Biomedical subjects

T H Joh

Publications and source records attributed to T H Joh.

At least 91 records · Page 5Linked to original sources

PubMed1991

In situ hybridization analysis of c-fos and c-jun expression in the rat brain following transient forebrain ischemia.

Early induction of the mRNAs encoding the c-Fos and c-Jun nuclear proteins was examined in rat brain by in situ hybridization at various timepoints following global forebrain ischemia by the method of four-vessel occlusion. All animals were subjected to 20 min of transient ischemia. This produced a pattern of proto-oncogene activation that was most intense in the granule cells of the dentate gyrus 30 min after ischemia, while the hilar cells in the dentate and the pyramidal cells of the CA3 region in the hippocampus showed a more delayed but robust expression of these immediate early genes at 1 h. The neurons of the CA1 region exhibited a more moderate hybridization signal at 1-2 h postischemia. Very little hybridization signal for either immediate early gene could be detected in animals perfused with fixative immediately following ischemia, suggesting that cellular energy levels may have to be restored to a certain level before efficient de novo mRNA synthesis can occur. In the cerebellum, a similar temporal pattern was observed: the granule cells exhibited a prompt but patchy expression of c-fos and c-jun that was followed by a delayed signal in the Purkinje cells. Without exception c-fos and c-jun appeared to be expressed in unison, although the time course of c-fos and c-jun mRNA accumulation and decay was different in various brain regions: invariably the cerebellum returned rapidly to its baseline with virtually no remaining signal at 3 h postischemia, while c-fos and c-jun activation in the hippocampus remained high at 3 h and returned to baseline by 6 h. Several other brain regions showed early production of c-fos and c-jun mRNAs, such as the medial habenula, piriform cortex, the amygdala, the centromedian, lateral posterior, paracentral, intermediodorsal and reuniens nuclei of the thalamus and the ventromedial and dorsal nuclei of the hypothalamus; in the brainstem, the trapezoid body and the noradrenergic neurons of the locus ceruleus as well as the adrenergic neurons in the ventrolateral medulla (C1 group) and nucleus tractus solitarius (C2 group) regions displayed slightly less intense hybridization signals. In addition, the ependyma of the lateral ventricles and the third ventricle showed a prompt albeit short-lived production of c-fos and c-jun mRNAs. Sham-operated animals as well as animals that had survived to one week postischemia showed either no or only trace levels of hybridization signal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Differential effect of functional olfactory bulb deafferentation on tyrosine hydroxylase and glutamic acid decarboxylase messenger RNA levels in rodent juxtaglomerular neurons.

Expression of the dopaminergic phenotype in olfactory bulb (OB) juxtaglomerular neurons (constituting a population of periglomerular and external tufted cells) is dependent upon functional innervation by peripheral olfactory receptors. Loss of functional input in rodents, by either peripheral deafferentation or deprivation of odorant access, results in a profound decrease in the expression of juxtaglomerular tyrosine hydroxylase (TH). We have examined the effects of such treatments on the expression of the neurotransmitter biosynthetic enzyme glutamic acid decarboxylase (GAD), which is colocalized with TH in the majority of TH-containing juxtaglomerular neurons. Following either chemically induced OB deafferentation in adult mice or unilateral odor deprivation in neonatal rats, steady-state OB GAD messenger RNA levels remained essentially unchanged as assessed by Northern blot analysis 20-40 days after treatment. These results were confirmed by in situ hybridization analysis, which demonstrated a profound loss of juxtaglomerular TH messenger RNA but no accompanying decrease in regionally colocalized GAD message. Since GAD is found in nearly all dopaminergic OB cells, the preservation of juxtaglomerular GAD message implies that olfactory receptor neurons exert a differential transneuronal regulation of TH and GAD gene transcription.

Animals↗

PubMed1991

Strain difference in phenylethanolamine N-methyltransferase activity and immunoreactivity of medulla oblongata of Sprague-Dawley and Long-Evans hooded rats.

Previously, we reported that retinal phenylethanolamine N-methyltransferase (PNMT) activity of Sprague-Dawley (SD) rats was significantly higher than that of Long-Evans (LE) hooded rats. However, there were no noticeable differences observed in PNMT-immunostaining patterns between the retinae of LE and SD rats. In order to examine this discrepancy, we extended this study to areas of the medulla oblongata harboring PNMT-containing cell bodies. In the present report, we demonstrate that the enzyme activity as well as the immunoreactivity of PNMT in medulla oblongata of SD rats were significantly higher than that observed in the LE strain.

