Human growth hormone-releasing hormone receptor (GHRHR) maps to a YAC at chromosome 7p15.
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Animals↗
Biomedical subjects
S C Chua
Publications and source records attributed to S C Chua.
Kinetics of styrene urinary metabolites: a study in a low-level occupational exposure setting in Singapore.
Biological monitoring of styrene exposure commonly involves measurement of styrene metabolites, mainly mandelic acid (MA) and phenylglyoxylic acid (PGA), in the urine of exposed subjects. Previous studies on the kinetics of styrene metabolites in urine were mostly conducted in a controlled environment on subjects exposed to high concentrations of styrene. In this study, we examined subjects exposed to low levels of styrene in a fiber-reinforced plastics (FRP) plant to see whether the excretion kinetics of styrene metabolites are similar under field conditions. Eight healthy Chinese male volunteers were exposed to styrene for 4 h with a mean environmental concentration of 11 ppm. Urine samples were collected continuously for 20 h after termination of the exposure and concentrations of urinary MA and PCA were determined. The results showed that MA was rapidly excreted in urine after the exposure, with a half-life of 2.1 h or 1.9 h when corrected with urine creatinine. The excretion of PGA followed that of MA and the half-life was 8.1 h or 5.1 h after correction with creatinine. The half-lives are considerably shorter compared to those in previous reports, suggesting that environmental factors, exposure conditions, or ethnic differences may affect the excretion kinetics of styrene metabolites. The fast excretion of styrene metabolites is also consistent with the observation that urine MA and PGA levels correlated better with the half-day time-weighted average (TWA) concentration of environmental styrene than with the whole-day TWA concentration. Our findings thus underscore the need for information on excretion kinetics in order to develop an appropriate biological monitoring scheme for specific exposure settings and subjects.
Adult↗
Utility of a C-jun microsatellite marker in determining gene dosage for fatty (fa).
The Zucker fatty (fa) mutation provides a genetic model for obesity and non-insulin dependent diabetes mellitus. The molecular pathogenesis of the metabolic phenotype of these animals is not known. Detailed molecular maps of the region surrounding the fa locus on rat chromosome 5 can be used for positional cloning experiments as well as to permit genotyping of animals from appropriate crosses before the confounding metabolic effects of obesity have occurred. We describe the development of a polymerase chain reaction (PCR) assay for a polymorphic simple sequence repeat (SSR) in the promoter region of the protooncogene c-Jun. This assay was used to position c-Jun 4.5cM proximal to the fa locus in 111 F2 progeny of a 13MBN fa/+ F1 intercross. Concurrent use of the c-Jun SSR with a previously described assay for a microsatellite in the glucose transporter, Glut1, permits rapid and accurate assessment of genotypes at the fa locus in animals of any age using minimal amounts of DNA. A strategy is described which minimizes the error rate in assigning genotype at the fatty locus for backcross and intercross progeny.
Animals↗
The little (lit) mutation cosegregates with the growth hormone releasing factor receptor on mouse chromosome 6.
The little (lit) autosomal recessive mutation in the mouse causes dwarfism due to isolated growth hormone deficiency. The in vitro physiology of pituitary growth hormone release in lit/lit animals suggests that an abnormality in the growth hormone releasing factor (GRF) receptor (Ghrfr) is a very likely candidate for the lit mutation. We mapped Ghrfr to the region around lit on Chromosome (Chr) 6 in 100 chromosomes of an FVB x Czech II interspecific backcross. Molecular markers were Neuropeptide Y (Npy), homeobox (Hoxa2), immunoglobulin kappa chain (Igk), wingless-related MMTV integration site (Wnt-2), cystic fibrosis (Cftr), carboxypeptidase A (Cpa), and Ghrfr. Map order and distances were as follows: Cen-II-Wnt-2-(0 cM)-Cftr-(6.0 cM)-Cpa-(8.0 cM)-Npy-(1.0 cM)-Hoxa2-(3.0 cM)-Ghrfr-(2.0 cM)-Igk. We mapped lit (by phenotype) relative to Hoxa2 and Igk on 72 F2 chromosomes of offspring of a B6CZ lit/ + x B6FVB lit/ + intercross and 18 chromosomes of offspring of a B6FVB lit/ + intercross. Map order and distances were as follows: Hoxa2-(2.1 cM)-lit/Ghrfr-(3.7 cM)-Igk. No recombinations between lit and Ghrfr were detected. Thus, Ghrfr maps to mouse Chr 6 and may be allelic with lit. Amplification of a short segment at the 3' end of the Ghrfr mRNA by reverse transcription coupled to the polymerase chain reaction showed a greatly diminished level of GRF receptor mRNA in the pituitaries of lit/lit mice as compared with lit/ + controls. Low level of message could reflect a primary molecular defect or be due to secondary hypoplasia of somatotropes.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Critical effects of aging and nutritional state on hypothalamic neuropeptide Y and galanin gene expression in lean and genetically obese Zucker rats.
