metabolite excreted in the bile was identified as the glucuromide ester of parent drug using base-hydrolysis of the conjugate metabolite followed by co-HPLC with standard compound, 19F-NMR and LC-MS methods. The mean urinary recoveries of free and total (free plus glucuronide ester) DW116 were 28.6 +/- 2.7% and 36.4 +/- 1.8% of the administered dose and the corresponding biliary recoveries were 14.4 +/- 5.5% and 37.0 +/- 7.6%, respectively. The mass balance study after a single (100mg/kg) oral administration of 14C-DW116 indicated complete recovery of radioactivity over a 7-day period, accounting for approximately 60-70% in feces and 30-40% in urine. 14C-DW116 extensively distributed during a prolonged process into all tissues with a rather slower penetration into the brain, lung, and muscle. The compound also readily crossed the placenta and was transferred to the fetus.

Administration, Oral↗

PubMed1997

GC-MS determination of organic acids with solvent extraction after cation-exchange chromatography.

We combined column and partition chromatography to isolate, purify, and quantify biological organic acids in urine and cerebrospinal fluid (CSF). Urine and CSF samples were introduced onto a preconditioned cation-exchange column (Dowex 50 W x 8 resin) to remove the biological interferences. The effluent with water was extracted with ethyl acetate two times (pH 1 and 3) and the organic acids were quantitatively converted into their trimethylsilyl derivatives for detection by gas chromatography-mass spectrometry. The good quality-control data were obtained through precision and accuracy tests. Inter- and intraassay CVs were 0.01-10.2% and 0.02-12.2%, respectively. Analytical recoveries compared favorably with results from the commonly used solvent extraction method. This method was used for the measurement of the 14 organic acids in the urine and CSF of healthy volunteers. The values obtained were in the range of the published data.

Adult↗

PubMed1996

Excretion study of furazabol, an anabolic steroid, in human urine.

Furazabol and 16-hydroxyfurazabol represented the most abundant peaks in urinary metabolic profiles for two men obtained by gas chromatography-mass spectrometry (GC-MS) after oral administration of 5 mg furazabol. The excreted amounts of unchanged furazabol were determined and its response in GC-MS was compared with that of the 16-hydroxy metabolite. The maximum excretion rates of these compounds were reached 2-3 h after oral administration. The half-lives of unchanged furazabol for two human subjects, were 1.87 and 1.29 h respectively and the recovered amount in 48 h was 24% (33% for one, 15% for the other). Also the spectrum of an unidentified metabolite is reported.

Administration, Oral↗

PubMed1996

Function and membrane topology of wild-type and mutated cytochrome P-450c21.

We have studied membrane topology of cytochrome P-450c21 (P450c21) using the approaches of mutagenesis and protease digestion. P450c21 is located at the cytoplasm with an N-terminal hydrophobic domain integrated into microsomal membranes. When this hydrophobic domain was replaced by a secretory signal peptide, P450c21 was translocated into the lumen and lost enzymic activity. No other topogenic sequence was detected in the bulk of the P450c21 peptide. A mutant protein with Pro-30 replaced by Leu (L30) corresponding to the mutation found in the diseased state was created. L30 protein lost 90% of enzymic activity, while a double mutant (L30R32) with an additional Leu-32 to Arg mutation had slightly higher residual enzymic activity. Apart from lower activity, L30 was also present in the cell at a lower level than wild-type P450c21. This lower level is probably due to increased degradation, as L30 is synthesized at a normal rate. Both L30 and L30R32 proteins, however, were integrated into membranes normally. Therefore the Pro-30 --> Leu mutation did not affect membrane integration, but affected the abundance and enzymic activity of P450c21.

Amino Acid Sequence↗

PubMed1996

The common I172N mutation causes conformational change of cytochrome P450c21 revealed by systematic mutation, kinetic, and structural studies.

We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169 173. Substitutions of Ile-171 and -172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower Vmax value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.

Amino Acid Sequence↗

PubMed1996

Variegated expression of a mouse steroid 21-hydroxylase/beta- galactosidase transgene suggests centripetal migration of adrenocortical cells.

