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Biomedical subjects

D L Wong

Publications and source records attributed to D L Wong.

At least 19 recordsLinked to original sources
PubMed1999

Phenylethanolamine N-methyltransferase gene expression. Sp1 and MAZ potential for tissue-specific expression.

Phenylethanolamine N-methyltransferase (PNMT) promoter-luciferase reporter gene constructs (pGL3RP863, pGL3RP444, and pGL3RP392) transfected into COS1, RS1, PC12, NIH/3T3, or Neuro2A cells showed the highest basal luciferase activity in the Neuro2A cells. DNase I footprinting with Neuro2A cell nuclear extract identified protected PNMT promoter regions spanning the -168/-165 and -48/-45 base pair Sp1/Egr-1 binding sites. Gel mobility shift assays and transient transfection assays using site-directed mutant PNMT promoter-luciferase reporter gene constructs indicated that the elevated basal luciferase activity in the Neuro2A cells was mediated by Sp-1. Furthermore, activation of the PNMT promoter by Sp1 depends on both its binding affinity for its cognate target sequences and its intracellular concentrations. When Sp1 levels were increased through an expression plasmid, luciferase reporter gene expression rose well beyond basal wild-type levels, even with either Sp1 binding element mutated. Finally, another transcription factor expressed in the Neuro2A cells competes with Sp1 by interacting with DNA sequences 3' to the -48 base pair Sp1 site to prevent Sp1 binding and induction of the PNMT promoter. The DNA consensus sequence, Southwestern analysis, and gel mobility shift assays with antibodies identify MAZ as the competitive factor. These findings suggest that Sp1 may potentially contribute to the tissue-specific expression of the PNMT gene, with the competition between Sp1 and MAZ conferring additional tissue-specific control.

Binding Sites↗
PubMed1999

Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors.

Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.

Androstanols↗
PubMed1998

Phenylethanolamine N-methyltransferase gene expression: synergistic activation by Egr-1, AP-2 and the glucocorticoid receptor.

The gene encoding the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), is transcriptionally activated by Egr-1, AP-2, and the glucocorticoid receptor (GR). Stimulation by AP-2 requires its synergistic interaction with an activated GR. The present studies show that the GR also cooperates with Egr-1 or the combination of Egr-1 and AP-2 to activate the PNMT promoter. Together Egr-1, AP-2, and the GR can induce PNMT promoter-mediated luciferase reporter gene expression beyond the sum of their independent contributions as well as synergistically activate the endogenous PNMT gene leading to marked increases in PNMT mRNA. Examination of the effects of mutation of the AP-2 or Egr-1 binding sites on PNMT promoter activation by DEX and the factor binding to the remaining intact site or by all three transcriptional activators showed changes in luciferase reporter gene expression which suggest that DNA structure may be altered thereby reducing or enhancing synergistic activation. It also appears that the -165 bp Egr-1 site may not be critical for the synergism observed between Egr-1, AP-2 and the GR. When the glucocorticoid response element (GRE) within the PNMT promoter was mutated, PNMT promoter activation by Egr-1 and DEX, AP-2 and DEX or all three showed both inhibition and enhancement, even when the GRE was completely eliminated. These observations indicate that induction of PNMT gene transcription may occur either through GR interaction with other transcriptional proteins after binding to its cognate GRE or through direct protein-protein interaction in the absence of GRE binding. While the mechanisms by which Egr-1 and the GR and Egr-1, AP-2 and the GR function cooperatively to stimulate PNMT promoter activity remain to be elucidated, this synergistic stimulation of the PNMT promoter by these factors may provide important in vivo and in vitro regulatory control of the PNMT gene.

Animals↗
PubMed1998

Electrospray ionization mass spectrometric characterization of photocrosslinked DNA-EcoRI DNA methyltransferase complexes.

We describe a novel strategy combining photocrosslinking and HPLC-based electrospray ionization mass spectrometry to identify UV crosslinked DNA-protein complexes. Eco RI DNA methyltransferase modifies the second adenine within the recognition sequence GAATTC. Substitution of 5-iodouracil for the thymine adjacent to the target base (GAATTC) does not detectably alter the DNA-protein complex. Irradiation of the 5-iodouracil-substituted DNA-protein complex at various wavelengths was optimized, with a crosslinking yield >60% at 313 nm after 1 min. No protein degradation was observed under these conditions. The crosslinked DNA-protein complex was further analyzed by electrospray ionization mass spectrometry. The total mass is consistent with irradiation-dependent covalent bond formation between one strand of DNA and the protein. These preliminary results support the possibility of identifying picomole quantities of crosslinked peptides by similar strategies.

Base Sequence↗
PubMed1998

Glucocorticoid-dependent action of neural crest factor AP-2: stimulation of phenylethanolamine N-methyltransferase gene expression.