Animals↗

PubMed1991

Species-specific distribution of aromatic L-amino acid decarboxylase in the rodent adrenal gland, cerebellum, and olfactory bulb.

Aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-dopa to dopamine, displayed species-specific differences in both activity and immunoreactivity in the cerebellum, olfactory bulb, and adrenal glands of three rodent species, the hamster, rat, and mouse. Specifically, in the hamster but not the rat or mouse, AADC immunoreactive cells were observed in the cerebellum and adrenal cortex. The unusual distribution of the enzyme was confirmed biochemically. AADC activity was greater in the adrenal gland and the cerebellum in the hamster than in the mouse or rat. In addition, by Western blot analysis, one band of appropriate molecular weight was observed both in the hamster adrenal gland and cerebellum. The rat adrenal gland displayed a similar immunoreactive protein on the Western blot; however, the protein could not be detected in the rat cerebellum by the technique utilized. Tyrosine hydroxylase (TH) immunoreactivity in these same tissues did not differ among the species. In the main olfactory bulb of the mouse, juxtaglomerular cells exhibited very limited immunoreactivity for AADC, but TH-immunoreactivity in these cells was robust. In contrast, juxtaglomerular cells in the rat displayed a similar intensity of immunostaining for both AADC and TH. AADC activity in the mouse, consistent with the reduced immunostaining for the enzyme, was 50% of that in the rat and the hamster. These data demonstrate that AADC protein, which is contained in cells of diverse function, also displays qualitative and quantitative species specific variations in both distribution and amount.

Adrenal Glands↗

PubMed1991

Genomic organization of the rat aromatic L-amino acid decarboxylase (AADC) locus: partial analysis reveals divergence from the Drosophila dopa decarboxylase (DDC) gene structure.

Aromatic L-amino acid decarboxylase (AADC) is responsible for the conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are important neurotransmitters. We characterized genomic clones derived from the rat AADC locus by Southern blot and nucleotide sequencing analyses to explore the exonal organization of the gene. Our results suggest that the rat AADC gene is relatively large, containing at least 12 exons and spanning at least 40 kb in the rat genome. In this study, nine exons corresponding to 71% of the published cDNA sequence were identified, the smallest of which was as short as 20 base pairs (bp). In the Drosophila dopa decarboxylase (DDC) gene, the sequences homologous to these nine exons are all present in the fourth exon. This implies that either multiple intron sequences have been added to the vertebrate AADC gene or alternatively, deleted from the invertebrate gene after the divergence of vertebrates and invertebrates during evolution.

Animals↗

PubMed1991

Effects of second messenger system activation on functional expression of tyrosine hydroxylase fusion gene constructs in neuronal and nonneuronal cells.

A genomic clone for rat tyrosine hydroxylase (TH) was isolated and a fragment containing 503 bp upstream of the transcription start site was sequenced. The BamHI/AluI fragment was inserted into a plasmid carrying the coding sequence for bacterial chloramphenicol acetyltransferase (CAT). Another construct with the 5' sequence truncated to -151 bp also was prepared. When these were introduced into several mammalian cell lines, including C6 glioma, BE(2) neuroblastoma, CV-1 or Ltk- fibroblasts, different basal levels of CAT expression were observed. In the fibroblast lines, THCAT constructs were not expressed unless the cells were treated with forskolin or TPA. However, the low basal expression was not correlated to endogenous expression as THCAT constructs expressed comparably in BE(2)C, HeLa, and C6 glioma. Treatment of any of the cell lines with forskolin, TPA, or a combination of the two agents stimulated the expression by at least two-fold in all cell lines and the maximally induced levels were at least 10-fold over promoterless controls. These data indicate that the essential promoter elements as well as those conferring responsivity to cyclic AMP reside within 151 bp of the transcription start site. However, the array of elements regulating cell-type expression lie, at least in part, beyond the 500-bp region examined. Further, a role for phosphorylation in the regulation of basal and induced transcription of TH is suggested.

Animals↗

PubMed1991

Differential and coordinate regulation of TH and PNMT mRNAs in chromaffin cell cultures by second messenger system activation and steroid treatment.

Primary cultures of chromaffin cells were prepared from bovine adrenal medullae and the levels of mRNA for tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) determined. The cells expressed moderate levels of TH mRNA and low levels of PNMT mRNA. The latter appeared to be more sensitive than TH mRNA to variations in the culture medium. The treatment of cultures with agents that activate signal transduction pathways, forskolin or phorbol esters, dramatically enhanced the expression of both mRNAs. The forskolin-induced increases in the steady-state levels of TH and PNMT mRNAs occurred rapidly and were apparent within 5 hours. These data suggest that the TH and PNMT genes can be regulated by second messengers. In contrast, dexamethasone treatment dramatically increased PNMT mRNA with no change in TH mRNA. The increase in PNMT mRNA was apparent within 6 hours of addition of the drug to the culture medium.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Characterization of recombinant bovine phenylethanolamine N-methyltransferase expressed in a mouse C127 cell line.

