Chromosomal-level genome of velvet bean (Mucuna pruriens) provides resources for L-DOPA synthetic research and development.

Mucuna pruriens, commonly called velvet bean, is the main natural source of levodopa (L-DOPA), which has been marketed as a psychoactive drug for the clinical management of Parkinson's disease and dopamine-responsive dystonia. Although velvet bean is a very important plant species for food and pharmaceutical manufacturing, the lack of genetic and genomic information about this species severely hinders further molecular research thereon and biotechnological development. Here, we reported the first velvet bean genome, with a size of 500.49 Mb and 11 chromosomes encoding 28,010 proteins. Genomic comparison among legume species indicated that velvet bean speciated ∼29 Ma from soybean clade, without specific genome duplication. Importantly, we identified 21 polyphenol oxidase coding genes that catalyse l-tyrosine to L-DOPA in velvet bean, and two subfamilies showing tandem expansion on Chr3 and Chr7 after speciation. Interestingly, disease-resistant and anti-pathogen gene families were found contracted in velvet bean, which might be related to the expansion of polyphenol oxidase. Our study generated a high-quality genomic reference for velvet bean, an economically important agricultural and medicinal plant, and the newly reported L-DOPA biosynthetic genes could provide indispensable information for the biotechnological and sustainable development of an environment-friendly L-DOPA biosynthesis processing method.

Kinetic approach for the enzymatic determination of levodopa and carbidopa assisted by multivariate curve resolution-alternating least squares.

A combination of kinetic spectroscopic monitoring and multivariate curve resolution-alternating least squares (MCR-ALS) was proposed for the enzymatic determination of levodopa (LVD) and carbidopa (CBD) in pharmaceuticals. The enzymatic reaction process was carried out in a reverse stopped-flow injection system and monitored by UV-vis spectroscopy. The spectra (292-600 nm) were recorded throughout the reaction and were analyzed by multivariate curve resolution-alternating least squares. A small calibration matrix containing nine mixtures was used in the model construction. Additionally, to evaluate the prediction ability of the model, a set with six validation mixtures was used. The lack of fit obtained was 4.3%, the explained variance 99.8% and the overall prediction error 5.5%. Tablets of commercial samples were analyzed and the results were validated by pharmacopeia method (high performance liquid chromatography). No significant differences were found (alpha=0.05) between the reference values and the ones obtained with the proposed method. It is important to note that a unique chemometric model made it possible to determine both analytes simultaneously.

Longitudinal behavioral and 6-[18F]fluoro-L-DOPA-PET assessment in MPTP-hemiparkinsonian monkeys.

Biochemical and behavioral criteria were established to determine the long-term stability of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced unilateral striatal dopamine deficiency in the vervet monkey. At time points over a 12-month period, post-MPTP striatal dopamine synthesis capacity was indexed with 6-[18F]fluoro-L-DOPA (FDOPA)-positron emission tomography. For the MPTP-treated subjects (n = 4), an intrasubject FDOPA influx rate constant (Ki) ratio method of right (lesioned) striatum/left (unlesioned) striatum values was used to assess changes in striatal activity. Striatal FDOPA Ki ratios differed less than 5% between studies conducted at 1-2, 5-7, and 9-11 months post-MPTP; these results indicated a stable MPTP-induced striatal lesion over this time period. At the 5-7 and 9-11 month time points, behavioral indices of the MPTP-induced deficits were obtained within a species-typical group setting. For three of the four subjects, persistent decrements in motoric, affiliative, and vigilance behavior were observed while the frequency of aggression toward group members was increased. At the 9-11 month time point, one subject showed a 30% improvement in the social measures, indicative of a partial recovery from the MPTP-induced behavioral decrements although its striatal FDOPAKi ratio remained unchanged. Thus, behavioral and noninvasive biochemical methods can provide complementary indices to assess individual differences in sensitivity to MPTP-induced deficits. Both types of data are required to determine lesion stability and, subsequently, the efficacy of interventions designed to restore normal function in this primate Parkinsonian model.

Assessment of renal dopaminergic system activity during cyclosporine A administration in the rat.

