永津 俊治

To clarity the relationship between collagenase-like peptidase (CL-peptidase) and bone metabolism, serum CL-peptidase activity and bone mineral density (BMD) in the lumbar spine, measured by dual energy X-ray absorptiometry (DEXA), were determined in 102 women with a mean age of 70 (41-94) years. The serum activity of CL-peptidase increased slightly with age up to the 8th decade, and then decreased. Next, we classified the subjects into the following 2 groups by age: a postmenopausal group 50-69 years, and an elderly group over 70 years. In the younger group serum CL-peptidase activity did not correlate with BMD, while in the elderly it did. Our result suggests that serum CL-peptidase activity may reflect bone resorption in elderly women.

Carboxypeptidase (CPase) was partially purified from Kidachi aloe (Aloe arborescens Miller var. natalensis Berger) by an FPLC system, and was administered intravenously to female ICR mice with inflammation. The enzyme preparation exhibited significant analgesic effects and inhibited vascular permeability in the abdominal region. It also revealed an antithermal burn action on the hind paw, when it was administered to female Wistar rats intravenously. Copyright © 1993 John Wiley &amp Sons, Ltd.

A carboxypeptidase was partially purified from Aloe arborescens Miller var. natalensis Berger on a scale suitable for pharmacological studies. The enzyme was most active and stable at pH 5.0. The enzyme had a broad specificity against various synthetic peptides, being capable of splitting C‐terminal proline. Its activity was inhibited almost completely by diisopropylfluorophosphate, strongly by transition metals, such as Fe3+, Hg2+ and Cu2+, and moderately by sulphydryl reagents. These results indicate that Aloe enzyme is a serine carboxypeptidase and appears to contain a sulphydryl group that may be involved in its inactivation. Copyright © 1993 John Wiley &amp Sons, Ltd.

The effects of naturally occurring tetrahydroisoquinolines on monoamine catabolism were examined by in vivo microdialysis study in the rat striatum, or by in vitro experiments using human brain synaptosomes as enzyme samples. By in vivo experiment, catechol isoquinolines, (R) and (S) enantiomer of 1-methyl- and 2-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol and N-methyl-norsalsolinol) markedly inhibited oxidation of serotonin by type A oxidase. On the other band, isoquinolines without catechol, 1,2,3,4-tetrabydroisoquinoline, 1-methyl-derivative and N-methyl-isoquinolinium ion, mainly inhibited dopamine metabolism by the oxidase. By in vitro experiments, norsalsolinol and salsolinol, and their N-methylated derivatives were found to inhibit monoamine oxidase. N-Methyl-norsalsolinol, (R) and (S) enantiomer of salsolinol, and N-methyl-salsolinols inhibited type A monoamine oxidase competitively to the substrate, kynuramine, and R enantiomers were more potent inhibitors than S enantiomers. The inhibition was reversible. Norsalsolinol induced positive cooperativity toward kynuramine. Both norsalsolinol and N-methyl-norsalsolinol inhibited type B oxidase non-competitively to the substrate, but their K(i) values were much higher than those to type A. These results are discussed in relation to possible involvement of the isoquinolines as neuro-toxic and neuro-protective agents to the pathogenesis of some neuro-degenerative diseases, such as Parkinson's disease, or to clinical features of some diseases, such as alcoholism.

A new pteridine compound, named umanopterin, was isolated from human urine both of cancer patients and non-cancer controls. The structure was confirmed to be 2-amino-4(4(3H)-oxo-6-[(1'R,2'R)-1',2',3'-trihydroxypropyl]pteridine, a diastereomer of neopterin. The amount of umanopterin relative to neopterin was about 10%, which was practically the same among the non-cancer controls and the patients of various cancers. A small amount of a threo diastereomer of biopterin, named orinapterin, was isolated from human urine for the first time. Its structure was shown to be 2-amino-4(3H)-oxo-6-[(1'S,2'S)-1',2'-dihydroxypropyl]pteridine. A non-enzymatic transformation of 7,8-dihydroneopterin and 7,8-dihydrobiopterin by a mechanism analogous to keto-enol tautomerism is postulated for the formation of umanopterin and orinapterin in human body.