Bovine phenylethanolamine N-methyltransferase (PNMT) cDNA was inserted into a bovine papilloma virus-based expression vector and used to transfect a mouse C127 cell line. The resultant recombinant bovine PNMT was characterized biochemically and immunochemically. Recombinant bovine PNMT activity, like the native bovine enzyme, was enhanced by phosphate ion in a concentration-dependent manner. Their molecular weights were shown to be identical by Western blot analysis. Antibodies raised against native bovine adrenal PNMT equally immunoprecipitated the activity of the recombinant and native enzymes. In addition, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine PNMT. These antibodies immunostained the recombinant enzyme protein in transfected cells and in their neurite-like processes. In addition, in situ hybridization with the bovine PNMT cDNA probe resulted in a labelling pattern similar to the immunostaining. The recombinant bovine PNMT as the native bovine enzyme exist in multiple-charge forms, but only one form is predominant. Taken together, our results suggest that recombinant bovine PNMT, expressed from bovine PNMT cDNA in a mouse cell line is enzymatically active and shares many common features with native bovine adrenal PNMT.

Adrenal Glands↗

PubMed1991

Molecular cloning and characterization of cDNA encoding tryptophan hydroxylase from rat central serotonergic neurons.

Tryptophan hydroxylase (TPH) from central serotonergic neurons in the dorsal raphe nucleus (DRN) and that from the endocrine pineal gland (PG) have been shown to exhibit difference biochemical characteristics. We further report here that the isoelectric point determined by chromatofocusing differs between TPH from the rat brainstem and PG. In addition, the levels of TPH mRNA are much greater in the PG than the DRN despite a higher enzymatic activity in the DRN. These data raise the question as to whether different forms of TPH may exist in the DRN and the PG. To address this question, we amplified TPH cDNAs by the polymerase chain reaction (PCR) using poly(A)+ RNA purified from both tissues. Several combinations of oligonucleotide primers encompassing different regions of the published coding sequence of rat pineal TPH were employed for this purpose. Subsequent analysis by gel electrophoresis and Southern blotting of PCR products indicated that DNA fragments of identical length were amplified from both sources. Furthermore, the nucleotide sequences of three independent subclones containing the putative full-length coding region of DRN TPH were determined and found to be identical to that of PG. In situ hybridization using the amplified cDNA as a probe demonstrated specific labeling within the DRN of the rat brain. These data support the hypothesis that tissue-specific differences in TPH characteristics result from differential post-translational events and clearly indicate that a TPH mRNA transcript identical in coding sequence to the PG form is expressed in the DRN.

Amino Acid Sequence↗

PubMed1991

Isolation of monoaminergic synaptosomes from rat brain by immunomagnetophoresis.

Monoaminergic synaptosomes have been isolated and purified from rat brain by immunomagnetophoresis. This novel technique uses magnetic beads to which Protein A is bound. Noradrenergic, dopaminergic, and serotonergic synaptosomes (previously cell-surface labelled with anti-dopamine-beta-hydroxylase, anti-tyrosine hydroxylase, and anti-tryptophan hydroxylase, respectively) may be isolated in a highly purified state. The synaptosomal subpopulations are recovered in a viable metabolic state and show glucose-stimulated respiration and Ca2(+)-dependent neurotransmitter release. A novel subtype of dopamine-beta-hydroxylase was found in dopaminergic terminals. No evidence for glutamate corelease from monoaminergic synaptosomes was obtained.

Animals↗

PubMed1991

Limited proteolysis of rat brain tyrosine hydroxylase defines an N-terminal region required for regulation of cofactor binding and directing substrate specificity.

Trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34-kDa fragment that is catalytically active but does not contain the regulatory phosphorylation sites. In this report, activation of TH by proteolysis was characterized further. Proteolysis results in a decrease in Kms for both substrate and cofactor. The increase in affinity for cofactor was identical to that produced by phosphorylation with cAMP-dependent protein kinase. Additionally, proteolysis of an N-terminal region containing the regulatory phosphorylation sites was sufficient to produce a decrease in Km for cofactor. Activation of substrate binding required more extensive proteolysis but also corresponded to N-terminal digestion. Moreover, this activation was coincident with a broadened substrate specificity. In combination, these data indicate that the N-terminus of tyrosine hydroxylase regulates cofactor binding and directs substrate specificity.

