Sample records for l-dopa decarboxylase activity

  1. Measuring L-dopa in plasma and urine to monitor therapy of elderly patients with Parkinson disease treated with L-dopa and a dopa decarboxylase inhibitor.

    Dutton, J; Copeland, L G; Playfer, J R; Roberts, N B


    We have established a method for measuring L-dopa in plasma and urine, including the metabolites dopamine and L-dopac, using separation by ion-pair reversed-phase HPLC and quantification with an electrochemical detector. The assay was applied to the therapeutic monitoring of elderly patients with established Parkinson disease being treated with L-dopa plus a dopa decarboxylase inhibitor. Plasma L-dopa was evaluated in relation to dosage and postdose sampling time in 71 outpatients with Parkinson disease. L-Dopa concentrations were greatest in the patients taking the highest dosages prescribed and decreased significantly with increasing time after postdose sampling. Comparison of plasma L-dopa concentrations with a published therapeutic range established by intravenous administration of L-dopa was helpful in assessing the suitability of each patient's drug dosage, assessing patients' compliance, and avoiding overdosage but was not useful in the overall clinical assessment of progression of disease or of the long-term therapeutic response. Urine measurements confirmed the plasma concentrations but showed no further advantage. The recommended time for sample collection is between 1.5 and 3 h after the first morning dose. Plasma is the preferred matrix but if blood sampling is difficult, particularly from elderly/infirm individuals, an untimed urine collection could be used.

  2. Treatment of idiopathic parkinsonism with L-dopa in the absence and presence of decarboxylase inhibitors: effects on plasma levels of L-dopa, dopa decarboxylase, catecholamines and 3-O-methyl-dopa.

    Boomsma, F; Meerwaldt, J D; Man in't Veld, A J; Hovestadt, A; Schalekamp, M A


    The effect of levodopa (L-dopa), alone or in combination with a peripheral decarboxylase inhibitor (PDI), on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD, = dopa decarboxylase), L-dopa, 3-O-methyl-dopa (3-OMD), dopamine (DA), noradrenaline, adrenaline and dopamine beta-hydroxylase has been studied. In healthy subjects and in patients with parkinsonism plasma ALAAD level fell after administration of L-dopa + benserazide, but returned to previous levels within 90 min. In a cross-sectional study blood was obtained, 2 h after dosing, from 104 patients with idiopathic parkinsonism, divided into four groups: no L-dopa treatment (group 1), L-dopa alone (group 2), L-dopa + benserazide (Madopar) (group 3) and L-dopa + carbidopa (Sinemet) (group 4). Plasma ALAAD, which was normal in groups 1 and 2, was increased 3-fold in groups 3 and 4, indicating that there was induction of ALAAD by the co-administration of PDI. Despite this induction of ALAAD, in groups 3 and 4, with half the daily L-dopa dose compared with group 2, plasma L-dopa and 3-OMD levels were 5 times higher, while plasma DA levels were not different. The DA/L-dopa ratio was decreased 5-fold in group 2 and 16-fold in groups 3 and 4 as compared with group 1. Neither 3-OMD levels nor 3-OMD/L-dopa ratios correlated with the occurrence of on-off fluctuations. In a longitudinal study of three patients started on Madopar treatment the induction of plasma ALAAD was found to occur gradually over 3-4 weeks. Further detailed pharmacokinetic studies in plasma and cerebrospinal fluid are required in order to elucidate whether the ALAAD induction by PDI may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  3. Oxidative Status of DJ-1-dependent Activation of Dopamine Synthesis through Interaction of Tyrosine Hydroxylase and 4-Dihydroxy-l-phenylalanine (l-DOPA) Decarboxylase with DJ-1*

    Ishikawa, Shizuma; Taira, Takahiro; Niki, Takeshi; Takahashi-Niki, Kazuko; Maita, Chinatsu; Maita, Hiroshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.


    Parkinson disease (PD) is caused by loss of dopamine, which is synthesized from tyrosine by two enzymes, tyrosine hydroxylase (TH) and 4-dihydroxy-l-phenylalanine decarboxylase (DDC). DJ-1 is a causative gene for the familial form of PD, but little is known about the roles of DJ-1 in dopamine synthesis. In this study, we found that DJ-1 directly bound to TH and DDC and positively regulated their activities in human dopaminergic cells. Mutants of DJ-1 found in PD patients, including heterozygous mutants, lost their activity and worked as dominant-negative forms toward wild-type DJ-1. When cells were treated with H2O2, 6-hydroxydopamine, or 1-methyl-4-phenylpyridinium, changes in activities of TH and DDC accompanied by oxidation of cysteine 106 of DJ-1 occurred. It was found that DJ-1 possessing Cys-106 with SH and SOH forms was active and that DJ-1 possessing Cys-106 with SO2H and SO3H forms was inactive in terms of stimulation of TH and DDC activities. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD. PMID:19703902

  4. The timing of administration, dose dependence and efficacy of dopa decarboxylase inhibitors on the reversal of motor disability produced by L-DOPA in the MPTP-treated common marmoset.

