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T Nagatsu

Publications and source records attributed to T Nagatsu.

At least 19 recordsLinked to original sources
PubMed1992

Mouse dopamine beta-hydroxylase: primary structure deduced from the cDNA sequence and exon/intron organization of the gene.

Genomic clones for mouse dopamine beta-hydroxylase (DBH) were isolated from two genomic libraries derived from DBA/2J and 129/SV mouse strains, by plaque hybridization with the human DBH cDNA probe. Subsequently, cDNA encoding mouse DBH was amplified with reverse transcription-polymerase chain reaction (RT-PCR) method using primers corresponding to 5'- and 3'-portions of the mouse DBH mRNA, subcloned into a plasmid vector, and subjected to nucleotide sequence analysis. The clone encoded a protein of 621 amino acids with a calculated molecular mass of 70,186 daltons. The predicted amino acid sequence of mouse DBH showed 87%, 80% and 79% identities with the rat, bovine and human enzymes, respectively. Several potential amino acid sequences that are involved in the posttranslational modification and catalytic function of DBH were identified in mouse DBH protein. Nucleotide sequence analysis of the overlapping genomic clones showed that the mouse DBH gene was composed of 12 exons about 17 kb in length. Typical TATA and CCAAT boxes were observed in the 5'-upstream region of the gene. Northern blot analysis of adrenal gland RNA detected a single size species of the mouse DBH mRNA.

Adrenal Glands↗
PubMed1992

Tissue-specific alternative splicing of the first exon generates two types of mRNAs in human aromatic L-amino acid decarboxylase.

Aromatic-L-amino-acid decarboxylase (AADC) is an enzyme that plays an essential role in synthesizing catecholamines and serotonin in neuronal and endocrine tissues. AADC has also been detected in other nonneuronal tissues including liver and kidney, although its physiological role in nonneuronal tissues has not yet been defined. Previously we have cloned a human AADC cDNA from a neuronal tissue (pheochromocytoma) [Ichinose, H., Kurosawa, Y., Titani, K., Fujita, K., & Nagatsu, T. (1989) Biochem. Biophys. Res. Commun. 164, 1024-1030] and the corresponding genomic DNA [Sumi-Ichinose, C., Ichinose, H., Takahashi, E., Hori, T., & Nagatsu, T. (1992) Biochemistry 31, 2229-2238]. Here we present isolation and characterization of AADC cDNA and genomic DNA from a nonneuronal tissue (human liver). The nonneuronal and neuronal AADC mRNAs differed only in the region corresponding to the untranslated first exon. The first exon for the nonneuronal-type mRNA was located 4.2 kilobases upstream to that for the neuronal-type mRNA and 22 kilobases from exon 2, to which it is spliced. Determination of the transcription initiation site indicated that the length of the nonneuronal-type exon 1 was 200 bp. A TATA box-like motif was located between positions -26 and -20 from the transcription initiation site. These results showed that an alternative usage of the first exon in the 5'-untranslated regions produces two types of mRNAs in AADC and suggested that alternative splicing would regulate the tissue-specific expression of AADC.

Adrenal Glands↗
PubMed1992

Multiple mRNA forms of human GTP cyclohydrolase I.

To isolate full length cDNA clones encoding human GTP cyclohydrolase I, the first and rate-limiting enzyme in tetrahydrobiopterin biosynthesis, a cDNA library generated from human liver was screened by plaque hybridization. Analysis of the clones, hybridized with rat cDNA fragment, by restriction mapping and partial sequencing showed the existence of three kinds of cDNAs. All three cDNAs were identical in their central and 5' regions. They were, however, found to diverge at 3' ends. Furthermore, the three species of mRNAs corresponding to the three cDNAs were detected in human liver by reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results indicate that, in humans, GTP cyclohydrolase I molecules are encoded by at least three distinct mRNAs.

Amino Acid Sequence↗
PubMed1992

Endogenous synthesis of N-methylsalsolinol, an analogue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, in rat brain during in vivo microdialysis with salsolinol, as demonstrated by gas chromatography-mass spectrometry.

N-Methylsalsolinol, an analogue of 1,2,3,6-tetrahydropyridine, is present in the brains of patients with Parkinson's disease. To determine the metabolic pathway for the synthesis of N-Methylsalsolinol in the brain, salsolinol was perfused through the striatum or the substantia nigra of the rat brain by in vivo microdialysis. N-Methylsalsolinol was detected in the brain dialysate samples during microdialysis with salsolinol using gas chromatography-mass spectrometry with selected-ion monitoring. These results demonstrate that endogenous N-methylation of salsolinol into N-methylsalsolinol occurs in the brain in vivo.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

Differential effect of self-stimulation on dopamine release and metabolism in the rat medial frontal cortex, nucleus accumbens and striatum studied by in vivo microdialysis.