Animals↗

PubMed1990

Evidence for decreased transport of tryptophan hydroxylase in Alzheimer's disease.

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of serotonin and a specific marker for serotonergic neurons. These neurons are affected in Alzheimer's disease (AD) in several ways: serotonin is decreased in axon terminals, serotonin neurons accumulate neurofibrillary protein, and these neurons are lost in AD brains. One subcellular mechanism which may underlie degeneration of neurons in AD is decreased axonal transport with accumulation of enzymes and their potentially toxic metabolites in the cell body. To determine whether there is a defect in axonal transport in serotonin neurons in AD we measured TPH activity, serotonin and its oxidative metabolite 5-hydroxyindoleacetic acid (5-HIAA) in dorsal raphe cell bodies from Alzheimer and control cases. TPH activity is increased 4.7-fold in raphe neuron cell bodies in Alzheimer brains. Serotonin and 5-HIAA are increased by 4.0- and 2.0-fold, respectively in Alzheimer compared to control raphe cell bodies. In contrast, in synaptic terminals of the amygdala 5-HT and 5-HIAA were decreased by 41% and 50%, respectively in the same AD cases. We propose that the accumulation of TPH and its products in the raphe perikarya in AD results from a diminished transport of TPH to axon terminals. The accumulation of oxidative metabolites of serotonin may contribute to the degeneration of serotonergic neurons in AD.

Aged↗

PubMed1990

Cell loss and class distribution of TH-I cells in the substantia nigra of the neurological mutant, weaver.

Eight-week-old homozygous weaver mutant mice and littermate wildtype controls were perfused with a buffered acrolein and paraformaldehyde solution. The brains were subsequently removed, blocked and sectioned on a vibratome. Representative sections through the midbrain were incubated overnight with an antibody for tyrosine hydroxylase. Visualization of the antibody was achieved using the peroxidase-antiperoxidase technique. The immunoreactive cells in the substantia nigra were examined to determine the subclasses of cells that are affected when dopaminergic neurons are lost in these mutants. Class distributions were determined and the data were subjected to chi 2 analyses. The results indicate a significant loss of tyrosine hydroxylase-immunoreactive cells in both the pars compacta and pars lateralis that is dependent upon the group and the region being studied. In addition, distributional shifts within the classes of labeled neurons suggest that there is an increase in the small neurons over the expected numbers in both divisions of the substantia nigra.

Animals↗

PubMed1990

Strain differences between albino and pigmented rats in monoamine-synthesizing enzyme activities of brain, retina and adrenal gland.

The present study compared the activities of some of the monoamine synthesizing enzymes in several brain regions, the retina as well as adrenal gland of albino Sprague-Dawley (SD) and Long-Evans hooded (LE) rats. Brainstem, hypothalamic and retinal tyrosine hydroxylase (TH) activity were significantly higher in LE than in SD. In addition to higher enzyme activity, a larger number of TH-immunoreactive perikarya as well as a higher concentration of TH-immunoreactive processes were observed in the retina of LE rats. There was no strain difference in TH activity of caudate nucleus (CN) and substantia nigra (SN). In contrast to brain regions and retina, adrenal TH activity was markedly higher in SD than in LE animals. Aromatic L-amino acid decarboxylase (AADC) activity of both the brainstem and adrenal gland in the LE strain was lower than in SD animals. No differences in the AADC activity of hypothalamus, SN and CN were found between LE and SD strains. Phenylethanolamine N-methyltransferase (PNMT) activity of the hypothalamus, retina and adrenal gland of LE strains was significantly lower than in SD rats. In spite of the difference in the enzyme activity, there were no marked morphological changes observed in PNMT-immunostaining patterns between the retina of LE and SD rats. Tryptophan hydroxylase activity of both the brainstem and hypothalamus did not exhibit strain differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1990

Neuropathology of immunohistochemically identified brainstem neurons in Parkinson's disease.