AP-2 is a vertebrate transcription factor expressed in neural crest cells and their derivative tissues, including the adrenal medulla, where epinephrine is produced. AP-2 is shown to stimulate expression of the gene encoding the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT). However, stimulation of the PNMT gene by AP-2 requires glucocorticoids and appears to be mediated through the interaction of AP-2 with activated type II glucocorticoid receptors. Mutation of AP-2 and/or glucocorticoid receptor binding elements within the PNMT promoter disrupts the ability of AP-2 and glucocorticoids to induce PNMT promoter activity. These findings suggest, in the case of PNMT, that AP-2 stimulates gene expression through a novel glucocorticoid-dependent mechanism.

Adrenal Medulla↗
PubMed1998

Vitamin B-6 deficiency and level of dietary protein affect hepatic tyrosine aminotransferase activity in cats.

Total activity [pyridoxal 5'-phosphate (PLP) added in the assay] of hepatic tyrosine aminotransferase (TAT) measured in cats at 0300, 0900, 1500 and 2100h was 10.3 +/-1.1, 14.0 +/- 0.7, 9.8 +/- 1.3 and 11.0 +/- 0.7 nkat/g liver, indicating little diurnal variation. Activity after 18 h of food deprivation was 10.0 +/- 0.3 nkat/g liver, also not different from cats that were eating ad libitum. These findings support the idea that cats have only limited changes in the activity of hepatic TAT compared with rats. Total TAT activity was measured in cats fed high protein (550 g/kg) and low protein (180 g/kg) diets for 4 wk. Cats fed a high protein diet had activities significantly higher (about twice) than cats fed the low protein diet. Hepatic TAT activity of vitamin B-6-deficient cats (diet without pyridoxine for 9 wk) was compared with cats given the same diet with 8 mg pyridoxine/kg. Total hepatic TAT activity in deficient cats was significantly (P < 0.05) lower per gram soluble or total protein (but not per gram liver) than control cats; holoenzyme activity and percentage of active enzyme in deficient cats were also significantly lower by 75 and 64%, respectively. The apparent Km of TAT from cats for tyrosine (2.1 mmol/L) was similar to that for rats (1.9 mmol/L), but higher for PLP in cats (0.16 micromol/L) than rats (0.034 micromol/L). Part of the reason for the higher plasma tyrosine in vitamin B-6-deficient cats than rats is the higher Km of TAT for PLP in cats than rats.

Animals↗
PubMed1997

Adrenergic differentiation potential in PC12 cells: influence of sodium butyrate and dexamethasone.

The ability of sodium butyrate and dexamethasone to promote adrenergic differentiation in PC12 cells was examined using the gene encoding the epinephrine biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), as a marker. Sodium butyrate and dexamethasone independently stimulated expression of PNMT mRNA in PC12 cells, and the combined action of these drugs led to synergistic activation of the PNMT gene. Despite the induction of the PNMT gene, epinephrine is not produced in these cells, in part due to the absence of a corresponding induction in PNMT enzymatic activity. Another contributing factor appears to be a reduction in the precursor catecholamines, norepinephrine and dopamine, in the presence of sodium butyrate. Thus, while sodium butyrate and dexamethasone can induce PNMT gene expression, treatment of PC12 cells with these drugs appears insufficient for full acquisition of the adrenergic phenotype.

Adrenal Glands↗
PubMed1996

Brain specific proteins binding to the 3' UTR of the 5-HT2C receptor mRNA.

The 5-HT2C receptor2 is a prominent serotonin receptor that is uniquely expressed in the central nervous system and has been implicated in a variety of psychiatric diseases. While characterizing the 5-HT2C receptor gene, we observed that the mRNA contains a long 3' untranslated region that binds multiple brain proteins. Two proteins, molecular weights 55 and 58 kDa, were of particular interest because they were detected only in brain regions known to express the 5-HT2C receptor abundantly, namely, the hippocampus and cortex. These proteins bind with high affinity to the 5-HT2C receptor mRNA at its extreme 3' end (Kd = 1.8 nM), and binding can be specifically competed by selected regions of the 3' UTR. Furthermore, binding of the 55 and 58 kDa proteins to the mRNA is directionally specific and shows preference for an AU-rich loop containing 6 to 7 nucleotides. These results suggest the possibility that these two brain specific proteins may play a role in the post-transcriptional regulation of the 5-HT2C receptor, and that post-transcriptional control of 5-HT2C receptor expression may be an important regulatory mechanism which has not been previously reported for this serotonin receptor subtype.

Animals↗
PubMed1996

Expression of phenylethanolamine n-methyltransferase in the embryonic rat heart.

