Biography:

One of their most recent publications is Radioimmunoassay for neopterin in body fluids and tissues. Which was published in journal Analytical Biochemistry.

More information about Toshiharu Nagatsu research including statistics on their citations can be found on their Copernicus Academic profile page.

Toshiharu Nagatsu's Articles: (18)

Radioimmunoassay for neopterin in body fluids and tissues

AbstractSpecific antibodies against d-erythroneopterin have been prepared in rabbits using a conjugate of d-erythroneopterin to bovine serum albumin (d-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished d-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, l-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for d-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of d-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak. Approximately 10 pmol/g tissue of neopterin was detected by this radioimmunoassay in rat tissues (liver, kidney, adrenal gland, and brain). About 35% less neopterin value was obtained when the samples from human urine were oxidized in alkaline solution, suggesting the presence of alkaline-labile reduced forms of neopterin. When the extract of rat liver was treated with alkaline phosphatase prior to the acid oxidation process, total neopterin values were increased approximately twofold. This indicates that approximately 50% of neopterin in rat liver is present as a phosphate ester.

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

AbstractA highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase

AbstractMelanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.

Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells

AbstractRelease of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.

Radioimmunoassay for neopterin in body fluids and tissues

AbstractSpecific antibodies against d-erythroneopterin have been prepared in rabbits using a conjugate of d-erythroneopterin to bovine serum albumin (d-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished d-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, l-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for d-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of d-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak. Approximately 10 pmol/g tissue of neopterin was detected by this radioimmunoassay in rat tissues (liver, kidney, adrenal gland, and brain). About 35% less neopterin value was obtained when the samples from human urine were oxidized in alkaline solution, suggesting the presence of alkaline-labile reduced forms of neopterin. When the extract of rat liver was treated with alkaline phosphatase prior to the acid oxidation process, total neopterin values were increased approximately twofold. This indicates that approximately 50% of neopterin in rat liver is present as a phosphate ester.

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

AbstractA highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase

AbstractMelanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.

Radioimmunoassay for neopterin in body fluids and tissues

AbstractSpecific antibodies against d-erythroneopterin have been prepared in rabbits using a conjugate of d-erythroneopterin to bovine serum albumin (d-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished d-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, l-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for d-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of d-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak. Approximately 10 pmol/g tissue of neopterin was detected by this radioimmunoassay in rat tissues (liver, kidney, adrenal gland, and brain). About 35% less neopterin value was obtained when the samples from human urine were oxidized in alkaline solution, suggesting the presence of alkaline-labile reduced forms of neopterin. When the extract of rat liver was treated with alkaline phosphatase prior to the acid oxidation process, total neopterin values were increased approximately twofold. This indicates that approximately 50% of neopterin in rat liver is present as a phosphate ester.

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

AbstractA highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.

Radioimmunoassay for neopterin in body fluids and tissues

AbstractSpecific antibodies against d-erythroneopterin have been prepared in rabbits using a conjugate of d-erythroneopterin to bovine serum albumin (d-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished d-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, l-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for d-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of d-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak. Approximately 10 pmol/g tissue of neopterin was detected by this radioimmunoassay in rat tissues (liver, kidney, adrenal gland, and brain). About 35% less neopterin value was obtained when the samples from human urine were oxidized in alkaline solution, suggesting the presence of alkaline-labile reduced forms of neopterin. When the extract of rat liver was treated with alkaline phosphatase prior to the acid oxidation process, total neopterin values were increased approximately twofold. This indicates that approximately 50% of neopterin in rat liver is present as a phosphate ester.

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

AbstractA highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase

AbstractMelanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.

Effects of myosin light-chain kinase inhibitor on catecholamine secretion from rat pheochromocytoma PC12h cells

AbstractRelease of dopamine from rat pheochromocytoma PC12h cells by high K+ (50 mM) was inhibited by a specific inhibitor of myosin light-chain kinase (ML-9) dose-dependently. The myosin light-chain kinase inhibitor also specifically inhibited the phosphorylation of a 20 KDa protein by myosin light-chain kinase. Myosin light chain kinase may play a stimulatory role in the release reaction of catecholamines from the rat pheochromocytoma cells.