Regional loss of immunohistochemically identified neurons in serial sections through the brainstem of 4 patients with idiopathic Parkinson's disease was compared with equivalent sections from 4 age-matched control subjects. In the Parkinson brains, the catecholamine cell groups of the midbrain, pons, and medulla showed variable neuropathological changes. All dopaminergic nuclei were variably affected, but were most severely affected in the caudal, central substantia nigra. The pontine noradrenergic locus ceruleus showed variable degrees of degeneration. There was also a substantial loss of substance P-containing neurons in the pedunculopontine tegmental nucleus. However, the most severely affected cell group in the pons was the serotonin-synthesizing neurons in the median raphe. In the medulla, substantial neuronal loss was found in several diverse cell groups including the adrenaline-synthesizing and neuropeptide Y-containing neurons in the rostral ventrolateral medulla, the serotonin-synthesizing neurons in the raphe obscurus nucleus, the substance P-containing neurons in the lateral reticular formation, as well as the substance P-containing neurons in the dorsal motor vagal nucleus. Lewy bodies were present in immunohistochemically identified neurons in many of these regions, indicating that they were affected directly by the disease process. These widespread but region- and transmitter-specific changes help account for the diversity of motor, cognitive, and autonomic manifestations of Parkinson's disease.

Aged↗

PubMed1990

Deduced amino acid sequence of bovine aromatic L-amino acid decarboxylase: homology to other decarboxylases.

Nucleotide sequence of cDNA for bovine aromatic L-amino acid decarboxylase (AADC) was analyzed. The deduced amino acid sequence of bovine AADC shows 57% identity to drosophila AADC and 37% to plant Catharanthus roseus AADC. The 7-amino acid sequence of the pyridoxal phosphate binding site is completely conserved among drosophila, pig and bovine AADC. AADC primary structure also shows high homology to that of feline glutamic acid decarboxylase.

Amino Acid Sequence↗

PubMed1990

Decrease in tyrosine hydroxylase, but not aromatic L-amino acid decarboxylase, messenger RNA in rat olfactory bulb following neonatal, unilateral odor deprivation.

Unilateral naris cauterization in rats results in occlusion of the affected naris and blockade of odorant access to ipsilateral olfactory receptor cells in the olfactory epithelium. These receptor cells project exclusively to the olfactory bulb (OB) and appear to regulate expression of the dopaminergic phenotype in a population of OB juxtaglomerular neurons. Unilateral odor deprivation results in a loss of normal stimulatory input to the OB and a marked and specific decrease in ipsilateral OB tyrosine hydroxylase (TH) expression. The expression of co-localized aromatic L-amino acid decarboxylase (AADC) is not similarly affected. We have used this procedure in neonatal rats to examine the effect of stimulus deprivation on OB TH and AADC mRNA levels. Both Northern blot and in situ hybridization analyses revealed a pronounced decrease in ipsilateral as compared to contralateral OB TH mRNA levels 40 days after naris closure. In contrast, the levels of OB AADC mRNA were unaltered by naris closure. By in situ hybridization histochemistry, both TH and AADC mRNAs were localized to OB juxtaglomerular neurons. Odor deprivation was associated with an apparent region-specific reduction in TH mRNA within the ipsilateral OB glomerular layer. By densitometric analysis, the loss of TH-specific message was quantitatively consistent with the decrease in TH activity, suggesting that the observed plasticity of OB dopaminergic neurons following functional deafferentation can be attributed to a selective, transneuronally-mediated down regulation of TH gene transcription.

Animals↗

PubMed1990

Transneuronal regulation of neuronal specific gene expression in the mouse olfactory bulb.

Peripheral afferent denervation (deafferentation) of the rodent main olfactory bulb produces a marked decrease in tyrosine hydroxylase (TH) activity and immunoreactivity in a population of juxtaglomerular dopaminergic neurons. Preservation of activity and immunostaining for aromatic L-amino acid decarboxylase implies that these cells do not die, but change phenotype. We now report that the steady-state level of TH mRNA markedly decreases in the adult mouse olfactory bulb in response to deafferentation. This reduction is permanent following intranasal irrigation with 0.17 M zinc sulphate (ZnSO4) but reversible following deafferentation produced by intranasal irrigation with 0.7% Triton X-100. The initial declines in TH activity, protein and mRNA of dopaminergic juxtaglomerular neurons observed after Triton X-100 treatment are all reversible as the steady-state level of TH mRNA gradually returns to control levels. Steady-state levels of mRNA for olfactory marker protein (OMP), a protein found in high concentrations in olfactory receptor neurons and their processes which innervate the olfactory bulb, were also monitored following deafferentation. Following treatment with either ZnSO4 or Triton X-100, the pattern of changes in steady-state levels of OMP mRNA was similar to that observed for TH. The steady-state level of PEP19 mRNA, a peptide previously localized to granule cells in the olfactory bulb, was not altered by deafferentation. These data indicate selective and parallel regulation of TH and OMP message and protein levels following deafferentation.

Afferent Pathways↗