Phenylethanolamine N-methyltransferase (PNMT), the final enzyme in the pathway for epinephrine biosynthesis, serves as a marker for tissues and cells producing epinephrine. The present study examines the developmental expression of PNMT in the rat embryo. A transient burst in PNMT mRNA expression begins on embryonic day 9.5 (E9.5), peaks between E10.0 and E11.0, and declines to barely detectable levels by E13.0. Regional localization of PNMT mRNA and enzyme activity demonstrates that PNMT is concentrated in the heart. PNMT has not previously been reported to be expressed at these early stages of development, and its presence in the developing heart suggests that this embryonic tissue may produce epinephrine. Because this catecholamine is known to increase cardiac output and promote the growth of cardiomyocytes, local production of epinephrine by the heart could play an important role in the development of cardiac structure and function.

Animals↗
PubMed1996

Role of Egr-1 in cholinergic stimulation of phenylethanolamine N-methyltransferase promoter.

The effects of the cholinergic agonist carbachol on phenylethanolamine N-methyltransferase promoter activity and Egr-1 mRNA expression in PC12-derived RS1 cells were examined to investigate the potential involvement of Egr-1 in the neural regulation of phenylethanolamine N-methyltransferase gene expression. Carbachol stimulated luciferase expression in cells transfected with a rat phenylethanolamine N-methyltransferase promoter-luciferase reporter gene construct and also elevated Egr-1 mRNA levels in untransfected cells. Maximum induction of Egr-1 mRNA by carbachol was rapid (0.5 h), whereas by comparison, peak luciferase activity was delayed (6 h). In addition, carbachol stimulation of both luciferase and Egr-1 mRNA expression could be completely inhibited by atropine but not hexamethonium. Furthermore, bethanechol but not nicotine could mimic the effects of carbachol, indicating that carbachol activation was mediated through muscarinic cholinergic receptors. Finally, carbachol failed to stimulate luciferase expression in cells transfected with a mutant construct, in which the Egr-1 binding element in the phenylethanolamine N-methyl-transferase promoter was mutated. These results suggest that carbachol activates the phenylethanolamine N-methyltransferase promotor through stimulation of Egr-1 expression, and are consistent with the potential involvement of Egr-1 in the cholinergic activation of the phenylethanolamine N-methyltransferase gene.

Animals↗
PubMed1996

Effects of botulinum toxin type A injections on aerodynamic measures of spasmodic dysphonia.

The effects of botulinum toxin type A (BOTOX) injections on aerodynamic measures of phonation were examined in 30 patients with adductor spasmodic dysphonia. Patients received bilateral injections of 2.5 U of botulinum toxin type A in each thyroarytenoid muscle. Measures of air pressure, average airflow, coefficient of variation (CV) of airflow, and laryngeal resistance were obtained before the injections and at 2 and 10 weeks after the injections. These measures were also obtained from 12 normal subjects. Compared with normal subjects, the spasmodic dysphonia patients had significantly higher preinjection values for air pressure, CV of airflow, and laryngeal resistance. At 2 and 10 weeks after the botulinum toxin type A injections, the spasmodic dysphonia patients showed a significant increase in the average airflow values and a significant decrease in the CV of airflow values and the laryngeal resistance values. At 10 weeks after the injections, the values for the three measures began to approach the preinjection values. It is concluded that these aerodynamic measures of phonation can provide useful measures of treatment outcome in adductor spasmodic dysphonia.

Adult↗
PubMed1996

Surgical anatomy of the epitympanic space.

A clear understanding of the surgical anatomy of the epitympanic space is crucial in modern otologic surgery. The tortuous facial nerve within the tight confines of this region presents important surgical challenges, especially when normal anatomy is obscured by disease. Microdissections of 20 human temporal bones were carried out with particular emphasis on the spatial relations of the facial nerve with various temporal bone landmarks. Measurements were made through a standard transmastoid approach to ensure clinical relevance. A high margin of safety can be achieved by observing consistent landmarks and relations in dealing with the facial nerve. We demonstrated the limitations of the transmastoid approach, particularly in accessing the labyrinthine segment of the facial nerve.

Ear, Inner↗
PubMed1996

Evaluation of vocal function in unilateral vocal fold paralysis following thyroplastic surgery.

Perceptual, acoustic, and aerodynamic measures of vocal function were examined, pre- and post-treatment, in nine patients who had received thyroplastic (type I) surgery for unilateral vocal fold paralysis. Following thyroplasty, the patients showed significant improvements in the following perceptual and acoustic measures of vocal function: perceived breathiness, maximum phonation time, s/z ratio, vocal shimmer, and signal-to-noise ratio. Significant post-treatment changes were also observed in the following aerodynamic measures of phonation: air pressure, average airflow, laryngeal resistance, and breath group duration. These findings suggest that a combination of perceptual, acoustic, and aerodynamic measures of vocal function may be useful for comparing the relative effectiveness of different treatment procedures (i.e., thyroplasty vs. Teflon injection) in unilateral vocal fold paralysis.