    Tayarani-Binazir, Kayhan A; Jackson, Michael J; Fisher, Ria; Zoubiane, Ghada; Rose, Sarah; Jenner, Peter


    Dopa decarboxylase inhibitors are routinely used to potentiate the effects of L-DOPA in the treatment of Parkinson's disease. However, neither in clinical use nor in experimental models of Parkinson's disease have the timing and dose of dopa decarboxylase inhibitors been thoroughly explored. We now report on the choice of dopa decarboxylase inhibitors, dose and the time of dosing relationships of carbidopa, benserazide and L-alpha-methyl dopa (L-AMD) in potentiating the effects of L-DOPA in the 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-treated common marmoset. Pre-treatment with benserazide for up to 3h did not alter the motor response to L-DOPA compared to simultaneous administration with L-DOPA. There was some evidence of a relationship between carbidopa and benserazide dose and increased locomotor activity and the reversal of motor disability. But in general, commonly used dose levels of dopa decarboxylase inhibitors appeared to produce a maximal motor response to L-DOPA. In contrast, dyskinesia intensity and duration continued to increase with both carbidopa and benserazide dose. The novel dopa decarboxylase inhibitor, L-AMD, increased locomotor activity and improved motor disability to the same extent as carbidopa or benserazide but importantly this was accompanied by significantly less dyskinesia. This study shows that currently, dopa decarboxylase inhibitors may be routinely employed in the MPTP-treated primate at doses which are higher than those necessary to produce a maximal potentiation of the anti-parkinsonian effect of L-DOPA. This may lead to excessive expression of dyskinesia in this model of Parkinson's disease and attention should be given to the dose regimens currently employed.

  5. Role of L-DOPA in spinal nociceptive reflex activity: higher sensitivity of Aδ versus C fibre-evoked nociceptive reflexes to L-DOPA.

    Schomburg, E D; Dibaj, P; Steffens, H


    The role of L-DOPA in spinal nociceptive reflex activity has been re-evaluated. In high spinal cats, with supraspinal loops being excluded, the onset of reflex facilitation induced by noxious radiant heat is delayed after injection of L-DOPA by 4 to 10 s, i.e. the early component of nociceptive reflex facilitation is blocked, while the late component persisted. Further investigations have shown that the early component of reflex facilitation induced by noxious radiant heat is mediated by Adelta-fibres and the late component by C-fibres. Therefore, it can be assumed that L-DOPA, like opioids, preferentially blocks the transmission in nociceptive reflex pathways from Adelta-fibres.

  6. Consequence of nigrostriatal denervation and L-dopa therapy on the expression of glutamic acid decarboxylase messenger RNA in the pallidum.

    Herrero, M T; Levy, R; Ruberg, M; Luquin, M R; Villares, J; Guillen, J; Faucheux, B; Javoy-Agid, F; Guridi, J; Agid, Y; Obeso, J A; Hirsch, E C


    To examine the consequences of nigrostriatal denervation and L-dopa treatment on the basal ganglia output system, we analyzed, by quantitative in situ hybridization, the messenger RNA coding for glutamic acid decarboxylase (Mr 67,000) (GAD67 mRNA) in pallidal cells from patients with Parkinson's disease (PD), monkeys rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) receiving or not receiving L-dopa, and their respective control subjects. In MPTP-treated monkeys, the expression of GAD67 mRNA was increased in cells from the internal pallidum, and this effect was abolished by L-dopa treatment. There were no differences in the levels of GAD67 mRNA between patients with PD, who were all treated with L-dopa, and control subjects. These results indicate that the level of GAD67 mRNA is increased in the cells of the internal pallidum after nigrostriatal dopaminergic denervation and that this increase can be reversed by L-dopa therapy.

  7. Interaction of Human Dopa Decarboxylase with L-Dopa: Spectroscopic and Kinetic Studies as a Function of pH

    Riccardo Montioli


    Full Text Available Human Dopa decarboxylase (hDDC, a pyridoxal 5′-phosphate (PLP enzyme, displays maxima at 420 and 335 nm and emits fluorescence at 384 and 504 nm upon excitation at 335 nm and at 504 nm when excited at 420 nm. Absorbance and fluorescence titrations of hDDC-bound coenzyme identify a single pKspec of ~7.2. This pKspec could not represent the ionization of a functional group on the Schiff base but that of an enzymic residue governing the equilibrium between the low- and the high-pH forms of the internal aldimine. During the reaction of hDDC with L-Dopa, monitored by stopped-flow spectrophotometry, a 420 nm band attributed to the 4′-N-protonated external aldimine first appears, and its decrease parallels the emergence of a 390 nm peak, assigned to the 4′-N-unprotonated external aldimine. The pH profile of the spectral change at 390 nm displays a pK of 6.4, a value similar to that (~6.3 observed in both kcat and kcat/Km profiles. This suggests that this pK represents the ESH+ → ES catalytic step. The assignment of the pKs of 7.9 and 8.3 observed on the basic side of kcat and the PLP binding affinity profiles, respectively, is also analyzed and discussed.