Changes in the extracellular levels of dopamine (DA) and its metabolites in the dopaminergic terminal regions, the medial frontal cortex (MFC), nucleus accumbens (NAC), and striatum (STR), were measured by microdialysis during self-stimulation of the medial forebrain bundle (MFB) in rats pretreated with the DA uptake inhibitor, nomifensine (1 mg/kg, i.p.). Self-stimulation of the MFB in nomifensine-pretreated rats caused an increase in the extracellular DA level in the MFC and NAC but not in the STR. Self-stimulation also increased the extracellular concentrations of the main DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) to a similar extent in the MFC and NAC and to a lesser extent in the STR. Thus, there was a regional difference in the neurochemical changes following self-stimulation with either the MFC or the NAC showing larger extracellular levels of DA, DOPAC, and HVA than the STR. Furthermore, these changes were observed on both hemispheres ipsilateral and contralateral to the stimulation. The results indicate that self-stimulation of the MFB preferentially activates the mesocorticolimbic DA systems, thereby bilateral increases in the release of DA and its metabolism being produced in their terminal regions, the MFC and NAC.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1992

Molecular cloning of genomic DNA and chromosomal assignment of the gene for human aromatic L-amino acid decarboxylase, the enzyme for catecholamine and serotonin biosynthesis.

Aromatic L-amino acid decarboxylase (AADC) catalyzes the decarboxylation of both L-3,4-dihydroxyphenylalanine and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are major mammalian neurotransmitters and hormones belonging to catecholamines and indoleamines. This report describes the organization of the human AADC gene. We proved that the gene of human AADC consists of 15 exons spanning more than 85 kilobases and exists as a single copy in the haploid genome. The boundaries between exon and intron followed the AG/GT rule. The sizes of exons and introns ranged from 20 to 400 bp and from 1.0 to 17.7 kb, respectively, while the sizes of four introns were not determined. Untranslated regions located in the 5' region of mRNA were encoded by two exons, exons 1 and 2. The transcriptional starting point was determined around G at position -111 by primer extension and S1 mapping. There were no typical "TATA box" and "CAAT box" within 540 bp from the transcriptional starting point. The human AADC gene was mapped to chromosome band 7p12.1-p12.3 by fluorescence in situ hybridization. This is the first report on the genomic structure and chromosomal localization of the AADC gene in mammals.

Amino Acid Sequence↗
PubMed1992

Comparison of characteristics of bovine aromatic L-amino acid decarboxylase with human enzyme.

Aromatic L-amino acid decarboxylase (AADC) was purified from bovine adrenal medulla and properties of this enzyme were compared with those of AADC from human pheochromocytoma. The molecular weights of the subunits were identical between human and bovine enzymes and estimated to be 50,000 by SDS-polyacrylamide gel electrophoresis. An isoelectric point of the human enzyme was 5.7, while the bovine enzyme showed several distinct bands at the region of pH 4.9-5.3 in the absence of urea. Multiplicity of the isoelectric point of bovine AADC disappeared in the presence of urea. These results showed that there were some differences between the properties of human and bovine AADC in spite of the high homology (88%) in their primary structures.

Adrenal Medulla↗
PubMed1992

Structure of the mouse tyrosine hydroxylase gene.

The mouse tyrosine hydroxylase (TH) gene was isolated from a genomic library by cross-hybridization with human TH cDNA probe. Nucleotide sequence analysis of two overlapping genomic clones showed that this gene is split into 13 exons distributed about 7.5 kb in length. The transcription initiation site was determined by primer extension analysis with mouse adrenal gland poly(A)+RNA. The structure of the mouse TH gene was similar to that of the human TH gene, but it contained neither the alternative splice donor site around the 3'-end of the first exon nor an independent exon corresponding to the second exon of the human TH gene. There were the canonical TATA and GC boxes, cyclic AMP responsive element (CRE), and AP1 binding site in the 5'-flanking region of the mouse TH gene.

Amino Acid Sequence↗
PubMed1992

Selective inhibition of complex I by N-methylisoquinolinium ion and N-methyl-1,2,3,4-tetrahydroisoquinoline in isolated mitochondria prepared from mouse brain.

