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N W Chong

Publications and source records attributed to N W Chong.

18 recordsLinked to original sources
PubMed2000

Characterization of the chicken serotonin N-acetyltransferase gene. Activation via clock gene heterodimer/E box interaction.

The abundance of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) mRNA in the chicken pineal gland exhibits a circadian rhythm, which is translated into a circadian rhythm in melatonin production. Here we have started to elucidate the molecular basis of the circadian rhythm in chicken AANAT (cAANAT). The 5'-flanking region of the cAANAT gene was isolated and found to contain an E box DNA element that confers strong luciferase reporter activity. In transfection experiments using chicken pineal cells, an E box mutation dramatically decreased reporter activity. Northern blot analysis indicated that several putative clock genes (bmal1, Clock, and MOP4) are co-expressed in the chicken pineal gland. bmal1 mRNA is expressed in a rhythmic manner in the chicken pineal gland, with peak levels at early subjective night, coincident with the increase in cAANAT expression. Co-transfection experiments in COS cells demonstrated that chicken BMAL1/CLOCK and human BMAL1/MOP4 heterodimers bound the AANAT E box element and enhanced transcription. These observations suggest that binding of clock gene heterodimers to the cAANAT E box is a critical element in the expression of the cAANAT gene in vitro.

ARNTL Transcription Factors↗
PubMed1998

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina.

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.

Animals↗
PubMed1998

Reduction of GABA and glutamate transporter messenger RNAs in the severe-seizure genetically epilepsy-prone rat.

The genetically epilepsy-prone rat is an animal model of inherited generalised tonic-clonic epilepsy that shows abnormal susceptibility to audiogenic seizures and a lowered threshold to a variety of seizure-inducing stimuli. Recent studies suggest a crucial role for glutamate and GABA transporters in epileptogenesis and seizure propagation. The present study examines the levels of expression of the messenger RNAs encoding the glial and neuronal glutamate transporters, GLT-1 and EAAC-1, and the neuronal GABA transporter, GAT-1, in paired male genetically epileptic-prone rats and Sprague Dawley control rats using the technique of in situ hybridization. In a parallel study, semiquantitative immunoblotting was used to assess GLT-1 and EAAC-1 protein levels in similarly paired animals. Animals were assessed for susceptibility to audiogenic seizures on six occasions, and killed seven days following the last audiogenic stimulus exposure. Rat brains were processed for in situ hybridization with radioactive 35S-labelled oligonucleotide probes (EAAC-1 and GAT-1), 35S-labelled riboprobes (GLT-1), and Fluorescein-labelled riboprobes (GLT-1 and GAT-1) or processed for immunoblotting using subtype-specific antibodies for GLT-1 and EAAC-1. Semiquantitative analyses were carried out on X-ray film autoradiograms in several brain regions for both in situ hybridization and immunoblotting studies. Reductions in GAT-1 messenger RNA were found in genetically epileptic-prone rats in all brain regions examined (-8 to -24% compared to control). Similar reductions in GLT-1 messenger RNA expression levels were seen in cortex, striatum, and CA1 (-8 to -12%) of genetically epileptic-prone rats; the largest reduction observed was in the inferior colliculus (-20%). There was a tendency for a reduced expression of EAAC-1 messenger RNA in most regions of the genetically epileptic-prone rat brain although this reached statistical significance only in the striatum (-12%). In contrast, no significant differences in GLT-1 and EAAC-1 protein between genetically epileptic-prone rats and control animals were observed in any region examined, although there was a tendency to follow the changes seen with the corresponding messenger RNAs. These results show differences in the messenger RNA expression levels of three crucial amino acid transporters. For the two glutamate transporters, GLT-1 and EAAC-1, differences in messenger RNA levels are not reflected or are only partially reflected in the expression of the corresponding proteins.

ATP-Binding Cassette Transporters↗
PubMed1996

Rapid identification of novel genes expressed in a circadian manner in rat suprachiasmatic nuclei.

The circadian clock in mammals is located in the suprachiasmatic nuclei (SCN). There is evidence that changes in gene expression are central to its mechanism. We are engaged in identifying the genes involved. The small size of the SCN, the large number of mammalian genes and the need to identify those differentially expressed over 24 h have required novel experimental procedures. mRNA differential display and an improved tissue micropunching method have been used to examine temporal changes in gene expression in the SCN of rats maintained in constant darkness. Several of the displayed cDNA species were found to be differentially expressed; they show no homology with published sequences. Riboprobes of these cDNA species were used for in situ hybridization. Emulsion-dipped sections confirmed that at least two of these differentially displayed mRNAs are expressed in a circadian manner.