  8. Relationship between L-DOPA-induced reduction in motor and exploratory activity and degree of DAT binding in the rat

    Susanne eNikolaus


    Full Text Available Purpose: The present study assessed the influence of L-DOPA administration on neostriatal dopamine (DA transporter (DAT binding in relation to motor and exploratory behaviors in the rat.Methods: Rats received injections of 5 mg/kg L-DOPA, 10 mg/kg L-DOPA or vehicle. Motor and exploratory behaviors were assessed for 30 min in an open field prior to administration of [123I]FP-CIT. DAT binding was measured with small animal SPECT 2 h after radioligand administration for 60 min. Results: Both L-DOPA doses significantly reduced DAT binding and led to significantly less head-shoulder motility and more sitting relative to vehicle. Moreover, 10 mg/kg L-DOPA induced less distance travelled and ambulation than 5 mg/kg L-DOPA. Analysis of time-behavior curves showed that L-DOPA-treated animals relative to vehicle exhibited (1 a faster rate of increase in duration of sitting, (2 a slower rate of increase in duration of head-shoulder motility, and (3 a slower rate of decrease in frequency of head-shoulder motility. Conclusions: The reductions of striatal DAT binding after L-DOPA challenges reflected elevated concentrations of synaptic DA. L-DOPA-treated animals showed less head-shoulder motility and more sitting than vehicle-treated animals, indicating an association between less behavioral activity and increased availability of striatal DA. The faster increase of sitting duration to a higher final level and the slower increase of head-shoulder motility to a lower final level relative to controls may be interpreted in terms on behavioral habituation to a novel environment.

  9. Direct Evaluation of L-DOPA Actions on Neuronal Activity of Parkinsonian Tissue In Vitro

    Plata, Víctor; Pérez-Ortega, Jesús E.; Galarraga, Elvira; Bargas, José


    Physiological and biochemical experiments in vivo and in vitro have explored striatal receptor signaling and neuronal excitability to posit pathophysiological models of Parkinson's disease. However, when therapeutic approaches, such as dopamine agonists, need to be evaluated, behavioral tests using animal models of Parkinson's disease are employed. To our knowledge, recordings of population neuronal activity in vitro to assess anti-Parkinsonian drugs and the correlation of circuit dynamics with disease state have only recently been attempted. We have shown that Parkinsonian pathological activity of neuronal striatal circuits can be characterized in in vitro cerebral tissue. Here, we show that calcium imaging techniques, capable of recording dozens of neurons simultaneously with single-cell resolution, can be extended to assess the action of therapeutic drugs. We used L-DOPA as a prototypical anti-Parkinsonian drug to show the efficiency of this proposed bioassay. In a rodent model of early Parkinson's disease, Parkinsonian neuronal activity can be returned to control levels by the bath addition of L-DOPA in a reversible way. This result raises the possibility to use calcium imaging techniques to measure, quantitatively, the actions of anti-Parkinsonian drugs over time and to obtain correlations with disease evolution and behavior. PMID:24151606

  10. Direct Evaluation of L-DOPA Actions on Neuronal Activity of Parkinsonian Tissue In Vitro

    Víctor Plata


    Full Text Available Physiological and biochemical experiments in vivo and in vitro have explored striatal receptor signaling and neuronal excitability to posit pathophysiological models of Parkinson's disease. However, when therapeutic approaches, such as dopamine agonists, need to be evaluated, behavioral tests using animal models of Parkinson's disease are employed. To our knowledge, recordings of population neuronal activity in vitro to assess anti-Parkinsonian drugs and the correlation of circuit dynamics with disease state have only recently been attempted. We have shown that Parkinsonian pathological activity of neuronal striatal circuits can be characterized in in vitro cerebral tissue. Here, we show that calcium imaging techniques, capable of recording dozens of neurons simultaneously with single-cell resolution, can be extended to assess the action of therapeutic drugs. We used L-DOPA as a prototypical anti-Parkinsonian drug to show the efficiency of this proposed bioassay. In a rodent model of early Parkinson's disease, Parkinsonian neuronal activity can be returned to control levels by the bath addition of L-DOPA in a reversible way. This result raises the possibility to use calcium imaging techniques to measure, quantitatively, the actions of anti-Parkinsonian drugs over time and to obtain correlations with disease evolution and behavior.