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-mu-M 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-mu-M, 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+.

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.

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 TPH. These data indicate that salsolinols might be naturally occurring inhibitors of indoleamine metabolism.

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.

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. Apomorophine-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.

A new pteridine compound, named umanopterin, was isolated from urine of cancer patients. The structure was confirmed to be 2-amino-4-hydroxy-6-[(1'R,2' R)-1',2',3'-trihydroxypropyl]-pteridine, a diastereomer of neopterin.

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.

Allostery of tyrosine hydroxylase was found by kinetical studies of partially purified tyrosine hydroxylase from clonal rat pheochromocytoma PC 12h cells. Postitive 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.

A new natural pteridine, named oncopterin, was isolated from urine of cancer patients by a combination of DEAE anion exchange, CM cation exchange, and reverse phase column chromatography. Because of its character as a strong base, oncopterin was effectively separated from other unconjugated pteridines by the cation exchange chromatography. The structure of oncopterin was confirmed to be 2-(3-aminopropyl)amino-4-hydroxy-6-[(1'R,2'S)-1',2'-dihydroxypropyl]pteridine, a N2-(3-aminopropyl) derivative of biopterin. Oncopterin could not be isolated from urine of healthy controls, and thus this compound is expected to be a promising biochemical marker of cancers and other

We stained chromaffin cells of the adrenal medulla and both the adrenergic and noradrenergic cells of the locus coeruleus of the monkey by using the antibodies for the N-terminal, intermediate and C-terminal peptides of human dopamine-beta-hydroxylase (hDBH). Immunoreactivities for both N- and C-terminals of hDBH were observed in the cells of either the adrenal medulla or the locus coeruleus of the monkey. In contrast, with the antibody for intermediate of hDBH, immunoreactivities were not observed in these cells. Together with our previous studies, it might be concluded that the antibody for the C-terminal of hDBH is primate specific and that for the N-terminal of hDBH is mammalian specific.

Serum dopamine-beta-hydroxylase (DBH, dopamine beta-monooxygenase ; EC 1.14.17.1) is derived from peripheral sympathetic nerves together with norepinephrine. Serum DBH shows circadian variations. We have developed a simple method to estimate the circadian variation, and have applied the method for the study of serum DBH in schizophrenic patients. We estimated the times at which the activity of serum DBH showed the maximum and minimum levels from the circadian rhythm in 40 normal controls. We then compared the activities at these two points between schizophrenic patients and normal controls, and evaluated the significance of the circadian variation of serum DBH activity as a biochemical marker in schizophrenia. From the circadian rhythm of the serum DBH activity (IU, mu-mol/min per liter serum) in 40 normal controls, 5:00 a.m. (minimum activity) and 13:00 p.m. (maximum activity) were selected as the two measurement points to estimate circadian variation. The serum DBH activity was then measured at these times in 40 normal controls and 166 volunteer patients with schizophrenia during 5 consecutive days. The mean serum DBH activities at 5:00 and 13:00 were significantly lower in 166 schizophrenic patients than those in 40 normal controls (25.65 +/- 0.91 vs 42.79 +/- 3.59 at 5:00 ; 25.80 +/- 0.92 vs 44.50 +/- 3.67 at 13:00), conforming to our previous report [Fujita, K. et al, (1978) J. Newochem, 30, 1569 - 1572]. The difference between the mean DBH levels at 13:00 and 5:00 was divided by the mean activity of the values at all 10 time points during the 5 days, and this value was defined as the circadian variation in serum DBH activity (%). The circadian variation in serum DBH activity (mean @ SEM) was 4.37 +/- 0.48 (%) in 40 normal controls and 1.05 +/- 0.33 (%) in 166 schizophrenic patients, being significantly smaller in schizophrenic patients than in normal controls (p < 0.001). Furthermore, in 6 from 40 normal controls and 78 from 166 schizophrenic patients whose serum DBH levels were less than 25 IU, the mean circadian variation in serum DBH activity was 6.33 +/- 1.95 (%) in normal controls and 1.02 +/- 0.55 (%) in schizophrenic patients (P < 0.05). Similarly, in schizophrenic patients and normal controls whose serum DBH levels were 25 IU and above, the circadian variation in DBH was 4.03 +/- 0.45 (%) in normal controls and 1.09 +/- 0.39 (%) in schizophrenic patients (p < 0.001). Significantly lower circadian variations were observed in three types of schizophrenia ; hebephrenic, Paranoid and catatonic forms. These results indicate that the circadian variation in serum DBH is significantly decreased in schizophrenic patients regardless of the mean serum DBH activity during 24 hours and of the types of disease. Therefore, the variation in DBH is considered to be useful as a biochemical marker in schizophrenia.