Animals↗
PubMed1996

Circadian variation of EAAC1 glutamate transporter messenger RNA in the rat suprachiasmatic nuclei.

Using in situ hybridization, we examined temporal changes of the EAAC1 glutamate transporter mRNA within the suprachiasmatic nuclei (SCN) of rats in constant darkness. Film autoradiographs showed that the SCN and supraoptic nuclei (SON) contained a marked density of hybridization signal. Analysis of silver grains per cell in emulsion-dipped sections indicated that cellular expression of EAAC1 mRNA in the SCN was elevated during the latter part of the subjective night and at the beginning of the subjective day, with a peak at circadian time 23.1 as determined by cosinor analysis. The times at which EAAC1 mRNA is highest correspond to the time points at which extracellular glutamate, a neurotransmitter that putatively mediates photic entrainment, has been reported to be low within the SCN. The presence of EAAC1 mRNA in the SCN and SON may partially explain the resistance of these nuclei to glutamate receptor-mediated excitotoxins; furthermore, the raised level preceding subjective dawn in the SCN may ensure sub-toxic levels of extracellular glutamate at the onset of photic stimulation during the LD cycle. In contrast, cellular expression of EAAC1 mRNA in the cingulate cortex and reticular thalamus remained constant at all time points studied. These results suggest that there is circadian control of the EAAC1 mRNA by the clock intrinsic to the SCN.

Amino Acid Transport System X-AG↗
PubMed1995

Structural requirements at the melatonin receptor.

1. High affinity, specific binding sites for the pineal hormone, melatonin (5-methoxy N-acetyltryptamine) can be detected in chick brain membranes by use of the radiolabelled agonist, 2-[125I]-iodomelatonin (2-[125I]-aMT). 2. The affinity of a number of analogues of melatonin at the 2-[125I]-aMT binding site was determined and compared with an analysis of their electronic structure and significant quantitative relationships obtained. 3. The best correlations indicated that binding affinity was correlated with delta E, the difference between the frontier orbital energies, and QNH, the electron density in the highest occupied molecular orbital of the side-chain nitrogen atom. 4. These findings suggest that ligand binding may involve hydrogen bonding between the 5-methoxy and amide moieties of melatonin and complementary amino acid residues, and charge transfer interactions between the indole ring of melatonin and an aromatic amino acid in the receptor binding site. 5. A molecular model of a putative binding site is proposed based on the predicted amino acid sequence of the cloned Xenopus laevis melanophore melatonin receptor and the quantitative structure-affinity relationships observed in the present study.

Animals↗
PubMed1994

Thermodynamic analysis of agonist and antagonist binding to the chicken brain melatonin receptor.

1. The binding of 2-[125I]-iodomelatonin to chicken brain membranes, and the inhibition of binding by melatonin, N-acetyltryptamine and luzindole, were examined at temperatures between 4 degrees C and 37 degrees C. 2. At all temperatures studied, the binding affinity (Kd or Ki) for 2-[125I]-iodomelatonin, melatonin (both agonists) and, to a lesser extent, N-acetyltryptamine (a partial agonist) was reduced by inclusion of guanosine triphosphate (GTP, 1 mM) in the assay. GTP did not affect the Ki for luzindole, a melatonin receptor antagonist. 3. The maximal density of binding sites (Bmax) was not affected by temperature but the Kd showed a peak at 21 degrees C with lower values at both higher and lower temperatures giving curvilinear van't Hoff plots (lnKA vs l/temperature). 4. Derived changes in entropy (delta S degree) and enthalpy (delta H degree) of binding for all of the melatonin ligands decreased as temperature increased. 5. The affinity, and thus the free energy of binding, delta G degree, of these ligands at the melatonin receptor have identical values at several temperatures yet at these temperatures delta S degree and delta H degree were very different, implying that more than one intermolecular force must be involved in the binding of ligand and receptor. 6. Conceivably, the large positive delta S degree observed at low temperatures, perhaps as a result of hydrophobic interactions, is compensated by a corresponding, but opposite, change in enthalpy at higher temperatures. However, it is not clear what type of binding force(s) would show such a temperature-dependence. 7. These studies suggest that caution must be exercised in the molecular interpretation of derived measures of delta S degree and delta H degree obtained from direct measurements of delta G degree.

Animals↗
PubMed1993

N-bromoacetyl 5-methoxytryptamine: an irreversible melatonin ligand?