5'-Flanking sequences (6.4 kb) of the mouse steroid 21-hyrodxylase (21-OHase) A gene linked to a LacZ reporter gene directed appropriate cell-specific expression in cultured Y1 adrenocortical tumor cells and in the adrenal cortex of transgenic mice. The transgene expression initiated at the same stage of adrenal development as the endogenous 21-OHase gene (embryonic day 11.5). Although the endogenous 21-OHase gene is expressed throughout the adrenal cortex, the 21-OHase/beta-gal transgene showed a strikingly variegated pattern of adrenocortical expression in all 10 transgene-expressing mouse lines examined. This presents as radial stripes of beta-gal staining transcending the classical zonal structure of the adrenal cortex but paralleling the columnar arrangement of cells of the zona fasciculata on the centripetal organization of the adrenocortical blood supply. To the extent that the variegated pattern of 21-OHase/beta-gal transgene expression depicts adrenocortical cell lineage, these results suggest that all cells within an individual stripe have a common clonal origin; the radial pattern of clonally derived cells argues that cellular migration maintains the adult adrenocortical cell population. Adrenal glands of developing embryos also exhibited a variegated pattern of 21-OHase/beta-gal transgene expression. However, this presented as islands of beta-gal reporter staining within the developing gland, suggesting that the rapid embryonic adrenal growth phase, which precedes the establishment of the classic adrenocortical zonal structure, may be governed by cellular mechanisms distinct from those responsible for maintenance of the adult adrenocortical cell population.

Adrenal Cortex↗

PubMed1995

Regulation of ferredoxin gene in steroidogenic and nonsteroidogenic cells.

Ferredoxin is an electron transport intermediate for all the mitochondrial cytochromes P450. It is especially abundant in steroidogenic organs where it functions in steroid biosynthesis. The regulation of ferredoxin gene expression was studied in both steroidogenic and nonsteroidogenic cell lines. In steroidogenic cell line Y1, the expression of ferredoxin was stimulated by cAMP and repressed slightly by angiotensin II and phorbol ester PMA. These drugs exhibited the same effect on the basal promoter of the ferredoxin gene, which includes one TATA box and an SP1 site. In human adrenocortical cell line H295, the stimulation of the ferredoxin gene by cAMP was blocked by cycloheximide, as observed in bovine adrenocortical cell culture. In nonsteroidogenic cell lines such as HeLa and COS-1, the stimulation of ferredoxin gene expression by cAMP was not observed, although basal expression was strong. Transfection studies showed that the ferredoxin promoter could not be stimulated by cAMP in nonsteroidogenic cells. Therefore the steroidogenic cell-specific regulation and the general expression pattern appears to be a property unique to the ferredoxin gene.

Adrenal Cortex↗

PubMed1995

Regulation of cholesterol side-chain cleavage cytochrome P450 in mouse testis Leydig cell line I-10.

We have characterized regulation of steroidogenesis in a mouse testis Leydig cell line, I-10. Progesterone secretion was increased in a time- and dose-dependent fashion by 8-Br-cAMP treatment. The amount of cholesterol side-chain cleavage enzyme (P450scc), the first and rate-limiting enzyme for the synthesis of steroids, as detected by immunoblotting, was also increased. The calcium ionophore A23187 decreased the amount of P450scc, but it did not interfere with cAMP stimulation of the accumulation of P450scc. This regulation of P450scc expression by cAMP and A23187 is at the post-transcriptional level because the amount of P450scc mRNA was not affected by either treatment. This result was further confirmed by direct measurement of transcription in the presence or absence of forskolin treatment. I-10, however, supported cAMP-dependent transcriptional activation of the exogenous gene, as shown by the increased expression of a reporter gene under the control of the -600 to -2,500 fragment of the P450scc gene. The ability for transcriptional activation of the exogenous but not endogenous P450scc gene makes I-10 a unique steroidogenic cell line.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Difference in transcriptional activity of two homologous CYP21A genes.

Steroid 21-hydroxylase (CYP21) deficiency is the major cause of congenital adrenal hyperplasia, a common genetic disease due to steroid imbalance. The main cause for the mutation of the CYP21A2 (c21B) gene is conversion of its nucleotide sequence to the neighboring homologous but nonfunctional c21A gene. In this report the transcriptional activities of the c21A and c21B genes have been analyzed. Transient transfection assays showed that transcription derived from the c21A gene was about 5-fold lower in strength than that of the c21B gene, although both sequences responded to cAMP normally in two adrenocortical cell lines. The normal response to cAMP could probably be attributed to equal activation of both genes by a transcription factor Nur77. The lower transcriptional activity of the c21A gene was attributed to sequence changes within 167 base pairs of the 5'-flanking region, which differs from the c21B gene by only four nucleotides at positions around -100. These four nucleotide changes render the c21A sequence to bind proteins less tightly than the -100 region of the c21B sequence, which binds proteins such as transcription factor Sp1 in electrophoretic mobility shift assays. The reduced transcription due to nucleotide changes at the regulatory region of the c21A gene, in combination with other mutations in the coding region, could play important roles in 21-hydroxylase deficiency.