We have investigated, by Northern blot analysis, the hypothalamic gene expression [messenger RNA (mRNA)] of two appetite stimulating neuropeptides, neuropeptide Y (NPY) and galanin (GAL) in lean (+/+) and genetically obese (fa/fa) Zucker rats at 11, 24 and 40 weeks of age and their responsiveness to food deprivation. At 11 weeks of age, hypothalamic NPY mRNA levels of fa/fa rats were similar to those observed in lean littermates. However, NPY mRNA levels of fa/fa rats were significantly greater than those of lean rats at 24 (+126%; P < 0.01) and 40 (+65%; P < 0.05) weeks of age. Food deprivation caused a significant increase in NPY mRNA levels in both lean and fa/fa Zucker rats at 11 and 24 weeks of age, but not at 40 weeks old rats. Hypothalamic GAL mRNA showed a different pattern of change. The relative content of GAL mRNA in 11 week old obese rats was significantly lower (-68%; P < 0.05) than that of lean rats, while GAL mRNA was significantly higher in 40 week old (+57%; P < 0.05) obese rats compared to their lean littermates. At 24 weeks of age, hypothalamic GAL mRNA levels did not differ between lean and obese rats. Food deprivation induced no change in hypothalamic GAL mRNA in lean rats of all 3 ages; however, it caused an increase of GAL mRNA in obese rats at 11 (+60%; P < 0.05) and 24 (+44%; P < 0.05) weeks, but not at 40 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
High-performance liquid chromatographic method for routine determination of vitamins A and E and beta-carotene in plasma.
A simple and reliable reversed-phase high-performance liquid chromatographic (HPLC) method for the routine determination of vitamins A and E and beta-carotene in plasma (or serum) with wavelength-programmed ultraviolet-visible absorbance detection is described. A 200-microliters aliquot of serum or plasma sample, after deproteinization with ethanol, and containing tocopherol acetate as internal standard, was extracted with butanol-ethyl acetate. Sodium sulphate was added for dehydration. Analytes of extracted samples were found to be stable for at least four days. A 10-microliters aliquot of this organic extract was used for HPLC analysis. The mobile phase was methanol-butanol-water (89.5:5:5.5, v/v) and the flow-rate was set at 1.5 ml/min. The analytes of interest were well separated from other plasma constituents within 22 min at 45 degrees C. The lowest detection limits of vitamins A and E and beta-carotene were 0.02, 0.5 and 0.1 microgram/ml, respectively. The recovery and reproducibility of the present method were around 90%. The method is sensitive, specific and can be used for epidemiological studies and for routine determination of vitamin deficiency. Several important factors that may affect the analysis are also discussed in this paper.
Carotenoids↗
Hypothalamic mechanism for cobalt protoporphyrin-induced hypophagia and weight loss: inhibition of the feeding response to NPY.
The mechanism whereby neurally or peripherally administered cobalt-protoporphyrin (CoPP) leads to transient hypophagia and prolonged weight reduction in normal and genetically obese animals is unknown. Neuropeptide Y (NPY) is a known endogenous stimulator of feeding behavior and is elevated in the hypothalamus of food-deprived rats. Accordingly, we examined the interaction between CoPP and NPY in the central nervous system. Concentrations of NPY mRNA in the hypothalami of rats treated intracerebroventricularly with vehicle or CoPP responded to decreased food intake with comparable increases. However, intracerebroventricular infusions of NPY elicited increased intake of food in vehicle-treated rats but were without effect in CoPP-treated animals. The results suggest that CoPP acts, at least in part, by blocking the feeding response to NPY.
Animals↗
A clinical perspective on peptides and food intake.
Viewed from a clinical perspective, it is difficult to generate enthusiasm for the likelihood of finding a peptide that could be helpful in the treatment of obesity. A deeper understanding of obesity, as will emerge from molecular biology, is more likely to point the way to a useful peptide than further evaluations of the clinical dilemmas posed by obesity. However, a clinical perspective may be useful in pointing the way to a system that needs to be examined by molecular biology and also to inject caution in the evaluation of early findings when peptides are used in treatment.
Adipose Tissue↗
Food deprivation and hypothalamic neuropeptide gene expression: effects of strain background and the diabetes mutation.