1,2,3,4-Tetrahydroisoquinoline (TIQ), which is structurally similar to MPTP, has been found in human brain and has been reported to inhibit the mitochondrial respiration as does 1-methyl-4-phenylpyridinium ion (MPP+). However, the potency of inhibition by TIQ is less than that of MPP+. In this study, we report the effects of N-methyl-1,2,3,4-tetrahydroisoquinoline (N-Me-TIQ) and N-methylisoquinolinium ion (N-Me-IQ+) on the mitochondrial electron transport system using mitochondria prepared from mouse brains. Five mM N-Me-TIQ and 500 microM N-Me-IQ+ inhibited complex I activity to 54% and 63% of the control, respectively. The IC50 of N-Me-TIQ and N-Me-IQ+ were approximately 6.5 mM and 650 microM, respectively. Neither substance inhibited complex II, III and IV activities. Kinetic analyses of N-Me-IQ+ on complex I activity revealed uncompetitive inhibition against NADH and non-competitive inhibition against ubiquinone. These inhibitory characteristics were the same to those of MPP+ and the inhibitory potency of N-Me-IQ+ on complex I activity was stronger than that of MPP+.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

Allosteric effect of tetrahydrobiopterin cofactors on tyrosine hydroxylase activity.

Allostery of tyrosine hydroxylase was found by kinetical studies of partially purified tyrosine hydroxylase from clonal rat pheochromocytoma PC12h cells. Positive cooperativity toward the cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4] and (6S)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6S)BH4], was observed. It is indicated that biopterin might be the regulatory factor of the enzyme polymers, which changes the affinity for the cofactor itself. Moreover, the stereochemical structure of (6R)BH4, the naturally-occurring cofactor, took an important role on the kinetical properties of the enzyme in concern with L-tyrosine.

Allosteric Regulation↗
PubMed1992

Inhibition of tryptophan hydroxylase by (R)- and (S)-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines (salsolinols).

The (R)- and (S)-enantiomers of salsolinol, the dopamine-derived tetrahydroisoquinolines, were found to inhibit the activity of tryptophan hydroxylase (TPH), prepared from serotonin-producing murine mastocytoma P-815 cells. Inhibition of TPH by salsolinols was found to be non-competitive with the substrate L-tryptophan. Tryptophan hydroxylase is composed of two elements with different kinetic properties in terms of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin and these two elements were inhibited by salsolinols in competitive and uncompetitive ways, respectively. Stereoselectivity of salsolinol was not observed, concerning the potency and the type of inhibition on PTH. These data indicate that salsolinols might be naturally occurring inhibitors of indoleamine metabolism.

Animals↗
PubMed1992

Organization and complete nucleotide sequence of the gene encoding mouse phenylethanolamine N-methyltransferase.

Phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) catalyzes the conversion of norepinephrine to epinephrine, the last step of catecholamine biosynthesis. We have previously reported molecular cloning of cDNA encoding human PNMT and chromosomal localization of its gene (Kaneda et al., J. Biol. Chem., 263 (1988) 7672-7677). In this report, we isolated the chromosomal gene encoding mouse PNMT by cross-hybridization with the human PNMT cDNA. Mouse PNMT gene spanned about 1.8 kb and consisted of 3 exons. Primer extension analysis showed two putative transcription initiation sites. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of the mouse PNMT mRNA in brain (pons and medulla oblongata) and adrenal gland. Subsequently cDNA encoding mouse PNMT was amplified by RT-PCR and cloned into the plasmid vector. Mouse PMNT gene contained the protein-coding region of 885 bp (295 amino acids) with the predicted molecular weight of 32,627. The deduced amino acid sequence of mouse PNMT revealed the major difference in the N-terminal region, as compared to the human and bovine PNMT sequences. In the 5'-terminal region of the mouse PNMT gene, we found the existence of 23 bp direct repeat sequences, which was not observed in the corresponding regions of the human and bovine PNMT genes. The presence or absence of the direct repeats caused the major difference in the PNMT sequences among species. The typical TATA, GC, and CACCC boxes as well as several sequences homologous to glucocorticoids response elements (GRE) were located in the 5'-flanking region of the mouse PNMT gene.

Amino Acid Sequence↗
PubMed1992

Analysis of the human tyrosine hydroxylase promoter-chloramphenicol acetyltransferase chimeric gene expression in transgenic mice.