  11. L-DOPA decarboxylase mRNA expression is associated with tumor stage and size in head and neck squamous cell carcinoma: a retrospective cohort study

    Geomela Panagiota-Aikaterini


    Full Text Available Abstract Background Head and neck squamous cell carcinoma (HNSCC represents one of the most commonly diagnosed malignancies worldwide. The DDC gene encodes L-DOPA decarboxylase, an enzyme catalyzing the decarboxylation of L-DOPA to dopamine. We have recently shown that DDC mRNA is a significant predictor of patients’ prognosis in colorectal adenocarcinoma and prostate cancer. The aim of the current study was to analyze the DDC mRNA expression in HNSCC patients. Methods 53 malignant tumors were resected from the larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissues of HNSCC patients, and were homogenized. Total RNA was isolated and converted into first-strand cDNA. An ultrasensitive real-time PCR method based on the SYBR Green chemistry was used for DDC mRNA quantification in head and neck tissue specimens. Relative quantification was performed using the comparative Ct (2-ddCt method. Results DDC mRNA levels were lower in squamous cell carcinomas (SCCs of the larynx and tongue than in adjacent non-cancerous tissue specimens. Furthermore, low DDC mRNA expression was noticed in laryngeal and tongue tumors of advanced TNM stage or bigger size, compared to early-stage or smaller tumors, respectively. No statistically significant differences were observed between SCCs resected from pharynx, buccal mucosa, or nasal cavity, and their normal counterparts. Conclusion This is the first study examining the DDC mRNA expression in HNSCC. According to our results, DDC mRNA expression may constitute a potential prognostic biomarker in tongue and/or larynx SCCs, which principally represent the overwhelming majority of HNSCC cases.

  12. Effect of selective and non-selective serotonin receptor activation on L-DOPA-induced therapeutic efficacy and dyskinesia in parkinsonian rats.

    Tronci, E; Fidalgo, C; Stancampiano, R; Carta, M


    Selective activation of 5-HT1 receptors has been shown to produce near to full suppression of L-DOPA-induced dyskinesia (LID) in animal models of Parkinson's disease; however, a reduction of the therapeutic effect of L-DOPA has been reported in several studies. Conversely, we recently found that increasing the serotonergic tone with chronic administration of the serotonin precursor 5-hydroxy-tryptophan (5-HTP) can reduce LID in 6-OHDA-lesioned rats, without affecting L-DOPA efficacy. To directly compare the effects of selective versus non-selective serotonin receptor activation, here we first tested different acute doses of the 5-HT1A/1B receptor agonist eltoprazine and 5-HTP on LID in order to identify doses of the individual compounds showing similar anti-dyskinetic efficacy in L-DOPA-primed dyskinetic rats. About 50% reduction of LID was observed with 0.1 mg/kg and 24 mg/kg of eltoprazine and 5-HTP, respectively; we then compared the effect of the two drugs, individually and in combination, on L-DOPA-induced stepping test in L-DOPA-naïve parkinsonian animals and LID over three weeks of L-DOPA treatment. Results showed that eltoprazine induced significant worsening of L-DOPA-mediated performance in the stepping test, while 5-HTP did not. Interestingly, combination of 5-HTP with eltoprazine prevented the reduction in the forelimb use induced by eltoprazine. Moreover, 5-HTP and eltoprazine given individually showed similar efficacy also upon chronic treatment, and had additive effect in dampening the appearance of LID when given in combination. Finally, chronic administration of eltoprazine and/or 5-HTP did not affect striatal serotonin innervation, compared to l-DOPA alone, as measured by serotonin transporter expression.

  13. Opposite effects of glutamate antagonists and antiparkinsonian drugs on the activities of DOPA decarboxylase and 5-HTP decarboxylase in the rat brain.

    Fisher, A; Starr, M S


    This study measured the activities of L-DOPA and 5-HTP decarboxylase (DDC and 5-HTPDC) in the substantia nigra and corpus striatum of reserpine-treated rats. Acute injection of the NMDA receptor antagonists CGP 40116 (5 mg/kg) and HA 966 (5 mg/kg), and to a lesser extent eliprodil (10 mg/kg), greatly elevated DDC in both structures, whilst having no effect on (nigra) or inhibiting (striatum) 5-HTPDC. L-DOPA (25 mg/kg) on its own inhibited both enzymes in either brain region. The weak NMDA receptor-channel blockers (and antiparkinsonian drugs) budipine (10 mg/kg), memantine (40 mg/kg) and amantadine (40 mg/kg) strongly increased DDC, whilst not affecting or decreasing 5-HTPDC activity in nigra and striatum. The L-DOPA-induced suppression of DDC was mostly reversed by all three antiparkinsonian drugs, whilst L-DOPA-induced inhibition of 5-HTPDC was only reversed by CGP 40116 (striatum only). It is concluded that glutamate exerts a differential physiological influence on the biosynthesis of dopamine and 5-HT in the brain, by tonically suppressing DDC and tonically stimulating 5-HTPDC. The L-DOPA-induced reduction in DDC may help to explain the eventual loss of efficacy of L-DOPA therapy in parkinsonian patients. It is suggested, however, that it may be possible to extend the lifetime of L-DOPA therapy with drugs which potentiate the activity of DDC, such as budipine and the 1-aminoadamantanes.