The tyrosine kinase inhibitor erbstatin did not inhibit nerve growth factor (NGF)-induced neurite out in PC12 cells but rather enhanced the effect of NGF. Erbstatin alone induced transient neurite formation and acetylcholinesterase activity. Other tyrosine kinase inhibitors such as methyl 2,5-dihydroxycinnamate, lavendustin C6, and herbimycin also induced transient differentiational changes in PC12 cells. Thus, our data suggest that inhibition of tyrosine kinase in one of the triggers for differentiation of PC12 cells.

Adriamycin (ADR) is effective against a wide range of human neoplasms. However, its clinical use is compromised by serious cardiac toxicity, possibly through induction of peroxidation in cardiac lipids. Ascorbic acid, a potent antioxidant, was examined for effect in reducing ADR toxicity in mice and guinea pigs. Ascorbic acid had no effect on the antitumor activity of ADR in mice inoculated with leukemia L1210 or Ehrlich ascites carcinoma, but it significantly prolonged the life of animals treated with ADR. ADR elevated lipid peroxide levels in mouse heart, and ascorbic acid prevented the elevation. The significant prevention of ADR-induced cardiomyopathy in guinea pigs by ascorbic acid was proved by electron microscopy. Ascorbic acid and the derivatives may delay general toxicity of ADR and also prevent the cardiac toxicity. The results also suggest the clinical efficacy of the combined treatment of ADR and ascorbic acid or the derivatives.

We have previously reported the distribution of human tyrosine hydroxylase (TH) transgene expression in dopaminergic neurons (ventral tegmental area and substantia nigra), adrenal gland, and non-catecholaminergic neurons in the forebrain of transgenic (Tg) mice. In this paper, we analysed the transgene expression in catecholaminergic (CAergic) neurons in the lower brainstem of Tg mice, by in situ hybridization and immunocytochemistry at the light and electron microscopic levels. High-level hybridization signals of the human TH mRNA were observed in the locus ceruleus and nucleus tractus solitarii of the Tg brain. Intense TH immunoreactivity was expressed specifically in the Tg brainstem, as was observed in non-Tg mice. These results reveal that the human TH transgene contains the regulatory elements responsible for the expression in three kinds of CAergic (dopaminergic, noradrenergic and adrenergic) neurons of the mouse brain.

Relationships among cytological features, doubling time, S-phase percentage, expression of myc-family oncogenes, DNA ploidy and biochemical properties were studied in thirteen small cell lung cancer cell lines. Six cell lines that grew slowly (average doubling time 99 h) and had lower S-phase percentages (average 32%) showed inconspicuous nucleoli (average area of 1.5-mu-m2), and the remaining seven cell lines that grew quickly (average doubling time 45 h) and had higher S-phase percentages (average 44%) showed large and prominent nucleoli (average area of 6.1-mu-m2). DNA index value obtained from flow cytometric DNA histograms showed that all cell lines except for H-69 cell line displayed aneuploidy. Ribbon-like cell arrangements were observed in the 7 cell lines that grew quickly, and in 1 cell line that grew slowly. Biochemically, six slow-growing cell lines and four fast-growing cell lines showed high levels of aromatic L-amino acid decarboxylase activity, while in the remaining three fast-growing cell lines its level was low. A high level of c-myc or N-myc oncogene expression was observed in all 7 cell lines that grew quickly, but not in any of the 6 cell lines that grew slowly. It appears that small cell lung cancer cell lines that grow quickly can be expected to have large nucleoli and ribbon-like cell arrangements and to express high levels of myc-family oncogenes, and that nucleolar size is a good indicator for growth characteristics.