Base Sequence↗

PubMed1995

Metastatic pleural cancer in radionuclide angiocardiography. A pulmonary time-activity curve mimicking left-to-right cardiac shunt.

A 55-year-old man, with a history of nephrectomy for renal cell carcinoma, was evaluated using radionuclide angiocardiography to exclude cardiac shunts as the cause of vascular bruits heard at auscultation. A pulmonary time-activity curve derived from the right lung showed a pattern typical of a large left-to-right shunt, whereas that from the left lung was normal. Increased systemic arterial flow to the lateral right chest was noted in early dynamic images. Subsequent studies revealed that this flow and the abnormal time-activity curve of the right lung were due to systemic arterial supply to metastatic pleural cancer, originating from the kidney. The authors conclude that special caution should be undertaken in the interpretation of the radionuclide angiocardiography for determining left-to-right cardiac shunts, especially in those showing a asymmetric pulmonary time-activity curve.

Carcinoma, Renal Cell↗

PubMed1994

Actions of two different cAMP-responsive sequences and an enhancer of the human CYP11A1 (P450scc) gene in adrenal Y1 and placental JEG-3 cells.

We have characterized three cis-acting elements of the human CYP11A1 gene. A proximal cAMP-responsive sequence (P-CRS) functioned in both adrenal Y1 and placental JEG-3 cells. An upstream cAMP-responsive sequence (U-CRS) and an enhancer, localized by transfections of deleted gene segments linked to a reporter gene to bases -1621 to -1503 and -1931 to -1822, respectively, functioned in Y1 but not JEG-3 cells. Both regions bind proteins only from Y1 cells as identified by footprinting analysis. U-CRS contains the TCAAGGTCA sequence that binds the nuclear receptor family of proteins. The cAMP-dependent transcription mediated by U-CRS, but not by P-CRS, was abolished in a cell line deficient in cAMP-dependent protein kinase. Therefore, P-CRS and U-CRS use different effectors to mediate cAMP response. Gel mobility shift, competition, and antibody supershift experiments showed that nucleotides -117 to -94, which contributed to P-CRS activity in transfection experiments, bound weakly to Sp1-like proteins. This feature is shared by many proximal regulatory elements of steroidogenic genes. Therefore, steroidogenic genes could be coordinately regulated through common regulatory elements such as P-CRS, U-CRS, and cell type-selective enhancers.

Adrenal Glands↗

PubMed1994

Identification of urinary metabolites of pyrilamine after oral administration to man.

1. The metabolism of pyrilamine, 2-[4-methoxybenzyl-(2-dimethylaminoethyl) amino] pyridine, was studied in adult male volunteers after a single oral dose of 50 mg. 2. Solvent extracts of urine obtained with or without enzyme hydrolysis were analysed by gc/ms after derivatization with MSTFA/TMSCI (N-methyl-N-trimethylsilyl-trifluoroacetamide/trimethyl chlorosilane). The structure of metabolites were determined based on EI mass spectra and confirmed with those of authentic standards. 3. Conjugated metabolites identified in the urine were pyrilamine, O-desmethylpyrilamine, and ring hydroxylated derivatives of pyrilamine. O-desmethylpyrilamine was also detected in low abundance as a free form. 4. These metabolites observed in human urine were quite different from those previously reported in rat.

Administration, Oral↗

PubMed1993

The N-terminal hydrophobic domain of P450c21 is required for membrane insertion and enzyme stability.

Microsomal cytochromes P-450 are known to be integrated into smooth endoplasmic reticulum through their hydrophobic sequences located at the N termini. The length requirement of the membrane insertion signal was determined by the generation of six plasmids encoding mutant P450c21 that lacked various portions of the N-terminal hydrophobic domains. When they were transcribed and translated in vitro in the presence of endoplasmic reticulum membranes, mutant protein lacking more than a third of the first hydrophobic domain gradually lost the ability to insert into the membrane and stayed mostly in the soluble fraction when the first N-terminal hydrophobic domain was removed. The steady-state amount of the truncated proteins was progressively reduced in parallel to the extent of their N-terminal deletions, due to their fast degradation. This process was accompanied by a decrease in the enzymatic activity. Therefore, the first hydrophobic domain of P450c21 not only serves as a membrane targeting and anchoring domain, but it is also important for the in vivo protein stability.

Animals↗

PubMed1993

Differential regulation of the CYP11A1 (P450scc) and ferredoxin genes in adrenal and placental cells.

