Norio Kaneda

Presence of tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline in Parkinsonian and normal human brains

1,2,3,4-Tetrahydroisoquinoline (TIQ) and 2-methyl-1,2,3,4-tetrahydroquinoline (2-Me-TQ) were identified for the first time by gas chromatography-mass spectrometry in the parkinsonian and normal human brains. TIQ, an analogue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was markedly increased in the parkinsonian brain and could be an endogenous neurotoxin to induce Parkinsons disease.

Isolation of a novel cDNA clone for human tyrosine hydroxylase: Alternative RNA splicing produces four kinds of mRNA from a single gene

Human tyrosine hydroxylase (TH) cDNA was isolated by molecular cloning. Lambda gt 11 cDNA library constructed from human pheochromocytoma was screened with a synthetic 23-mer oligonucleotide complementary to rat TH mRNA. We found a novel type of cDNA clone whose N-terminal sequence is similar to but clearly distinct from each of the three types (type 1, 2 and 3) of TH cDNA reported by Grima et al. [Nature (1987) 326, 707-711]. It contains both the 12-bp insert characteristic of type 2 cDNA and the 81-bp sequence of type 3. This novel cDNA clone was designated as type 4. Southern blot analysis of human genomic DNA indicated that TH is encoded by a single gene. This suggests that the four different forms of TH mRNA are produced by alternative RNA splicing from a single primary transcript.

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.

Acute effects of 1-methyl-4-phenylpyridinium ion (MPP+) on dopamine and serotonin metabolism in rat striatum as assayed in vivo by a micro-dialysis technique

The acute effect of 1-methyl-4-phenylpyridinium ion (MPP+), a neurotoxin derived from 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), was examined by the in vivo micro-dialysis technique. A dialysis cannula was implanted into rat striatum, and the changes in the concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the perfusate every 20 min after administration of MPP+ were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED). After MPP+ administration the levels of DOPAC, HVA and 5-HIAA were markedly decreased. On the contrary the level of DA was markedly increased and reached a maximum 40 min after beginning of the MPP+ administration. By postmortem analysis of the striatal tissue MPP+ was proved to cause the inhibition of monoamine oxidase (MAO), especially MAO-B. These results suggest that the acute biochemical changes induced by MPP+ in vivo were MAO inhibition and release of DA.

Simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue by high-performance liquid chromatography with electrochemical detection

A simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue was developed by using high-performance liquid chromatography with electrochemical detection. These compounds are analysed in a single chromatographic run within 30 min with a simple sample clean-up procedure. The detection system consists of two electrochemical detector cells aligned in series: a glassy-carbon electrode for catecholamines and serotonin, and a platinum electrode for acetylcholine and choline. For the detection of the latter compounds, they were converted enzymatically into hydrogen peroxide through a column reactor with immobilized acetylcholinesterase and choline oxidase. A column of boronic acid gel was placed just ahead of the immobilized enzyme column to remove catecholamines, which caused interfering responses on the platinum electrode. Two equivalent analytical columns and a column switching were employed to speed up the serotonin assay. Simultaneous determination of these major neurotransmitters in rat brain regions was successfully carried out with the system described.

Analysis of dansyl amino acids by reverse-phase high-performance liquid chromatography☆

Abstract All 24 dansyl amino acids were separated by reverse-phase high-performance liquid chromatography on Develosil C8-5, using a linear gradient made from Tris-HCl buffer (pH 7.75) and methanol. A linear relationship between the amount of sample and peak area was found over the range of 6 to 300 ng (0.02–1 nmol) of dansyl derivatives. An application of this method to the NH 2 -terminal analysis of lysozyme is described.

Synthesis of l-3,4-Bihydroxyphenylalanine by Tyrosine Hydroxylase cDNA-Transfected C6 Cells: Application for Intracerebral Grafting

In the present study, we obtained genetically manipulated nonneuronal cells which synthesize a catecholamine precursor for future use in intracerebral grafting. Human type 1 tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was inserted into eukaryotic expression vector pKCRH2 and was co‐transfected into C6 cells with plasmid pSV2neo. Expression of the TH minigene was screened by immuno‐histochemical staining with TH antibody and immunoblot‐ting analysis. Several clones of the C6 transfectahts that produce TH molecules were obtained. These cells showed TH activity, and the product, L‐3,4‐dihydroxyphenylalanine (L‐DOPA), was detected intracellulary due to the ajbsence of L‐amino acid decarboxylase (EC 4.1.1.28) activity. It was found that a large amount of L‐DOPA was released from the cells into the culture medium. These transfectants were transplanted into rat brain, and the expression of TH was examined immunohistochemically. On the 10th day following transplantation, a mass of C6 cells which was heavily stained with TH antibody was observed in the brain. These findings may provide us with an opportunity to investigate the effects of intracerebral transplantation of nonneuronal cells that produce catecholamine or its precursor.

Expression of four types of human tyrosine hydroxylase in COS cells

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1–4) which have structural diversity only in the N‐terminal region. We attempted expression of the type 1–4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The K m values of the four types for L‐tyrosine and 6‐methyl‐5,6,7,8‐tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.

Tetrahydrobiopterin-dependent production of l-DOPA in NRK fibroblasts transfected with tyrosine hydroxylase cDNA: future use for intracerebral grafting

In the present study, tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was transfected into cultured fibroblasts and the production of L-3,4-dihydroxyphenylalanine (L-DOPA) was determined. Type 2 TH cDNA was transfected into fibroblasts (NRK-49F) derived from the normal rat kidney, and the expression of the TH minigene was screened by immunocytochemical staining and immunoblotting analysis with TH antiserum. Several clones of the NRK transfectants that produce TH molecules were obtained. The expressed TH molecules showed high enzyme activity in a complete assay system in vitro. However, L-DOPA was not detected in the cultured cells due to the possible absence of de novo synthesis of (6R)-L-erythro-tetrahydrobiopterin (BH4) in these cells. When BH4 was added to the medium, a large amount of L-DOPA was detected not only in the cells but also in the medium. These findings may aid in regulating the amount of L-DOPA secretion from cells after they are transplanted into the brain.