The effects of N-bromoacetyl 5-methoxytryptamine (BraMT) and some related bromoacyl melatonin and tryptamine derivatives on 2-[125I]iodomelatonin (2-[125I]aMT) binding in chicken brain membranes were examined. All analogues displaced specific binding to chicken brain membranes in a concentration-dependent manner. Preincubation of chicken brain membranes with BraMT (10(-8)M) did not significantly alter either 2-[125I]aMT binding site affinity (Kd) or density (Bmax). SDS polyacrylamide gel electrophoresis of chicken brain membrane proteins preincubated with 2-[125I]BraMT indicated that numerous proteins were labelled. The incorporation of radiolabel into these proteins was not blocked by melatonin, 2-iodomelatonin or N-(1,4 dinitrophenyl)-5-methoxytryptamine (ML-23), but was reduced by BraMT. In contrast to previous reports on hamster and rat brain membranes, the present results provide no evidence that BraMT or 2-[125I]BraMT affinity labels specific melatonin binding proteins in chicken brain.

5-Methoxytryptamine↗
PubMed1993

Antigonadal effects of two novel melatonin analogues in adult Djungarian hamsters.

This study examined the effects of two novel melatonin analogues in adult Djungarian hamsters housed under long photoperiod (LD 16:8). Daily injection (10 micrograms, s.c.) of either melatonin, 5-methoxy N-butanoyltryptamine (bMT), or 5-methyl N-butanoyltryptamine (5-MebT) 3 hr before lights off for 8 weeks led to a significant decrease in paired testis weight compared to vehicle-injected controls. The reduction in testis weight was of similar magnitude with melatonin and bMT, but 5-MebT was not as effective. The affinity of the analogues was determined in competition experiments using chicken brain membranes and 2-[125I]iodomelatonin (2-[125I]aMT). Replacing the N-acetyl side-chain of melatonin with an N-butanoyl group increased affinity for the chicken brain 2-[125I]aMT binding site, but exchanging the 5-methoxy group of melatonin for a 5-methyl group reduced affinity. These studies show that these analogues not only inhibit 2-[125I]aMT binding in vitro but also mimic melatonin's antigonadal activity in vivo.

Animals↗
PubMed1992

The ontogeny of 2-[125I]iodomelatonin binding sites in chicken brain.

The characteristics of the binding sites for 2-[125I]iodomelatonin were studied in chicken brain membranes during development. Specific binding, defined using cold melatonin (1 microM), was detected as early as 8-day-old embryos. Scatchard analysis of saturation experiments showed that 2-[125I]iodomelatonin binds to a single class of site at all ages tested (8-day-old embryos to 3-month-old chicks). Binding affinity (Kd) did not change during development (18-31 pM), but the maximal number of binding sites (Bmax) increased until embryonic day 18, and then remained relatively constant until 30 days of age. A further increase in Bmax was seen at 3 months of age. Guanosine 5'-triphosphate (GTP, 1 mM) inhibited 2-[125I]iodomelatonin binding at all ages suggesting that the melatonin binding site is coupled to a guanine nucleotide binding protein at a very early stage of development. Competition experiments with a number of melatonin analogues indicated that the binding site detected in the brain at embryonic day 8 was pharmacologically identical to that observed 15 days after hatching.

Aging↗
PubMed1992

Characterization of 2-[125I]iodomelatonin binding sites in the brain of a marsupial, Bennett's wallaby (Macropus rufogriseus rufogriseus).

1. Specific high affinity binding of 2-[125I]iodomelatonin was detected in the brain of the pouch young of a marsupial, Bennett's wallaby. 2. Binding was rapid, stable, saturable and reversible. 3. Scatchard analysis indicated a single class of high affinity binding sites with an equilibrium dissociation constant (Kd) of 68 +/- 13 pM, a maximal number of binding sites (Bmax) of 0.7 +/- 0.1 fmol/mg protein and a Hill coefficient (nH) of 1.12 +/- 0.10. 4. Specific binding was inhibited by GTP (1 mM) indicating that the melatonin receptor is coupled to a guanine nucleotide binding protein, and by melatonin and closely related analogues with a potency order identical to that reported previously in the brain of eutherian mammals, birds and a reptile. 5. These studies suggest that the melatonin receptor is well-conserved through evolution.

Animals↗
PubMed1991

Pharmacological identity of 2-[125I]iodomelatonin binding sites in chicken brain and sheep pars tuberalis.

2-[125I]Iodomelatonin was used to compare the binding and pharmacological characteristics of the melatonin receptor sites found in chicken brain and sheep pars tuberalis. Scatchard analysis and kinetic experiments showed that 2-[125I]iodomelatonin binds to a single class of site in both tissues with high affinity (Kd 20-34 pM). Competition experiments, using 21 analogues of melatonin, gave inhibition constants (Ki) for the two sites which were significantly correlated (r = 0.985, n = 21, P less than 0.0001). We conclude that the 2-[125I]iodomelatonin binding sites in sheep pars tuberalis and chicken brain have identical binding and pharmacological characteristics.