  14. [Effect of iontophoretically administered L-DOPA and ketamin on the impulse activity of the somatomotor cortex neurons during the conditioned placing movements].

    Khorievin, V I


    Interactions between the neuronal dopamine and NMDA glutamate receptors were investigated by iontophoresis of L-DOPA and ketamin to 19 single cortical neurons of the somatomotor cortex neurons in cats performing the operant reflex. L-DOPA producing bursts of 8-12 impulses that led to the significant increase of the total number of spikes related to the movements and insignificant decreased the power or the average number of spikes in a conditional response. Effects of ketamin on impulse activity were multidirectional resulting in most cases (16 cells) in the significant increase of the response power and in other three cells producing the suppression of discharges related to movements. It is supposed that following the ketamin iontophoresis the facilitation of conditional responses was caused by the blocking of the NMDA-glutamatergic transmission to intracortical inhibitory neurons, whereas inhibition of conditioned responses of other cortical cells was produced by blocking NMDA-glutamatergic transmission to pyramidal neurons. The total number of spikes associated with the performance of movements following the joint application of L-DOPA and ketamin was significantly higher compared with the effect of the isolated L-DOPA iontophoresis. These data indicate the NMDA-glutamatergic transmission, which plays an important role in shaping the cortical neuron responses in the performance of behavioral reactions may be modulated by L-DOPA.

  15. Early L-dopa, but not pramipexole, restores basal ganglia activity in partially 6-OHDA-lesioned rats.

    Marin, C; Bonastre, M; Mengod, G; Cortés, R; Giralt, A; Obeso, J A; Schapira, A H


    The most appropriate time for the initiation of dopaminergic symptomatic therapy in Parkinson's disease remains debatable. It has been suggested that early correction of basal ganglia pathophysiological abnormalities may have long-term beneficial effects. To test this hypothesis, we investigated the early and delayed actions of L-dopa and pramipexole, using a delayed-start protocol of treatment. The effects of early and delayed administration of these drugs on motor response, development of dyskinesias, neurogenesis and molecular markers in basal ganglia were studied in rats with a unilateral and partial 6-hydroxydopamine-induced nigrostriatal lesion. Ten days after lesioning, rats received treatment with: a) L-dopa methyl ester (25mg/kg with 6.25mg/kg of benserazide, i.p., twice a day); b) pramipexole (0.5mg/kg, sc, twice a day) or c) saline for 4weeks. Four weeks after treatment initiation, rats from the saline group were distributed in three groups that then received the following treatments: d) L-dopa, e) pramipexole or f) saline, for 4weeks more. Three animals in each treatment arm received 5-bromo-2-deoxyuridine injections (200mg/kg) 3days before starting treatment. When compared with delayed-start L-dopa, early-start L-dopa treatment induced a lower rotational response (ppramipexole, early-start pramipexole induced a higher rotational response (ppramipexole treatments. Our data support a possible restoration of basal ganglia physiological mechanisms by early-start L-dopa therapy.

  16. Herbs containing L- Dopa: An update.

    Ramya, Kuber B; Thaakur, Santhrani


    L-Dopa is the drug of choice in the treatment of Parkinson's disease but it has dose related adverse effects such as nausea, vomiting, orthostatic hypotension, end of dose deterioration, on off phenomena and on chronic therapy motor complications synonymous to parkinsonism. Mucuna pruriens (M.P) commonly known as velvet beans or cowitch are used in case of spasms associated with Parkins onism. Clinical efficacy of seeds of this plant was confirmed and the efficacy was contributed to its L-Dopa content. M.P extract showed twice the antiparkinsonism activity compared with synthetic L-Dopa. There is sufficient L-Dopa in broad bean (Vicia faba) pods. One study proved its efficacy in Parkinsonism. Ginkgo biloba extract showed protective effect in vivo and invitro. 50% ethanolic extract of Plumbago zeylanica was effective in rats. The following plants were reported to have L-Dopa but their protective effect is yet to be established in animal models. Vigna aconitifolia, Vigna unguiculata, Vigna vexillata, Prosopis chilensis, Pileostigma malabarica, Phanera vahlis, Parkinsonia acculeata, Macuna urens, Canvavalia glandiata, Cassia floribanda, Casia hirsute and Dalbergia retusa etc.

