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T H Joh

Publications and source records attributed to T H Joh.

At least 55 records · Page 3Linked to original sources
PubMed1996

A transgenic mouse model to study transsynaptic regulation of tyrosine hydroxylase gene expression.

Previous studies demonstrated that 9 kb of the rat tyrosine hydroxylase (TH) 5' flanking sequence directed appropriate spatiotemporal expression of a lacZ reporter gene to catecholaminergic cells in the CNS of transgenic mice. In the present study, specificity of transgene expression was further extended to demonstrate cell type-specific functional regulation of lacZ expression using manipulations known to alter endogenous TH expression. Alterations in lacZ reporter expression should parallel changes in endogenous TH levels if the DNA elements mediating these functional changes of TH expression in vivo reside within the 9 kb of the TH promoter region. Naris closure induced an activity-dependent decrease of TH expression in dopaminergic periglomerular cells in the olfactory bulb that was paralleled by down-regulation of lacZ expression in the transgenic mice. Densitometry and image analysis were used to quantify lacZ expression following acute reserpine administration (5 mg/kg s.c.), which up-regulates endogenous TH. At 48 h postinjection, analysis of OD values indicated a significant increase of X-gal staining in the locus coeruleus and ventral tegmental area but not in the substantia nigra or olfactory bulb of reserpine-treated transgenic animals. These data showed that the 9-kb sequence also mediates cell type-specific transsynaptic regulation of reporter gene expression. Analysis of this transgenic animal offers a useful model system to study in vivo regulation of TH gene expression.

Animals↗
PubMed1995

Identification of a negative regulatory element in the 5'-flanking region of the human dopamine beta-hydroxylase gene.

Transient transfection experiments indicate that a 5'-flanking upstream domain, residing between -437 and -262 bp of the human dopamine beta-hydroxylase (DBH) gene, has a cell type-specific silencer function. This domain contains a putative silencer motif (which we designate DBH negative regulatory element, DNRE), showing sequence homology with the neural-restrictive silencer element (NRSE or RE-1) recently characterized in type II sodium channel, SCG10 and synapsin I genes. When the DNRE was placed at the proximal 262 bp of the homologous (DBH) promoter, it exhibited strong silencer activity both in DBH-expressing SK-N-BE(2)C as well as in DBH-nonexpressing HeLa cells. In addition, the DNRE also exhibited modest silencer activity upon a heterologous tk (herpes simplex virus thymidine kinase) promoter in both cell lines. Electrophoretic mobility shift assay demonstrated that nuclear extracts from both SK-N-BE(2)C and HeLa cells contain protein(s) that specifically bind to the DNRE. Formation of this DNRE/protein complex was specifically inhibited by an excess of unlabeled DNRE or NRSE. Finally, a similar sequence motif residing in the corresponding upstream area of the rat DBH gene also had a negative regulatory function, indicating that the silencer function of the DNRE is conserved in human and rat DBH genes.

Base Sequence↗
PubMed1995

Innervation-independent changes in the mRNAs encoding tyrosine hydroxylase and the norepinephrine transporter in rat adrenal medulla after high-dose reserpine.

To determine whether a trans-synaptic mechanism triggered the effects of reserpine on adrenomedullary mRNAs encoding the norepinephrine transporter and tyrosine hydroxylase, we administered 10 mg/kg reserpine to rats after unilateral splanchnicotomy, and examined their adrenal medullas using quantitative in situ hybridization. Splanchnicotomy did not alter the decrease in norepinephrine transporter mRNA that follows reserpine administration, but diminished the reserpine-induced increase in tyrosine hydroxylase mRNA by almost 80%. Despite the latter effect, reserpine still induced a significant increase in tyrosine hydroxylase mRNA in denervated adrenal medullas, compared to vehicle-treated adrenal medullas. These results show that a trans-synaptic mechanism does not trigger the decrease in adrenomedullary norepinephrine transporter mRNA following reserpine. In addition, an innervation-independent mechanism mediates a portion of the reserpine-induced increase in adrenomedullary tyrosine hydroxylase mRNA.