We have used a novel method to identify genes expressed in the hypothalamus which may be potentially involved in controlling food intake and energy metabolism. We assumed that food deprivation, a powerful stimulus of food intake, would stimulate the activity of neural pathways involved in feeding behavior which should be reflected in an increase in the synthesis of any relevant neuropeptide and its messenger RNA. A study of 5 neuropeptides in 5 strains of mice has identified neuropeptide Y (NPY) as a gene whose expression in the hypothalamus is controlled by nutritional status, suggesting that hypothalamic NPY neurons are a link in the neural network regulating feeding behavior and energy metabolism. In addition, we have studied the effect of the diabetes mutation on neuropeptide gene expression during fasting and refeeding. Our findings suggest that abnormal NPY and enkephalin gene expression in the hypothalamus may be two important determinants of the expression of the diabetes mutation.
Animals↗
Food deprivation and age modulate neuropeptide gene expression in the murine hypothalamus and adrenal gland.
We have developed a rapid and simple method for quantitative Northern blot analysis of rare messenger RNA species from single mouse hypothalami and adrenals. This technique has allowed us to study the effects of food deprivation on neuropeptide gene expression in the mouse hypothalamus and adrenal gland. The potential modulatory effects of sex and age were also investigated. Food deprivation induced a two-fold increase in the amount of hypothalamic neuropeptide Y (NPY) mRNA, but did not increase NPY mRNA in the brainstem. Age had a significant effect on levels of NPY messenger RNA levels in the hypothalamus. However, there were no gender-associated effects. Sexually immature females (6 weeks old) had higher levels of NPY expression than mature females (9 weeks old). In contrast, in the adrenal gland, increasing maturity was associated with higher levels of NPY mRNA. As in the hypothalamus, fasting caused approximately two-fold increases in NPY mRNA over levels in the ad libitum fed state for both mature and immature mice. Thus, hypothalamic NPY neurons are responsive to nutritional deprivation and developmental status, suggesting that NPY neurons may be important in energy homeostasis.
Adrenal Glands↗
Structural and functional analysis of the promoter region of the gene encoding mouse steroid 11 beta-hydroxylase.
The mouse gene encoding adrenal steroid 11 beta-hydroxylase (11 beta-OHase) has been cloned and the nucleotide sequence of its 5' end has been determined. The coding regions sequenced are homologous (75%) to the sequence of bovine 11 beta-OHase cDNA. The 5'-flanking region of the 11 beta-OHase gene contains a potential cAMP response element (TGACGTGA) located 56 base pairs upstream of the transcription initiation site (position -56) and two motifs at positions -249 and -148 which are similar to an element postulated to be required for the expression of 21-hydroxylase. Transfection of mouse Y1 adrenocortical tumor cells and MA-10 testicular Leydig cells with plasmids containing the 11 beta-OHase promoter linked to a growth hormone reporter gene showed that the 11 beta-OHase promoter can direct cell-specific expression. Deletion analyses of the 5'-flanking region suggest that multiple sequence elements, one of which is located between positions -425 and -338 and a second between positions -338 and -123, interact to produce full levels of promoter activity. Mutant Y1 cells defective in cAMP-dependent protein kinase activity do not express growth hormone driven by the 11 beta-OHase promoter, indicating that expression of 11 beta-OHase in Y1 cells requires an intact cAMP second messenger system. Moreover, mutation of the putative cAMP response element at position -56 abolishes expression. These experiments thus present a useful system for the investigation of cis-acting elements involved in the transcriptional regulation of 11 beta-OHase.
Amino Acid Sequence↗
Cloning of cDNA encoding steroid 11 beta-hydroxylase (P450c11).
We have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11 beta-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage lambda vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11 beta-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia.
Adrenal Cortex↗
Determination of mandelic acid and phenylglyoxylic acid in the urine and its use in monitoring of styrene exposure.
This paper describes a sensitive biological monitoring method for assessing exposure to styrene. Two major metabolites of styrene, mandelic acid (MA) and phenylglyoxylic acid (PGA), were measured in urine using reversed-phase high-performance liquid chromatography with a variable wavelength UV detector. The urine sample (200 microL) was saturated with 60 mg of sodium chloride and spiked with 20 microL of internal standard (O-methyl hippuric acid). Hydrochloric acid (6N HCl) was added for acidification followed by extraction with ethyl acetate. The extract (0.5 mL) was dried and reconstituted with the mobile phase. The mobile phase used was water-methanol (90:10) with 0.5% acetic acid. The filtrate (5 microL) was injected into the HPLC with a C18 column. The detection limits for MA and PGA were estimated to be 5 mg/L and 0.5 mg/L, respectively. The average recovery was 96% for MA and 84% for PGA. The between-days coefficients of variation for both metabolites were generally less than 11%. The average within-day variations were usually less than 5%. The method was verified with urine samples collected from workers exposed to styrene. Excellent correlations were observed between environmental styrene exposure and urinary MA (r = 0.92) and PGA (r = 0.85), determined by using the present method. The procedure is sensitive and reproducible, and can be applied to occupational health measurement of styrene exposure.
Chromatography, High Pressure Liquid↗