  17. The neurochemistry of Parkinson's disease: effect of L-dopa therapy.

    Lloyd, K G; Davidson, L; Hornykiewicz, O


    Post-mortem brain material from control and Parkinson's disease patients was examined to elucidate further the neurochemistry of this disease and to determine the mechanism of action of L-dopa as a therapeutic agent. The activities of L-aromatic amino acid decarboxylase (dopa D), tyrosine hydroxylase, monoamine oxidase and catechol-O-methyl transferase were examined; in addition the tissue levels of dopa, 3-O-methyldopa, dopamine (DA) and homovanillic acid (HVA) were determined. In the non-dopa-treated Parkinsonian patients, the greatest decreases were detected for striatal DA and dopa D, with homovanillic acid and tyrosine hydroxylase levels showing a lesser change. The activities of monoamine oxidase and catechol-O-methyl transferase in the striatal nuclei were not different from the controls. The putamen was consistently the most severely affected region. Dopa and 3-O-methyldopa were detectable in all brain areas only in those patients treated with L-dopa shortly before death. The mean concentrations of DA in the striatum of these patients were 1) 9 to 15 times higher than those in non-dopa-treated patients, 2) related to the time before death of the last dose of L-dopa and 3) greater in the striatum of patients clinically classified as "good responders" as compared to "poor responders." Although L-dopa therapy increased homovanillic acid levels in all brain areas, a preferential increase was observed in the striatum. It was concluded that L-dopa's principal therapeutic effects in Parkinson's disease are consistent with its transformation to DA in the striatum.

  18. L-DOPA is an endogenous ligand for OA1.

    Vanessa M Lopez


    Full Text Available Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE. How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1 expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.

  19. A Role for Mitogen- and Stress-Activated Kinase 1 in L-DOPA-Induced Dyskinesia and ∆FosB Expression

    Feyder, Michael; Södersten, Erik; Santini, Emanuela


    BACKGROUND: Abnormal regulation of extracellular signal-regulated kinases 1 and 2 has been implicated in 3,4-dihydroxy-l-phenylalanine (L-DOPA)-induced dyskinesia (LID), a motor complication affecting Parkinson's disease patients subjected to standard pharmacotherapy. We examined the involvement ...

  20. Delayed O-methylation of l-DOPA in MB-COMT-deficient mice after oral administration of l-DOPA and carbidopa.

    Tammimäki, Anne; Aonurm-Helm, Anu; Männistö, Pekka T


    1. Catechol-O-methyltransferase (COMT) is involved in the O-methylation of l-DOPA, dopamine, and other catechols. The enzyme is expressed in two isoforms: soluble (S-COMT), which resides in the cytoplasm, and membrane-bound (MB-COMT), which is anchored to intracellular membranes. 2. To obtain specific information on the functions of COMT isoforms, we studied how a complete MB-COMT deficiency affects the total COMT activity in the body, peripheral l-DOPA levels, and metabolism after l-DOPA (10 mg kg(-1)) plus carbidopa (30 mg kg(-1)) administration by gastric tube in wild-type (WT) and MB-COMT-deficient mice. l-DOPA and 3-O-methyl-l-DOPA (3-OMD) levels were assayed in plasma, duodenum, and liver. 3. We showed that the selective lack of MB-COMT did not alter the total COMT activity, COMT enzyme kinetics, l-DOPA levels, or the total O-methylation of l-DOPA but delayed production of 3-OMD in plasma and peripheral tissues.

  1. Impairment of Serotonergic Transmission by the Antiparkinsonian Drug L-DOPA: Mechanisms and Clinical Implications

    Cristina Miguelez


    Full Text Available The link between the anti-Parkinsonian drug L-3,4-dihydroxyphenylalanine (L-DOPA and the serotonergic (5-HT system has been long established and has received increased attention during the last decade. Most studies have focused on the fact that L-DOPA can be transformed into dopamine (DA and released from 5-HT terminals, which is especially important for the management of L-DOPA-induced dyskinesia. In patients, treatment using L-DOPA also impacts 5-HT neurotransmission; however, few studies have investigated the mechanisms of this effect. The purpose of this review is to summarize the electrophysiological and neurochemical data concerning the effects of L-DOPA on 5-HT cell function. This review will argue that L-DOPA disrupts the link between the electrical activity of 5-HT neurons and 5-HT release as well as that between 5-HT release and extracellular 5-HT levels. These effects are caused by the actions of L-DOPA and DA in 5-HT neurons, which affect 5-HT neurotransmission from the biosynthesis of 5-HT to the impairment of the 5-HT transporter. The interaction between L-DOPA and 5-HT transmission is especially relevant in those Parkinson’s disease (PD patients that suffer dyskinesia, comorbid anxiety or depression, since the efficacy of antidepressants or 5-HT compounds may be affected.

