A quantitative-trait analysis of human plasma-dopamine beta-hydroxylase activity: evidence for a major functional polymorphism at the DBH locus

Dopamine-beta-hydroxylase (D beta H) catalyzes the conversion of dopamine to norepinephrine and is released from sympathetic neurons into the circulation. Plasma-D beta H activity varies widely between individuals, and a subgroup of the population has very low activity levels. Mounting evidence suggests that the DBH structural gene is itself the major quantitative-trait locus (QTL) for plasma-D beta H activity, and a single unidentified polymorphism may account for a majority of the variation in activity levels. Through use of both sequencing-based mutational analysis of extreme phenotypes and genotype/phenotype correlations in samples from African American, European American (EA), and Japanese populations, we have identified a novel polymorphism (--1021C-->T), in the 5' flanking region of the DBH gene, that accounts for 35%--52% of the variation in plasma-D beta H activity in these populations. In EAs, homozygosity at the T allele predicted the very low D beta H-activity trait, and activity values in heterozygotes formed an intermediate distribution, indicating codominant inheritance. Our findings demonstrate that --1021C-->T is a major genetic marker for plasma-D beta H activity and provide new tools for investigation of the role of both D beta H and the DBH gene in human disease.     

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

Release 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.     

Selective inhibition of vascular smooth muscle cell proliferation by coptisine isolated from Coptis rhizoma, one of the crude drugs composing Kampo medicines Unsei-in.

Acceleration of vascular smooth muscle cell (VSMC) proliferation is closely linked to the pathogenesis of vascular diseases. We, therefore, focused on traditional Japanese herbal medicines (Kampo medicines) used to ameliorate the impairment of microcirculation or blood stasis and screened them for their ability to inhibit rat VSMC proliferation. Among them, Unsei-in was found to effectively suppress VSMC proliferation, and Coptis rhizome was the responsible constituent crude drug. The extract of Coptis rhizome inhibited VSMC proliferation with the GI(50) value of 4.4 microg/ml, which was much lower than those against the proliferation of 3Y1, dRLh-84, B16, and HeLa cells. The Coptis rhizome extract inhibited the progression of VSMC arrested at G(0)/G(1) phase from G(0)/G(1) to S phase, but not that of 3Y1 cells. Biological assay-guided fractionation revealed that an alkaloid of Coptis rhizome, coptisine, was the active ingredient in selectively preventing VSMC proliferation with GI(50) of 3.3 microM (1.2 microg/ml). When the structurally-related isoquinoline alkaloids of protoberberine class were studied for their inhibitory activities, berberine decreased the VSMC proliferation with GI(50) of 95.1 microM (35.4 microg/ml), about 30 times higher concentration than coptisine, while palmatine failed to show any activity. This study provides evidence that coptisine, an ingredient of Unsei-in, prevents VSMC proliferation selectively at lower concentrations compared with various cells or other structurally related alkaloids.     

Inhibition of monoamine oxidase by 3,4-dihydroxyphenyl L-alanine and its analogues

L-3,4-Dihydroxyphenylalanine (DOPA) was found to inhibit type A monoamine oxidase in human placental mitochondria. The inhibition proved to be noncompetitive with the substrate, kynuramine, and the inhibition was completely reversible. D-DOPA was found to inhibit monoamine oxidase in the same way, and the apparent Ki values of L- and D-DOPA were obtained to be 154 microM and 133 microM, respectively. L-alpha-Methyl-DOPA was found to inhibit the MAO activity competitively with the substrate, but studies with other analogues of DOPA revealed that the inhibition required an amino and a carboxyl group at alpha-position. The substitution of a hydroxy group at 3 or 4 position of catechol ring with a methoxy group was found to abolish the inhibition of the MAO activity. In addition to type A MAO in human liver and placental mitochondria, type B MAO in liver mitochondria was inhibited by L-DOPA, but type B MAO was less sensitive to L-DOPA. These results were discussed in terms of its possible regulation of the level of biogenic amines in the brain.     

Studies on Polyoxins,Antifungal Antibiotics

Three strains of soil Streptomyces which belong to Streptomyces cacaoi were found to produce a new antifungal antibiotic complex, polyoxin complex. Polyoxin A and B were isolated in pure forms out of it. They are amphoteric compounds with the molecular formulas, C₂₃H₃₂N₆O₁₄ and C₁₇H₂₅N₅O₁₃ respectively. They showed very specific activities against phytopathogenic fungi.     

Isolation and characterization of a cDNA clone encoding human aromatic L-amino acid decarboxylase

The nucleotide sequence of a cDNA clone that includes the entire coding region of human aromatic L-amino acid decarboxylase gene is presented. A human pheochromocytoma cDNA library was screened using an oligonucleotide probe which corresponded to a partial amino acid sequence of the enzyme purified from the human pheochromocytoma. The isolated cDNA clone encoded a protein of 480 amino acids with a calculated molecular mass of 53.9 kDa. The amino acid sequence Asn-Phe-Asn-Pro-His-Lys-Trp around a possible cofactor (pyridoxal phosphate) binding site is identical in human, Drosophila, and pig enzymes.     

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.