Adrenal Medulla↗
PubMed1995

Differential in vivo regulation of mRNA encoding the norepinephrine transporter and tyrosine hydroxylase in rat adrenal medulla and locus ceruleus.

To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5-kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine-secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady-state levels of norepinephrine transporter mRNA in the adrenal medulla by approximately 65% and in the locus ceruleus by approximately 25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine depletion.

Adrenal Medulla↗
PubMed1994

Increased c-fos expression in nucleus of the solitary tract correlated with conditioned taste aversion to sucrose in rats.

The pattern of neuronal activation in the rat nucleus of the solitary tract (NTS) in response to a standard gustatory stimulus was examined using c-Fos-like immunoreactivity (c-FLI) before and after conditioned taste aversion (CTA) formation. While unconditioned oral infusions of sucrose solution did not induce c-FLI in the NTS, after three pairings of sucrose with lithium chloride injections, sucrose induced c-FLI in the medial intermediate NTS 1 h after oral infusion. Extinction of the CTA by repeated infusions of sucrose alone reversed the induction of c-FLI.

Animals↗
PubMed1994

Gene expression in central cholinergic neurons in response to axotomy and deafferentation.

Although the molecular and cellular responses to injury in the central nervous system (CNS) have been widely investigated, few studies have examined the potential variations between direct and indirect neuronal injury. To differentiate between the response to axotomy and deafferentation, two central cholinergic populations were analyzed: the horizontal limb of the diagonal band of Broca (HLDB) and the interneurons in the corpus striatum (CS). At time points from one hour to eight weeks postinjury the levels of choline acetyltransferase (ChAT) mRNA and protein were assessed by in situ hybridization and immunohistochemistry. Also examined was the expression of the immediate early gene product, c-fos. One week post axotomy, neurons in the HLDB exhibited an increase in the levels of ChAT mRNA without a concomitant increase in ChAT protein, followed by a steady decrease reaching a nadir in both parameters at eight weeks. In contrast, a transient increase occurred at one week postdeafferentation in the levels of both ChAT mRNA and protein in the interneurons of the CS. Axotomized neurons in the HLDB did not exhibit either c-fos mRNA or protein expression, while robust fos induction occurred after one hour in deafferented neurons in the CS. These data demonstrate that the molecular and cellular responses differ following direct and indirect neuronal injury. Furthermore, they suggest that in these central cholinergic populations deafferentation may result in cellular hyperactivity and cell survival while axotomy results in decreased cellular activity and subsequent cellular regression.

Animals↗
PubMed1994

Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli.

Recombinant mouse tryptophan hydroxylase (TPH) was expressed in large quantities in Escherichia coli strain MC 1061, using a bacterial expression vector, pKS, containing the full coding region of mouse TPH. Specific polyclonal antiserum to the subunit of the recombinant mouse TPH was produced in rabbit by injecting the TPH band cut from SDS-polyacrylamide slab gels. The resultant antiserum recognized a single identical protein band (MW = 54,000) from rat dorsal raphe area, pineal gland, and brain stem by Western blot analysis. The specific activity of recombinant mouse TPH obtained was equivalent to that of TPH purified from rat brain. The recombinant mouse TPH was stable for 3 days at 4 degrees C but lost 25% of the original activity for the same period at -20 degrees C. A serotonin concentration greater than 1 mM inhibited TPH activity under our assay conditions in a concentration-dependent fashion. The recombinant mouse TPH exhibited a charge isozyme corresponding to that of pineal gland TPH as applied to chromatofocusing column chromatography. Taken together, our results show that recombinant mouse TPH, expressed in large quantities in E. coli is not only enzymatically highly active but also shares many biochemical and immunochemical properties with native TPH.

Animals↗
PubMed1994

5' upstream DNA sequence of the rat tyrosine hydroxylase gene directs high-level and tissue-specific expression to catecholaminergic neurons in the central nervous system of transgenic mice.