  2. Effects of L-DOPA/benserazide co-treatment on colonic excitatory cholinergic motility and enteric inflammation following dopaminergic nigrostriatal neurodegeneration.

    Pellegrini, C; Antonioli, L; Colucci, R; Tirotta, E; Gentile, D; Ippolito, C; Segnani, C; Levandis, G; Cerri, S; Blandini, F; Barocelli, E; Ballabeni, V; Bernardini, N; Blandizzi, C; Fornai, M


    The mainstay therapy for Parkinson's disease (PD) relies on L-3,4-dihydroxyphenylalanine (L-DOPA) plus a DOPA-decarboxylase inhibitor. However, their effects on colonic dysmotility and inflammation observed in PD are undetermined. This study examined the effects of L-DOPA plus benserazide (BE) on colonic motility and inflammation in rats with central nigrostriatal dopaminergic denervation. Neurodegeneration was induced by 6-hydroxydopamine (6-OHDA) injection into the medial forebrain bundle (MFB). 6-OHDA animals were treated orally with L-DOPA/BE for 28 days, starting 28 days after 6-OHDA injection. At the end of treatment, in vivo colonic transit was evaluated by a radiologic assay. Electrically stimulated (ES) cholinergic contractions were recorded in vitro from colonic preparations, while acetylcholine release was measured in the incubation medium. Choline acetyltransferase (ChAT) and glial fibrillary acidic protein (GFAP) expression as well as eosinophil and mast cell density were examined in the colonic wall by immunohistochemistry. Colonic TNF and IL-1β levels were also assayed. 6-OHDA animals displayed: 1) decrease in in vivo colonic transit; 2) impairment of ES-stimulated cholinergic contractions; 3) decreased acetylcholine release from myenteric nerves; 4) decrease in ChAT and increase in GFAP myenteric immunopositivity; 5) increase in eosinophil and mast cell density; 6) increase in TNF and IL-1β levels. Treatment with L-DOPA/BE elicited an improvement of in vivo and in vitro colonic motor activity, a normalization of acetylcholine release, ChAT immunopositivity, as well as pro-inflammatory cytokine patterns, ganglionic GFAP levels, eosinophil and mast cell density. Under dopaminergic nigrostriatal denervation, treatment with L-DOPA/BE ameliorated colonic motility through a normalization of myenteric cholinergic neurotransmission, along with an improvement of colonic inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. L-dopa dose and the duration and severity of dyskinesia in primed MPTP-treated primates.

    Kuoppamäki, M; Al-Barghouthy, G; Jackson, M J; Smith, L A; Quinn, N; Jenner, P


    Most patients with Parkinson's disease (PD) develop dyskinesia and other motor complications after prolonged L-dopa use. We now report on the relationship between L-dopa dose and the duration and severity of dyskinesia in L-dopa-primed MPTP-treated primates with marked nigral degeneration mimicking late stage PD. With increasing doses of L-dopa, locomotor activity increased and motor disability declined. The duration of dyskinesia following L-dopa administration increased dose-dependently, and showed a linear correlation with total locomotor activity. In addition, the time-course of dyskinesia paralleled closely that of locomotor activity in a dose-dependent manner. In contrast, severity of dyskinesia showed a non-linear correlation with total locomotor activity, low doses of L-dopa eliciting severe dyskinesia for short periods of time. The threshold for dyskinesia induction and the antiparkinsonian effects of L-dopa appear very similar in primed MPTP primates mimicking late stage PD. Reducing individual doses of L-dopa to avoid severe dyskinesia can markedly compromise the antiparkinsonian response. Our results extend the relevance of the dyskinetic MPTP-treated primate in studying the genesis of involuntary movements occurring in L-dopa treated patients with PD.

  4. From L-dopa to dihydroxyphenylacetaldehyde: a toxic biochemical pathway plays a vital physiological function in insects.

    Christopher Vavricka

    Full Text Available One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD family based on extremely high sequence homology (∼70% with dopa decarboxylase (Ddc, was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects.