Animals↗
PubMed1991

Guanine nucleotides regulate 2-[125I]iodomelatonin binding sites in chick retinal pigment epithelium but not in neuronal retina.

The characteristics of the binding sites labeled by the radioligand 2-[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 +/- 55 pM and a total number of binding sites (Bmax) of 25.4 +/- 2.2 fmol/mg of protein for neuronal retina. For RPE the KD was 34.1 +/- 2.2 pM and the Bmax 59.5 +/- 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin greater than 2-chloromelatonin greater than melatonin greater than 6-chloromelatonin greater than 6-hydroxymelatonin greater than N-acetylserotonin greater than 6-methoxyharmalan greater than 5-hydroxytryptamine. Linear regression of log Ki values from neuronal retina and RPE gave a highly significant correlation (r = 0.994, n = 8; p less than 0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.

Adenine Nucleotides↗
PubMed1990

L-NG-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro.

1. The effect of L-NG-nitro arginine (L-NOARG) was compared with that of L-NG-monomethyl arginine (L-NMMA) on vasodilatation of the isolated aorta of the rabbit and perfused mesentery of the rat in response to acetylcholine (ACh) and sodium nitroprusside (NP). 2. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) produced concentration-related contraction of the rabbit aorta precontracted with phenylephrine (700-900 nM). Similarly, L-NOARG (10-200 microM) and L-NMMA (30-100 microM) elevated perfusion pressure of the noradrenaline (NA, 0.6-2.5 mM)-preconstricted rat mesentery preparation. 3. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) caused concentration-related inhibition of the vasodilator effect of ACh (0.01-1.0 microM) on the rabbit aorta without influencing responses to NP (0.03-0.5 microM). L-NOARG methyl ester (30 microM) also inhibited ACh-induced vasorelaxation with similar potency to NOARG. L-arginine (30-150 microM) but not D-arginine (100 microM) caused graded reversal of the inhibitory effect of both L-NOARG (15 microM) and L-NMMA (30 microM). Complete reversal of the effect of both inhibitors was achieved with 150 microM L-arginine. L-Alanine (50 microM), L-arginosuccinic acid (5 microM), L-citrulline (50 microM), L-methionine (50 microM) and L-ornithine (50 microM) failed to reverse the inhibitory effect of L-NOARG (15 microM). 4. L-NOARG (10-200 microM) and L-NMMA (30-100 microM) inhibited the vasodilator effect of ACh (0.006-18.0 nmol) in the rat mesentery without affecting vasodilatation due to NP (1.1-11.1 nmol). L-Arginine (100 microM) but not D-arginine (100 microM) produced partial reversal of the effect of L-NOARG (30 microM) and L-NMMA (30 microM). 5. L- and D-N'-butyloxycarbonyl No-nitro arginine (100 microM) produced modest (approximately 20%) inhibition of the effect of ACh on the rabbit aorta; this effect was not reversible with L-arginine (100 microM). L-Namonocarbobenzoxy arginine (L-NMCA, 5O microM), L-N-NG-dicarbobenzoxy arginine (L-NDCA, 5 microM) and L-NG-tosyl arginine (50 microM) were inactive. 6. These results identify L-NOARG as a potent, L-arginine reversible inhibitor of endothelium-dependent vasodilatation. The available data suggests that L-NOARG, like L-NMMA, inhibits endothelial nitric oxide (NO) biosynthesis.

Acetylcholine↗
PubMedDate not supplied

Melatonin synthesis pathway: circadian regulation of the genes encoding the key enzymes in the chicken pineal gland and retina.

The mRNAs encoding three enzymes of the melatonin synthesis pathway (tryptophan hydroxylase (TPH), arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyl-transferase (HIOMT)) are expressed with a day/night rhythm in the chicken pineal gland and retina. TPH and AANAT mRNA levels reach their peak at night. HIOMT mRNA levels peak at night in the retina, but during the day in the pineal gland. In this tissue, the rhythm of TPH, AANAT and HIOMT mRNA levels persisted in constant darkness (DD), both in vivo and in vitro, indicating that the three genes are controlled by the circadian oscillator of the chicken pineal. In the retina, the rhythms of TPH and AANAT mRNA levels also persisted in DD in vivo, suggesting that they are driven by a circadian oscillator. In contrast, the rhythm of HIOMT mRNA in the retina appeared to be controlled only by light. The clones of chicken AANAT and HIOMT genes that we have isolated should help us to understand the molecular mechanisms of: 1) their transcriptional regulation by circadian oscillators and by light; 2) their tissue-specific expression in the pineal gland and the retina.

ARNTL Transcription Factors↗