We have recently reported the production of transgenic (Tg) mice carrying the human tyrosine hydroxylase (TH) gene, and have described tissue-specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. This paper describes the transgene expression in non-catecholaminergic (nCAergic) neurons in the brain of Tg mice by immunocytochemistry and in situ hybridization. In adult Tg mice, human TH was atypically expressed in the olfactory (typically, the anterior olfactory nucleus and pyriform cortex) and visual (typically, n. suprachiasmaticus and n. parabigeminalis) systems, in addition to typical CAergic neuron-rich nuclei in the brain. These results suggest the possibility that TH plays some novel roles in sensory systems.

The cDNAs for tyrosine hydroxylase were cloned from a mouse brain cDNA library by plaque hybridization. Since the longest cDNA clone lacked approximately 150 bp sequence of its N-terminal region, additional 5' region was obtained using polymerase chain reaction. Nucleotide sequence determination of cDNAs revealed that mouse tyrosine hydroxylase m-RNA encodes 498 amino acids with a calculated molecular mass of 55,990. The amino acid sequence of mouse tyrosine hydroxylase is highly homologous to rat (97%) and human (92%) enzymes.

The application of high-performance liquid chromatography to the study of biogenic amine-related enzymes is reviewed. Biogenic amines include catecholamines (dopamine, norepinephrine and epinephrine), indoleamines (serotonin and melatonin), imidazoleamines (histamine), polyamines (putrescine, spermidine and spermine) and acetylcholine. Three particular aspects are covered. The first aspect is the assay of enzyme activities of biogenic amine-related enzymes, such as tyrosine hydroxylase, tryptophan hydroxylase, aromatic L-amino acid decarboxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase. The introduction of highly sensitive assays of biogenic amines with electrochemical detection or fluorescence detection have made possible the non-isotopic assay of these activities, replacing the previously used radioisotopic methods. The second aspect is the purification of these enzymes. Since biogenic amine-synthesizing enzymes are generally unstable, rapid and efficient purification of these enzymes is very useful. The third aspect is the assay of biogenic amines (for example, acetylcholine and polyamines) using post-column derivatization with biogenic amine oxidases and electrochemical detection.

A carcinogenic, food-derived heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1) was found to reduce the enzymatic activity of tyrosine hydroxylase in clonal rat pheochromocytoma PC12h cells, by its supplement to the culture medium. The reduction was observed with 10-mu-M Trp-P-1, and at this concentration the amount of cell protein and the activity of a non-specific enzyme, beta-galactosidase, were not affected. The mechanism of the reduction of the enzyme activity was clarified by kinetical studies. The amine reduced the affinity of tyrosine hydroxylase to a cofactor, tetrahydrobiopterin. The alteration of the enzymatic properties by Trp-P-1 was discussed in relation to the possible effect on catecholamine metabolism in the brain.

The suprachiasmatic nucleus (SCN) has been identified as a major circadian pacemaker. Methamphetamine has been shown to modify the behavior of circadian rhythms. We detected extracellular serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the SCN in freely moving rats, using a microdialysis method, to investigate biochemical effects of methamphetamine in the SCN. Methamphetamine infusion into the SCN dose-dependently increased extracellular 5-HT and decreased extracellular 5-HIAA.

1. Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. 2. The structures of TH from various species have been elucidated. 3. We have cloned and determined the sequences of four types of human TH cDNA and human TH genomic DNA. 4. We have compared the amino acid sequences of TH from various species. 5. The results indicate that the amino acid sequences of TH are highly conserved among various species, and that TH consists of the regulatory domain containing serine residues which are phosphorylated by protein kinases and of the catalytic domain where the substrates, tyrosine and oxygen, and the cofactor, tetrahydrobiopterin, are bound. 6. Comparison of amino acid sequences among TH from various species can give us useful information on the functional importance of each amino acid residue.