Adult↗
PubMed1996

Neural stimulation of Egr-1 messenger RNA expression in rat adrenal gland: possible relation to phenylethanolamine N-methyltransferase gene regulation.

Metrazole, which reflexively activates the splanchnic nerve to the adrenal medulla, was used to investigate the physiological role of Egr-1 in the neural regulation of phenylethanolamine N-methyltransferase (PNMT) gene transcription in the rat adrenal gland. A single dose of this drug (70 mg/kg s.c.) rapidly and transiently induced Egr-1 mRNA, with a maximum 22.0-fold increase at 30 min after treatment, followed by a 3.7-fold increase in PNMT mRNA at 8 hr. In contrast, cocaine (15 mg/kg i.p.), which activates the hypothalamic-pituitary-adrenal axis, increased Egr-1 mRNA only 3-fold at 30 min, although it elevated PNMT mRNA comparably. Consistent with their mechanisms of activation, cocaine increased corticosterone levels 7.7-fold at 30 min, whereas metrazole modestly elevated this endogenous corticosteroid 2.5-fold. The cholinergic agonists nicotine (2 mg/kg l.p.) and muscarine (0.1 mg/kg i.p.) also elevated Egr-1 mRNA, with a peak 12- to 15-fold increase being apparent at 30 min after treatment, followed by a 1.7-to 2.0-fold rise in PNMT mRNA at 8 hr. In vitro, metrazole did not increase Egr-1 mRNA above levels observed with carbachol alone (100 microM) in PC-12-derived RS1 cells pretreated with this cholinergic agonist. Finally, splanchnic denervation partially blocked the metrazole-induced rise in Egr-1 mRNA (50% control), while having no effect on cocaine-induced changes in Egr-1 mRNA. These results provide further support for the involvement of Egr-1 in the neural regulation of PNMT gene expression in the rat adrenal gland.

Adrenal Glands↗
PubMed1995

Differential activation of the rat phenylethanolamine N-methyltransferase gene by Sp1 and Egr-1.

The rat phenylethanolamine N-methyltransferase (PNMT) gene contains overlapping consensus elements for the Sp1 and Egr-1 transcription factors located at -45 bp and -165 bp in the PNMT promoter. In the present study, we show that Sp1 and Egr-1 can specifically bind to these overlapping elements, that this binding appears to be mutually exclusive, and that binding site occupancy is dependent upon the concentration of each factor and its binding affinity for each site. Egr-1 binds to the -165 bp site with relatively high affinity (IC50 = 14 nM) and to the -45 bp site with relatively low affinity (IC50 = 1360 nM), whereas Sp1 binds to both sites with intermediate affinities (IC50 = 210 and 140 nM, respectively). Consistent with the DNA-binding data, Egr-1 stimulates PNMT promoter activity primarily through interaction with the -165 bp site, while Sp1 stimulates PNMT promoter activity by interacting with both the -45 bp and the -165 bp sites. These results show that Sp1 and Egr-1 are capable of differentially activating PNMT gene expression, thereby suggesting that different stimuli may control the activity of the PNMT gene by selectively regulating Sp1 and/or Egr-1.

Animals↗
PubMed1995

Role of transcription factor Egr-1 in phorbol ester-induced phenylethanolamine N-methyltransferase gene expression.

Transfection of PC12-variant RS1 cells with an Egr-1 expression construct has previously been shown to stimulate phenylethanolamine N-methyltransferase (PNMT) promoter activity, thus suggesting a putative role of Egr-1 as a factor regulating PNMT gene expression. To elucidate the physiological implication of this finding, the effects of phorbol 12-myristate 13-acetate (PMA) on PNMT promoter activity and Egr-1 expression were examined. PMA stimulated luciferase expression in RS1 cells transfected with a rat PNMT promoter-luciferase reporter gene construct, and also elevated both Egr-1 mRNA and Egr-1 protein levels in the untransfected cells. Further study on the concentration dependence of PMA action showed that the stimulation of luciferase expression correlated with the elevation of Egr-1 mRNA level. Finally, the stimulatory action of PMA on luciferase expression was dramatically diminished in the cells transfected with a mutant construct in which the Egr-1 binding site in PNMT promoter was mutated. These findings suggest that PMA-stimulated PNMT gene expression requires the enhancement of Egr-1 expression, thus providing further evidence for the physiological role of Egr-1 in the regulation of PNMT gene expression in the adrenergic cell.

Animals↗