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P Y Woon

Publications and source records attributed to P Y Woon.

10 recordsLinked to original sources
PubMed2006

Genetic control of plasma lipid levels in a cross derived from normoglycaemic Brown Norway and spontaneously diabetic Goto-Kakizaki rats.

AIMS/HYPOTHESIS: Dyslipidaemia is a main component of the insulin resistance syndrome. The inbred Goto-Kakizaki (GK) rat is a model of spontaneous type 2 diabetes and insulin resistance, which has been used to identify diabetes-related susceptibility loci in genetic crosses. The objective of our study was to test the genetic control of lipid metabolism in the GK rat and investigate a possible relationship with known genetic loci regulating glucose homeostasis in this strain. MATERIALS AND METHODS: Plasma concentration of triglycerides, phospholipids, total cholesterol, HDL, LDL and VLDL cholesterol were determined in a cohort of 151 hybrids of an F2 cross derived from GK and non-diabetic Brown Norway (BN) rats. Data from the genome-wide scan of the F2 hybrids were used to test for evidence of genetic linkage to the lipid quantitative traits. RESULTS: We identified statistically significant quantitative trait loci (QTLs) that control the level of plasma phospholipids and triglycerides (chromosome 1), LDL cholesterol (chromosome 3) and total and HDL cholesterol (chromosomes 1 and 5). These QTLs do not coincide with previously identified diabetes susceptibility loci in a similar cross. The significance of lipid QTLs mapped to chromosomes 1 and 5 is strongly influenced by sex. CONCLUSION/INTERPRETATION: We established that several genetic loci control the quantitative variations of plasma lipid variables in a GKxBN cross. They appear to be distinct from known GK diabetes QTLs, indicating that lipid metabolism and traits directly relevant to glucose and insulin regulation are controlled by different gene variants in this strain combination.

Animals↗
PubMed2005

Genetic and genomic studies of PADI4 in rheumatoid arthritis.

OBJECTIVES: Strong genetic association of rheumatoid arthritis (RA) with PADI4 (peptidyl arginine deiminase) has previously been described in Japanese, although this was not confirmed in a subsequent study in the UK. We therefore undertook a further study of genetic association between PADI4 and RA in UK Caucasians and also studied expression of PADI4 in the peripheral blood of patients with RA. METHODS: Seven single-nucleotide polymorphisms (SNP) were genotyped using polymerase chain reaction (PCR)-restriction fragment length polymorphism in 111 RA cases and controls. A marker significantly associated with RA (PADI4_100, rs#2240339) in this first data set (P = 0.03) was then tested for association in a larger group of 439 RA patients and 428 controls. PADI4 transcription was also assessed by real-time quantitative PCR using RNA extracted from peripheral blood mononuclear cells from 13 RA patients and 11 healthy controls. RESULTS: A single SNP was weakly associated with RA (P = 0.03) in the initial case-control study, a single SNP (PADI4_100) and a two marker haplotype of that SNP and the neighbouring SNP (PADI4_104) were significantly associated with RA (P = 0.02 and P = 0.03 respectively). PADI4_100 was not associated with RA in a second sample set. PADI4 expression was four times greater in cases than controls (P = 0.004), but expression levels did not correlate with the levels of markers of inflammation. CONCLUSION: PADI4 is significantly overexpressed in the blood of RA patients but genetic variation within PADI4 is not a major risk factor for RA in Caucasians.

Arthritis, Rheumatoid↗
PubMed2000

Bacterial artificial chromosome libraries for mouse sequencing and functional analysis.

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.

Animals↗
PubMed1998

An improved approach for construction of bacterial artificial chromosome libraries.

Presented here are improved methodologies that enable the generation of highly redundant bacterial artificial chromosome/P1-derived artificial chromosome libraries, with larger and relatively uniform insert sizes. Improvements in vector preparation and enhanced ligation conditions reduce the number of background nonrecombinant clones. Preelectrophoresis of immobilized high-molecular-weight DNA removes inhibitors of the cloning process, while sizing DNA fragments twice within a single gel effectively eliminates small restriction fragments, thus increasing the average insert size of the clones. The size-fractionated DNA fragments are recovered by electroelution rather than the more common melting of gel slices with subsequent beta-agarase treatment. Concentration of the ligation products yields a 6- to 12-fold reduction in the number of electroporations required in preparing a library of desirable size. These improved methods have been applied to prepare PAC and BAC libraries from the human, murine, rat, canine, and baboon genomes with average insert sizes ranging between 160 and 235 kb.

Animals↗
PubMed1998

Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.

A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614,384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.

Animals↗
PubMed1995

Cholinergic regulation of amylase gene expression in the rat parotid gland. Inhibition by two distinct post-transcriptional mechanisms.