1. Administration of cyclosporine A (CsA; 50 mg kg-1 day-1, s.c.) for 14 days produced an increase in both systolic (SBP) and diastolic (DBP) blood pressure by 60 and 25 mmHg, respectively. The urinary excretion of dopamine, DOPAC and HVA was reduced from day 5-6 of CsA administration onwards (dopamine from 19 to 46%, DOPAC from 16 to 48%; HVA from 18 to 42%). In vehicle-treated rats, the urinary excretion of dopamine and DOPAC increased (from 7 to 60%) from day 5 onwards; by contrast, the urinary excretion of HVA was reduced (from 27 to 60%) during the second week. 2. No significant difference was observed between the Vmax and Km values of renal aromatic L-amino acid decarboxylase (AAAD) in rats treated with CsA for 7 and 14 days or with vehicle. 3. Km and Vmax of monoamine oxidase types A and B did not differ significantly between rats treated with CsA for 7 and 14 days or with vehicle. 4. Maximal catechol-O-methyltransferase activity (Vmax) in homogenates of renal tissues obtained from rats treated with CsA for 7 or 14 days was significantly higher than that in vehicle-treated rats; Km (22.3 +/- 1.5 microM) values for COMT did not differ between the three groups of rats. 5. The accumulation of newly-formed dopamine and DOPAC in cortical tissues of rats treated with CsA for 14 days was three to four times higher than in controls. The outflow of both dopamine and DOPAC declined progressively with time and reflected the amine and amine metabolite tissue contents. No significant difference was observed between the DOPAC/dopamine ratios in the perifusate of renal tissues obtained from CsA- and vehicle-treated rats. In addition, no significant differences were observed in k values or in the slope of decline of both DA and DOPAC between experiments performed with CsA and vehicle-treated animals. 6. The Vmax for the saturable component of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in renal tubules from rats treated with CsA was twice that of vehicle-treated animals. Km in CsA- and vehicle-treated rats did not differ. 7. The decrease in the urinary excretion of sodium and an increase in blood pressure during CsA treatment was accompanied by a reduction in daily urinary excretion of dopamine. This appears to result from a reduction in the amount of L-DOPA made available to the kidney and does not involve changes in tubular AAAD, the availability of dopamine to leave the renal cells and dopamine metabolism. The enhanced ability of the renal tissues of CsA-treated animals to synthesize dopamine, when exogenous L-DOPA is provided, results from an enhanced activity of the uptake process of L-DOPA in renal tubular cells.

Assessment of renal dopaminergic system activity in the nitric oxide-deprived hypertensive rat model.