REGULATION OF TYROSINE HYDROXYLASE

ABSTRACTBiosynthesis of catecholamines in dopaminergic or noradrenergic neurons is mainly controlled by the regulation of tyrosine hydroxylase (TH). Short-term regulation of TH may be mediated through activation and probably removal of feed-back inhibition of the catecholamine after its phosphorylation by cyclic AMP-dependent protein kinase and the resultant conformational changes. Either pre-synaptic or post-synaptic receptor activity would be important for the acute regulation of TH. Long-term regulation depends on the synthesis of TH molecules in the cell bodies of catecholaminergic neurons and subsequent increase at nerve terminals, and this enzyme induction mechanism appears to be due to transsynaptic stimulation of acetylcholine receptors and to be mediated through cyclic AMP-dependent protein kinase.

Review articleIsoquinoline neurotoxins in the brain and Parkinson's disease

AbstractParkinson's disease is thought to be caused by some unknown endogenous or exogenous factors interacting with genetic dispositions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is an exogenous neurotoxin producing parkinsonism in humans, monkeys and various animals as the result of monoamine oxidase type B (MAO-B)-catalyzed conversion of it to the 1-methyl-4-phenyl-pyridinium ion (MPP+), which selectively kills the nigrostriatal dopaminergic neurons. Various isoquinoline derivatives were found in the brain of patients with Parkinson's disease. Isoquinoline derivatives have neurochemical properties similar to those of MPTP and they are considered to be the endogenous neurotoxins which cause Parkinson's disease. Among them, tetrahydroisoquinoline (TIQ), 1-benzyl-TIQ, and (R)-1,2-dimethyl-5,6-dihydroxy-TIQ [(R)-N-methyl-salsolinol)] have the most potent neurotoxicity. TIQs, like MPTP, may be activated via N-methylation by N-methyltransferase and oxidation by MAO. TIQs as well as MPP+ inhibit complex I of the electron transport system in mitochondria, thereby reducing ATP formation and producing oxygen radicals. Although the properties of TIQs are similar to those of MPTP, the neurotoxicity of TIQs is weaker than that of MPTP. Since Parkinson's disease is a slowly progressing neurodegenerative disease, long term neurotoxic effects of IQs remain to be further examined in primates.

Review articleAmine-related neurotoxins in Parkinson's disease: Past, present, and future

AbstractParkinson's disease (PD) is an aging-related movement disorder caused by a deficiency of the neurotransmitter dopamine (DA) in the striatum of the brain as a result of selective degeneration of nigrostriatal DA neurons. The molecular basis of the cell death of DA neurons is unknown, but one hypothesis is the presence of some amine-related neurotoxins that kill specifically nigrostriatal DA neurons over a long period of time. This neurotoxin hypothesis of PD started in the 1980s when 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was discovered to produce acutely PD-like symptoms. Two groups of natural MPTP-like and amine-related neurotoxins have been investigated as endogenous candidate compounds: isoquinolines (IQs) and beta-carbolines. These neurotoxins are speculated to cause oxidative stress, mitochondrial dysfunction, apoptotic cell death, and PD symptoms. However, since PD is a neurodegenerative disorder that progresses slowly over a period of many years, a long-term study may be required to elucidate the neurotoxicity of such neurotoxins in relation to PD.

ReviewL-dopa therapy for Parkinson's disease: Past, present, and future

AbstractDopamine (DA) supplementation therapy by L-dopa for Parkinson's disease (PD) was established around 1970. The dose of L-dopa can be reduced by the combined administration of inhibitors of peripheral L-amino acid decarboxylase (AADC), catechol O-methyltransferase (COMT), or monoamine oxidase B (MAO B). DA in the striatum may be produced from exogenously administered L-dopa by various AADC-containing cells, such as serotonin neurons. The long-term administration of L-dopa in PD patients may produce L-dopa–induced dyskinesia (LID), which may be due to chronic overstimulation of supersensitive DA D1 receptors. L-dopa may be used in combination with various new strategies such as gene therapy or transplantation in the future.

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