Tyrosine hydroxylase (TH), the first and rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed within central and peripheral catecholaminergic cells. To delineate DNA sequences necessary for tissue-specific expression of the rat TH gene, transgenic mice were produced containing 0.15 kb, 2.4 kb, and 9.0 kb of 5' flanking sequence fused to the E. coli lacZ (beta-galactosidase) reporter gene. The reporter gene expression in the transgenic animals was monitored by both X-gal histochemical staining and beta-galactosidase immunohistochemistry and compared to TH mRNA and protein expression. Transgenic mice bearing 9.0 kb, but not the smaller constructs with either 2.4 kb or 0.15 kb of 5' flanking sequence, fused to lacZ were able to direct high level expression of beta-galactosidase at levels equivalent to the endogenous TH in central catecholaminergic cells, and to a lesser degree to adrenal gland. Previously, 4.8 kb of 5' flanking region was reported to contain some tissue-specific element(s) determined by chloramphenicol acetyltransferase (CAT) assay using regional brain dissections and was not able to demonstrate cellular localization of the CAT expression [2]. Using histological procedures which allow for spatial resolution, this study demonstrated that the crucial catecholaminergic neuron-specific DNA element(s) resides between -9 kb and -2.4 kb of the 5' flanking region of the rat TH gene; this assertion is substantiated by the high-level of tissue-specific expression of lacZ in catecholaminergic cells.

Adrenal Glands↗
PubMed1994

Early induction of rat brain tryptophan hydroxylase (TPH) mRNA following parachlorophenylalanine (PCPA) treatment.

Tryptophan hydroxylase (TPH) is the first and presumably rate-limiting enzyme in serotonin (5-HT) biosynthesis. End-product inhibition of rate-limiting enzymes is common and 5-HT is known to inhibit TPH activity in vivo. However, it is not known whether levels of 5-HT could also be involved in the regulation of the TPH gene. In order to determine whether TPH gene regulation is dependent on the 5-HT concentration, 5-HT levels were reduced by the administration of parachlorophenylalanine (PCPA). PCPA is a potent, specific and irreversible inhibitor of TPH activity which drastically reduces 5-HT concentration in the 5-HT neurons and terminals. When PCPA was administered, TPH activity in both cell bodies and nerve terminal areas, was reduced to 10% of control values and recovered to the control levels by day 7 in raphe nucleus, and within 14 days in the hypothalamus. In serotonergic terminal areas, 5-HT could not be detected immunohistochemically at day 1, but slowly recovered within 2 weeks. At all time points examined, aromatic L-amino acid decarboxylase (AADC) levels were not changed either in the cell body or terminal areas. The steady state levels of TPH mRNA estimated by in situ hybridization increased at day 1 and returned to control levels by day 4. AADC message levels were not altered throughout the periods. These data suggest that a decrease in 5-HT concentration may lead to an up-regulation of TPH gene transcription, by an, as yet, unknown mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A 6.1 kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice.

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.

Animals↗
PubMed1994

Drastic and selective hyperinnervation of central serotonergic neurons in a lethal neurodevelopmental mouse mutant, Anorexia (anx).

The autosomal recessive lethal anorexia mutation in mice (anx/anx) causes starvation in preweanlings. In addition, this murine neurodevelopmental mutant shows other distinct phenotypic characteristics and dysfunctional behaviors. Previous studies strongly suggested that the mutation results in elevated serotonergic stimulation, because these traits are characteristic of such overstimulation and because brain serotonin is believed to have an inhibitory effect on feeding behavior. In this report, we show extensive serotonergic hyperinnervation in normal target fields (hippocampus, cortex, olfactory bulb and cerebellum) of mutant mice. Despite the extensive hyperinnervation, the normal laminar organization of the brain was retained. The specificity of the mutation to the serotonergic system was confirmed by demonstration of normal catecholaminergic innervation in the central nervous system (CNS), and this specificity was especially striking in a common target field, the cerebellum. Serotonergic hyperinnervation in these mutant preweanling mice may represent the underlying etiology of increased serotonergic stimulation which leads to anorexic starvation, abnormal behavior, and premature death.