1. The present paper reports changes in the urinary excretion of dopamine, 5-hydroxytryptamine and amine metabolites in nitric oxide deprived hypertensive rats during long-term administration of NG-nitro-L-arginine methyl ester (L-NAME). Aromatic L-amino acid decarboxylase (AAAD) activity in renal tissues and the ability of newly-formed dopamine to leave the cellular compartment where the synthesis of the amine has occurred were also determined. 2. Twenty four hours after exposure to L-NAME, both systolic (SBP) and diastolic (DBP) blood pressure were increased by 20 mmHg; heart rate was slightly decreased. During the next 13 days both SBP and DBP increased progressively reaching 170 +/- 3 and 116 +/- 3 mmHg, respectively. 3. Baseline urinary excretion of L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) during the 4 day period of stabilization averaged 4.4 +/- 0.5, 13.8 +/- 0.3, 37.4 +/- 0.8, 180.0 +/- 2.7 and 206.1 +/- 6.7 nmol day-1, respectively. The urinary excretion of L-DOPA, dopamine and DOPAC, but not that of 3-MT and HVA, were increased from day 6-8 of L-NAME administration onwards (L-DOPA, up to 13.4 +/- 2.1; dopamine, up to 23.0 +/- 1.6; DOPAC, up to 62.8 +/- 3.7 nmol day-1). Baseline daily urinary excretion of 5-hydroxytryptamine and 5-hydroxyindolacetic acid (5-HIAA) averaged 73.5 +/- 1.1 and 241.7 +/- 5.4 nmol day-1, respectively. During the first week of L-NAME administration, the urinary excretion of both 5-hydroxytryptamine and 5-HIAA did not change significantly; however, as was found with dopamine and DOPAC, changes in the urinary excretion of 5-hydroxytryptamine were evident during the second week of L-NAME administration. 4. In experiments performed on homogenates of isolated renal tubules, the decarboxylation of L-DOPA to dopamine was dependent on the concentration of L-DOPA used (10 to 5000 microM) and saturable at 1000 microM. AAAD activity as determined in homogenates (Vmax, in nmol mg-1 protein h-1; Km in microM) was significantly (P < 0.01) higher in rats given L-NAME for 14 days (Vmax = 25 +/- 2; Km = 72 +/- 10) than in control rats (Vmax = 14 +/- 1; Km = 63 +/- 7), rats given L-NAME for 7 days (Vmax = 15 +/- 1; Km = 69 +/- 5) and rats given L-NAME plus L-arginine (Vmax = 13 +/- 1; Km = 60 +/- 3) for 14 days. 5. A considerable amount of the total dopamine formed from added L-DOPA in kidney slices escaped into the incubation medium. The application of the Michaelis-Menten equation to the net transport of newly-formed dopamine allowed the identification of a saturable (carrier-mediated transfer) and a non-saturable component (diffusion). No significant differences in the diffusional rate of transfer(0.14 +/- 0.02 micro mol-1) were observed between the four experimental groups. However, the saturable outward transfer of dopamine (Vmax, in micromol mg-1 protein h-1; Km in microM) was higher in control animals(Vmax= 2.3 +/- 0.2; Km = 568 +/- 67) than that in rats treated with L-NAME for 14 days (Vmax = 0.8 +/- 0.02;Km = 241 +/- 21), but similar to that observed in rats receiving L-NAME plus L-arginine (Vmax= 2.4+/- 0.2; Km= 618 +/- 61); the saturable dopamine outward rate of transfer in rats given L-NAME for 7days (Vmax = 3.9 +/- 0.2; Km = 1006 +/- 32) was higher than in controls.6. In conclusion, the present studies show that the hypertensive response resulting from the long-term administration of L-NAME is accompanied by an increased urinary excretion of dopamine and 5-hydroxytryptamine, which appears to follow an enhanced activity of renal AAAD. The observation that the increased AAAD activity can be reversed by the administration of L-arginine to L-NAME treated rats favours the view that the adaptational response which results in an enhanced AAAD activity probably involves a decrease in the generation of nitric oxide.

Assessment of dopamine and its metabolites in the intracellular and extracellular compartments of the rat striatum after peripheral administration of L-[11C]dopa.

Success in the synthesis of L-3,4-[beta-11C]dihydroxyphenylalanine (L-[11C]DOPA) and its application to positron emission tomography encouraged us to perform radioactive metabolite analyses in rats in an early phase after peripheral injection of L-[11C]DOPA. Following intravenous injection of [11C]DOPA, the radioactivity associated with DOPA and its metabolites was determined in the striatum after decapitation and in striatal extracellular fluid using in vivo brain microdialysis. Without pretreatment, 70-80% of 11C-radioactivity taken up into the striatum was associated with acidic metabolites of dopamine (DA) from 2 to 30 min after administration of L-[11C]DOPA with or without 300 micrograms/kg of unlabelled L-DOPA. In contrast, 80-90% of 11C-radioactivity in the striatum was associated with DOPA and DA after pretreatment with benserazide (25 mg/kg, i.p.) followed by administration of L-[11C]DOPA with or without unlabelled L-DOPA. The radioactivity in the DOPA fraction decreased with time (from 35% of 11C-radioactivity in the striatum at 5 min to 10% at 30 min), but that in the DA fraction increased (from 57% to 68%). The 11C-radioactivity in the extracellular fluid determined by brain microdialysis was less than 0.4% of that in the whole striatum and no radioactivity was present in the DA fraction. These results suggest that, in an early phase after administration of L-[11C]DOPA, [11C]DA is the main metabolite and is localized exclusively in the intracellular compartment within this time frame.