Stimulation of the beta-adrenergic or cholinergic muscarinic receptors are the principal mechanisms by which parotid salivary secretion is regulated in vivo. In this study we have examined the effects of cholinergic stimulation on amylase gene expression in dispersed rat parotid cells. [3H]Leucine incorporation into amylase and total protein was inhibited by carbamylcholine. Within 5 min of its addition, 10 microM carbamylcholine induced a 50-60% reduction in the rate of amylase synthesis which was sustained for more than 2 h. Blockade of the muscarinic receptor with atropine 8 min after addition of 10 microM carbamylcholine reversed the carbamylcholine-induced inhibition of amylase synthesis. When cells were exposed to carbamylcholine for 2 h before addition of atropine, there was only a slight reversal of inhibition. Carbamylcholine had no significant effect on the rate of total RNA synthesis but caused a progressive loss of amylase mRNA. After 2 h, amylase mRNA in cells treated with 10 microM carbamylcholine was 46% of control levels. Actinomycin D (5 micrograms/ml) lowered amylase mRNA by 8%; cycloheximide and phorbol 12-myristate 13-acetate had no effect. Isoprenaline (isoproterenol; at a concentration of 10 microM), which is an inducer of amylase gene transcription, elevated the amylase mRNA content by 30% after 2h. The calcium ionophore A23187 mimicked the effect of carbamylcholine by inhibiting [3H]leucine incorporation into amylase and lowering amylase mRNA content. The results suggest that acute stimulation of the muscarinic cholinergic receptor inhibits amylase biosynthesis in parotid cells not only by rapid attenuation of translation but also by causing a gradual loss of amylase mRNA, apparently by a Ca(2+)-dependent destabilization of the mRNA.

Amylases↗
PubMed1993

Adrenergic regulation of RNA synthesis in the rat parotid gland.

Adrenergic regulation of RNA synthesis by in vivo stimulated parotid glands and dispersed parotid lobules was studied by a combination of in vivo and in vitro methods. Following a single intraperitoneal injection of isoproterenol, [3H]uridine incorporation into RNA was increased by 50% after the first hour. Amylase mRNA content was also elevated within 1 hr and was 2-3-fold higher than control values at 4 hr. An increase in the rate of total protein synthesis was detectable after 2 hr, and maximal rates were achieved 6 hr after isoproterenol administration. In dispersed parotid lobules, both isoproterenol and epinephrine stimulated [3H]uridine incorporation and at optimal concentrations increased incorporation by almost 200%. Phenylephrine (10 microM) caused a slight increase of about 20% whereas methoxamine (10 microM) had no effect. Stimulation by epinephrine was reversed by propranolol, but not by either phentolamine or prazosin. The increase in RNA synthesis induced by isoproterenol or epinephrine was dose dependent and half-maximal stimulation required 5.0 x 10(-8) M isoproterenol and 7.9 x 10(-7) M epinephrine. Dibutyryl cyclic AMP also stimulated [3H]uridine incorporation, whereas 8-bromo cyclic GMP, A23187 and phorbol myristate acetate had no effect. The importance of protein phosphorylation in mediating the observed stimulation was evaluated using protein kinase and phosphatase inhibitors. N-[2-(Methylamino)ethyl]-5-isoquinolinesulphonamide, an inhibitor of cyclic nucleotide-dependent protein kinases, substantially diminished the isoproterenol-induced stimulation. Okadaic acid treatment of lobules increased [3H]uridine incorporation. Furthermore, okadaic acid synergistically potentiated the stimulatory effect of a suboptimal concentration of isoproterenol. The results demonstrate that activation of the beta-adrenergic receptor induces the synthesis of certain RNA species in the parotid gland and that protein phosphorylation by a cyclic AMP-dependent protein kinase is a key event in the signal transduction pathway.

Amylases↗
PubMed1993

Stimulation of RNA synthesis in rat parotid lobules by phorbol myristate acetate.

Incubation of parotid lobules with phorbol 12-myristate 13-acetate (PMA) transiently stimulated the incorporation of [3H]uridine into RNA. At 100 nM PMA, the rate of RNA synthesis during the first hour was 30% above control rates. The Ca2+ ionophore A23187 had no effect on either basal or PMA-stimulated RNA synthesis. Stimulation by 100 nM PMA was reversed by the protein kinase C inhibitor staurosporin (10 nM). When PMA was added together with isoproterenol or okadaic acid, both of which are potent activators of RNA synthesis, the increase in RNA synthesis was additive rather than synergistic. The results suggest that in the rat parotid gland, protein kinase C induces the rapid transcription of certain cellular genes by a mechanism that is independent of the beta-adrenergic receptor-activated pathway.

Alkaloids↗