The regulation of the genes encoding cholesterol side-chain cleavage enzyme (P450scc) and ferredoxin, two components in the first step of steroid synthetic pathways, was studied by RNA analyses of endogenous and transfected genes. cAMP rather than calcium was the major secondary messenger that stimulated expression of both P450scc and ferredoxin genes in human placental JEG-3 cells. The effect of cAMP on P450scc expression was abolished by cycloheximide in JEG-3 cells, but it was superinduced in mouse adrenal Y1 cells. For ferredoxin expression, both reagents have synergistic effect in Y1 and JEG-3 cells. To test the mechanism of regulation, DNA segments containing regulatory elements of the P450scc and ferredoxin genes were connected to reporter genes and analyzed in cotransfection experiments. The results showed that the proximal cAMP-responsive sequences of both P450scc and ferredoxin genes were stimulated by cAMP early in both Y1 and JEG-3 cells, requiring no new protein synthesis. This indicates a common mechanism for the regulated expression of both genes. P450scc possessed an additional upstream cAMP-responsive sequence that also responded to cAMP induction in a different manner from the proximal element. The presence of additional upstream regulatory elements makes it possible for the P450scc gene to be further regulated.

Adrenal Glands↗

PubMed1992

Amplification of P450c21 expression in cultured mammalian cells.

We describe in this paper an investigation of mammalian expression systems for P450c21 (21-hydroxylase). Four different promoters, the SV40 early and late promoters, MMTV-LTR, and CMV immediate early promoter were tested for their ability to drive the expression of P450c21 in cultured COS-1 cells. With the exception of MMTV-LTR, all drove the expression of similar levels of functional 21-hydroxylase. In addition, the Rat-1 cell line was tested and shown to be suitable for the stable expression of functional P450c21. We have established cell lines derived from Rat-1 either normal or mutant P450c21 stably expressed together with amplifiable markers. The expression of P450c21 was further increased by selection in methotrexate.

Animals↗

PubMed1992

Synthesis of deuterium-labeled 5 alpha-androstane-3 alpha, 17 beta-diol and its 17 beta-glucuronide.

Using unlabeled androsterone as starting material, 5 alpha-[16,16-2H2]androstan-3 alpha-ol-17-one was synthesized by exchange using deuterated potassium methoxide. This labeled androsterone product was reduced by sodium borodeuteride, which gave predominantly trideuterated 5 alpha-androstane-3 alpha, 17 beta-diol. The labeled androstanediol was conjugated with glucuronide by using the Koenig-Knorr reaction with methyl-1-bromo-1-deoxy-2,3,4-tri-O-acetyl-alpha-D-glucopyranosuronate . The dominant product was identified by thermospray high-performance liquid chromatography/mass spectrometry (MS) and electrospray MS as 5 alpha-[16,16,17-2H3]androstane-3 alpha, 17 beta-diol, 17 beta-glucuronide.

Androstane-3,17-diol↗

PubMed1992

Transcription of the human ferredoxin gene through a single promoter which contains the 3',5'-cyclic adenosine monophosphate-responsive sequence and Sp 1-binding site.

We have investigated the functional elements involved in cAMP-stimulated transcription of the human ferredoxin gene. Unlike the bovine gene, the human gene lacked a second upstream RNA initiation site as demonstrated by sequence analysis of the exon boundary, lack of upstream RNA, and analysis of the promoter. The presence of a single promoter was determined by testing the ability of various gene segments to drive the expression of the chloramphenicol acetyltransferase gene after transfection into a mouse adrenal cell line Y1. Full promoter activity was conferred by a DNA fragment spanning -209 to +55, although the -94 to +55 fragment already provided some promoter activity. Transcription from the -94 to +55 segment was stimulated by 2-fold when 8-bromo-cAMP was added to the cell. Footprinting analyses showed two GC boxes at -50 to -70 and -87 to -108 were protected by proteins from both Y1 and HeLa cells. Competition experiments showed that a protein with a recognition sequence indistinguishable from Sp1 bound to these sites. When connected to a heterologous TATA box, the sequence at -76 to -42, which contained the proximal GC box, was able to confer a high level of basal transcription and cAMP stimulation. This sequence does not show sequence homology with the known cAMP-responsive element. Mutations or deletion of the Sp 1-binding site showed diminished basal transcription and defined the cAMP responsive sequence to be from -76 to -62. Therefore the cAMP-responsive sequence of the human ferredoxin gene was located at -76 to -62, which was adjacent to the Sp 1-binding site.

Amino Acid Sequence↗