Animals↗
PubMed1994

Cyclic AMP-dependent protein kinase regulates basal and cyclic AMP-stimulated but not phorbol ester-stimulated transcription of the tyrosine hydroxylase gene.

To define the precise role of cyclic AMP (cAMP)-dependent protein kinase (PKA) in transcriptional regulation of the tyrosine hydroxylase (TH) gene, we performed transient cotransfection analyses of a reporter construct containing the upstream 2,400 bp sequence of the rat TH gene with expression plasmids encoding a heat-stable specific inhibitor of PKA (PKI), a mutant regulatory subunit of PKA, or the catalytic subunit of PKA. Inhibition of PKA activity by expression of either PKI or mutant regulatory subunit blocked cAMP-stimulated induction and reduced basal transcription of the TH-reporter construct. Expression of the catalytic subunit of PKA induced the expression of the TH-reporter construct up to 50-fold in a dose-dependent manner. Primer extension analysis confirmed that PKA-mediated induction of TH-reporter expression occurred at the correct transcription initiation site. Expression of PKI did not affect induction following phorbol ester treatment, suggesting that PKA and protein kinase C (PKC) induce TH transcription by independent mechanisms. Finally, a double mutation within the cAMP response element (CRE) of TH2400-CAT diminished its basal and forskolin-stimulated transcription to the level of the promoterless plasmid, pBLCAT3, but did not alter the induction following treatment with phorbol ester, indicating that the CRE is not required for PKC-mediated transcriptional induction. Our results indicate that PKA, via the CRE, plays a crucial role for basal and cAMP-inducible transcription of the TH gene.

Base Sequence↗
PubMed1994

A single transmitter regulates gene expression through two separate mechanisms: cholinergic regulation of phenylethanolamine N-methyltransferase mRNA via nicotinic and muscarinic pathways.

ACh regulates the gene encoding phenylethanolamine N-methyltransferase (PNMT) in bovine adrenal chromaffin cells. In addition to stimulating catecholamine release from these cells, cholinergic agents elevate transcription of the PNMT gene. Carbachol, which activates both nicotinic and muscarinic receptors, produces 12-19-fold increases in PNMT mRNA and a 22-fold increase in epinephrine release. Selective nicotinic and muscarinic antagonists (hexamethonium and atropine) each partially reduce carbachol-stimulated increases in PNMT mRNA while a combination of both eliminates > 90% of the carbachol response, thus indicating that separable nicotinic and muscarinic components contribute to the cholinergic increase in PNMT mRNA. Muscarine alone produces a dose-dependent increase (mean sixfold) in steady state PNMT mRNA levels and stimulates the rate of transcription fivefold. Only atropine and the m3-m4-selective muscarinic antagonist 4-diphenylacetoxy-4-methyl-piperidine (4-DAMP) reduce the response to muscarine, strongly suggesting that the m4 receptor is crucial for PNMT mRNA activation. In these chromaffin cells, muscarine inhibits adenylate cyclase, antagonist bind with affinities characteristic of m4 receptors, and cDNA hybridization detects only m4 mRNAs (Fernando et al., 1991). Nicotine also induces a dose-dependent increase (mean of 8.5-fold) in PNMT mRNA levels. The importance of voltage-gated Ca2+ channels in the nicotine effect is demonstrated by the stimulatory effects of calcium ionophores on PNMT mRNA levels (two-to fivefold increase) and the ability of the L- and N-type channel blockers nifedipine and omega-conotoxin to decrease the nicotine response (by 60% and 40%, respectively). Nuclear "run-on" assays further reveal that nicotine enhances transcription of the PNMT gene (approximately fourfold). Thus, this study provides the first demonstration that both nicotinic and muscarinic stimulation modify genomic responses of bovine adrenergic chromaffin cells and identifies possible mechanisms.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

The cAMP-dependent protein kinase regulates transcription of the dopamine beta-hydroxylase gene.

Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to norepinephrine, and is expressed specifically in neurons and neuroendocrine cells that release norepinephrine and epinephrine. In the present study, we used DBH-expressing human neuroblastoma SK-N-BE(2)C and rat pheochromocytoma (PC12) cell lines to investigate the role of cAMP-dependent protein kinase (PKA) in transcriptional regulation of the DBH gene. Coexpression of the catalytic subunit of PKA (PKAc) robustly stimulated the transcriptional activity of the DBH gene in a dose-dependent manner. Conversely, coexpression of a specific inhibitor of PKA abrogated forskolin- and cAMP-mediated but not phorbol ester-mediated transcriptional induction of DBH. Deletion of the cAMP response element (CRE) dramatically reduced the stimulatory effect of PKA, indicating that the CRE mediates the induction of DBH by PKA. In DBH-nonexpressing HeLa and C6 glioma cell lines, coexpression of PKAc changed the transcriptional activity of the DBH promoter to a minimal degree, indicating that basal and PKA-mediated transcription of the DBH gene occur in a cell type-specific manner. Finally, both basal and cAMP-stimulated transcription of the DBH gene are diminished in three PKA-deficient PC12 cell lines, compared to wild-type cells. Based on these data, we conclude that PKA, via the CRE, plays an important role in basal and cAMP-inducible transcription, but is not required for phorbol ester-mediated induction, of the DBH gene in noradrenergic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Acute effects of reserpine on tryptophan hydroxylase activity and mRNA in rat brain.

The present study examined changes in tryptophan hydroxylase (TPH) activity in the rat brainstem 1-4 days after i.p. reserpine administration. TPH enzyme activity was significantly decreased day 1 posttreatment and followed by a small but significant increase on days 2-4. Maximal TPH activity on day 4 was elevated only 42% above control level. This small increase in TPH activity was not preceded by any change of in situ hybridization signal of TPH message on day 1. In contrast, adrenal tyrosine hydroxylase (TH) activity in these same reserpinized animals was 2.5-fold higher than control on 4 day posttreatment. In addition, TH message was correspondingly increased day 1 posttreatment. Taken together, these results indicate a trend of small increase in TPH activity and no alteration in message after reserpine treatment unlike catecholaminergic systems.

Adrenal Glands↗
PubMed1993

Neuron-specific expression of the human dopamine beta-hydroxylase gene requires both the cAMP-response element and a silencer region.

Dopamine beta-hydroxylase (DBH), the enzyme catalyzing the conversion of dopamine to norepinephrine, is specifically expressed in adrenergic and noradrenergic neurons in the central nervous system. DNase I hypersensitive sites were found in the 5'-flanking region of the DBH gene in noradrenergic human neuroblastoma SK-N-BE(2)C cells, but not in DBH-nonexpressing HeLa cells. We report here that the 4.3-kilobase upstream sequence of the human DBH gene confers cell type-specific expression as assessed by transient expression assay. Furthermore, deletional and mutational analyses revealed two genetic regulatory elements required for the regulation of cell type specificity. First, deletion of the cAMP-response element (CRE) abolished > 95% of the transcriptional activity by the DBH upstream promoter, thus implicating the CRE as an essential positive genetic element. Second, deletion of a region between -490 and -263 base pairs resulted in 10-fold increase of reporter gene activity only in HeLa cells, indicating that this region contains a cell-specific silencer. A 13-base pair fragment residing within that region shows 77% sequence identity with the neuron-specific silencer motif recently identified in two neuronal genes, i.e. SCG10 and type II sodium channel genes. We propose that the interplay between the CRE and this neuron-specific silencer region plays an important role in the tissue-specific expression of the DBH gene in noradrenergic cells.

Animals↗