To investigate cis-elements responsible for catecholaminergic (CAnergic) neuron-specific expression of the tyrosine hydroxylase (TH) gene, we produced lines of transgenic mice carrying 5.0-kb, 2.5-kb and 0.2-kb fragments from the 5'-flanking region of the human TH gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and designated them as TC 50, TC 25, and TC 02, respectively, and reporter gene expression in transgenic mice was analyzed by CAT assay by immunocytochemistry with anti-CAT antibody. High-level CAT expression was observed in the brain and adrenal gland using the 5.0-kb promoter of the TC 50 mice, but ectopic expression was consistently observed in several somatic tissues, e.g. thymus, colon, and testis. In brain, expression was achieved in CAnergic neurons with the largest construct (5.0 kb), but not with 2.5 kb or 0.2 kb of 5' flanking sequence. However, TC 50 mice also expressed CAT immunoreactivity in non-CAnergic neurons. In the TC 25 line CAT immunoreactivity was detected only in some non-CAnergic neurons. In the TC 02 line no CAT immunoreactivity was detected in any of the tissues examined. These results indicate that the 5.0-kb DNA fragment of the TH gene upstream region contains activity to express CAT in CAnergic neurons and surprisingly, lacks some regulatory elements attenuating ectopic expression, and that the 2.5-kb and 0.2-kb fragment are not sufficient for the proper expression. We discuss the presence of the tissue-specific regulatory elements in the structure portion of the TH gene and/or 3'-flanking region.

Animals↗
PubMed1992

The activity of dipeptidyl peptidase II and dipeptidyl peptidase IV in mice immunized with type II collagen.

We investigated the activity of peptidases in the serum of mice with experimental polyarthritis that was induced by the injection of type II collagen, an experimental model of human rheumatoid arthritis. The activity of dipeptidyl peptidase II (DPP II) was increased and that of dipeptidyl peptidase IV (DPP IV) was decreased resulting in the significant increase of the serum DPP II/DPP IV ratio in the polyarthritic mice compared with that of controls. These results indicate that the DPP II/DPP IV ratio is a novel index of disease activity in mice with collagen-induced polyarthritis and may be useful in assessing the activity of rheumatoid arthritis in humans.

Animals↗
PubMed1992

N-methylation of dopamine-derived 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, (R)-salsolinol, in rat brains: in vivo microdialysis study.

N-Methylation of (R)-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-salsolinol] derived from dopamine was proved by in vivo microdialysis study in the rat brain. The striatum was perfused with (R)-salsolinol and N-methylated compound was identified in the dialysate using HPLC and electrochemical detection with multichanneled electrodes. N-Methylation of (R)-salsolinol was confirmed in three other regions of the brain, the substantia nigra, hypothalamus, and hippocampus. In the substantia nigra, the amount of N-methylated (R)-salsolinol was significantly larger than in the other three regions. These results indicate that around dopaminergic neurons, particularly in the substantia nigra, (R)-salsolinol was methylated into N-methyl-(R)-salsolinol, which has a chemical structure similar to that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the selective dopaminergic neurotoxin. N-Methylation of tetrahydroisoquinolines and beta-carbolines have already been proven to increase their toxicity to dopaminergic neurons and N-methylation might be an essential step for these alkaloids to increase their toxicity. On the other hand, after perfusion of (R)-salsolinol, release of dopamine and 5-hydroxytryptamine was observed and inhibition of monoamine oxidase was indicated. (R)-Salsolinol and its derivatives may be candidates for being dopaminergic neurotoxins.

Animals↗
PubMed1992

Inhibition of tyrosine hydroxylase by R and S enantiomers of salsolinol, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline.

Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.

Adrenal Gland Neoplasms↗
PubMed1992

Tetrahydrobiopterin-dependent functional recovery in 6-hydroxydopamine-treated rats by intracerebral grafting of fibroblasts transfected with tyrosine hydroxylase cDNA.

Fibroblasts (NRK-49F) were transfected with human type 2 tyrosine hydroxylase (TH; EC 1.14.16.29) cDNA, to clarify the mechanism involved in amelioration of parkinsonism by intracerebral grafting of catecholaminergic neurons and to investigate its possible use as a donor material. These genetically manipulated fibroblasts did not develop into a mass of tissue, and survived well in the host striatum. Expression of the TH minigene in the cells was successful even when they were transplanted into the host brain. Intracerebral microdialysis revealed that a measurable amount of L-3,4-dihydroxyphenylalanine (L-DOPA) was not spontaneously released from the implanted cells into the host striatum. However, release of a large amount of L-DOPA from the cells was observed when (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) was perfused through a dialysis probe. Finally, we investigated whether these BH4-dependent L-DOPA-secreting fibroblasts are able to ameliorate the abnormal behavior of 6-hydroxydopamine-treated rats. Apomorphine-induced rotating behavior was not reversed by the grafting alone, whereas a marked reduction in drug-induced circling was observed temporarily after BH4 was perfused around the implanted cells. These findings indicate that TH cDNA-transfected non-neuronal cells might be able to be used as donor material for intracerebral grafting and ameliorate the abnormal behavior of rats with experimental Parkinson's disease.

Animals↗