  5. Alpha and beta EEG power reflects L-dopa acute administration in parkinsonian patients

    Jean-Marc eMelgari


    Full Text Available Aim. To evaluate the effect of an acute L-dopa administration on eye-closed resting state electroencephalographic (EEG activity of cognitively preserved Parkinsonian patients. Methods. We examined 24 right-handed patients diagnosed as uncomplicated probable Parkinson’s disease (PD. Each patient underwent UPDRS-part-III evaluation before and 60 minutes after an oral load of L-dopa-methyl-ester/carbidopa 250/25 mg. Resting condition eyes-closed EEG data were recorded both pre- and post L-dopa load. Absolute EEG power values were calculated at each scalp derivation for Delta, Theta, Alpha and Beta frequency bands. UPDRS scores (both global and subscale scores and EEG data (power values of different frequency bands for each scalp derivation were submitted to a statistical analysis to compare Pre e Post L-Dopa conditions. Finally, a correlation analysis was carried out between EEG spectral content and UPDRS scores. Results. Considering EEG power spectral analysis, no statistically significant differences arose on Delta and Theta bands after L-dopa intake. Conversely, Alpha and Beta rhythms significantly increased on centro-parietal scalp derivations, as a function of L-dopa administration. Correlation analysis indicated a significant negative correlation between Beta power increase on centro-parietal areas and UPDRS subscores (Rigidity of arms and Bradykinesia. A minor significant negative correlation was also found between Alpha band increase and resting tremor. Conclusions. Assuming that a significant change in EEG power spectrum after L-dopa intake may be related to dopaminergic mechanisms, our findings are consistent with the hypothesis that dopaminergic defective networks are implicated in cortical oscillatory abnormalities at rest in non-demented PD patients.

  6. 5-Hydroxy-tryptophan for the treatment of L-DOPA-induced dyskinesia in the rat Parkinson's disease model.

    Tronci, Elisabetta; Lisci, Carlo; Stancampiano, Roberto; Fidalgo, Camino; Collu, Maria; Devoto, Paola; Carta, Manolo


    The serotonin system has recently emerged as an important player in the appearance of L-DOPA-induced dyskinesia (LID) in experimental models of Parkinson's disease, as it provides an unregulated source of L-DOPA-derived dopamine release in the dopamine-depleted striatum. Accordingly, toxin lesion or pharmacological silencing of serotonin neurons suppressed LID in the rat and monkey models of Parkinson's disease. However, 5-HT1 receptor agonists were also found to partially reduce the therapeutic effect of L-DOPA. In this study, we evaluated whether enhancement of the serotonin tone induced by the administration of the serotonin precursor 5-hydroxy-tryptophan (5-HTP) could affect induction and expression of LID, as well as the therapeutic effect of L-DOPA, in 6-OHDA-lesioned rats. Drug naïve and L-DOPA-primed 6-OHDA-lesioned rats were chronically treated with a daily injection of L-DOPA (6 mg/kg plus benserazide, s.c.) alone, or in combination with 5-HTP (24-48 mg/kg, i.p.). The abnormal involuntary movements (AIMs) test, as well as the stepping and the motor activity tests, were performed during the chronic treatments. Results showed that 5-HTP reduced the appearance of LID of about 50% at both tested doses. A partial reduction of the therapeutic effect of L-DOPA was seen with the higher but not with the lower dose of 5-HTP. 5-HTP 24 mg/kg was also able to reduce the expression of dyskinesia in L-DOPA-primed dyskinetic rats, to a similar extent than in L-DOPA-primed rats. Importantly, the antidyskinetic effect of 5-HTP 24 mg/kg does not appear to be due to a competition with L-DOPA for crossing the blood-brain barrier; in fact, similar L-DOPA striatal levels were found in L-DOPA only and L-DOPA plus 5-HTP 24 mg/kg treated animals. These data further confirm the involvement of the serotonin system in the appearance of LID, and suggest that 5-HTP may be useful to counteract the appearance of dyskinesia in Parkinson's disease patients.

  7. Induction of aromatic-L-amino acid decarboxylase by decarboxylase inhibitors in idiopathic parkinsonism.

    Boomsma, F; Meerwaldt, J D; Man in 't Veld, A J; Hovestadt, A; Schalekamp, M A


    We evaluated the effect of administration of L-dopa, alone or in combination with a peripheral decarboxylase inhibitor, on plasma levels of aromatic-L-amino acid decarboxylase (ALAAD). After single-dose administration of L-dopa plus benserazide (Madopar) in healthy subjects and in chronically treated patients with parkinsonism, plasma ALAAD followed for 2 to 3 hours fell, but returned to predosing levels within 90 minutes. Four groups of patients with idiopathic parkinsonism were studied during chronic treatment: Group I, no L-dopa treatment (n = 31); Group II, L-dopa alone (n = 15); Group III, L-dopa plus benserazide (n = 28); and Group IV, L-dopa plus carbidopa (Sinemet, n = 30). Plasma ALAAD 2 hours after dosing was normal in Groups I and II. ALAAD was increased threefold in Groups III and IV, suggesting induction of ALAAD by the coadministration of a peripheral decarboxylase inhibitor. In a study of 3 patients in whom L-dopa/benserazide was started, plasma ALAAD rose gradually over 3 to 4 weeks. Further detailed pharmacokinetic studies of L-dopa, dopamine, and ALAAD in plasma and cerebrospinal fluid are required to determine if the apparent ALAAD induction by a peripheral decarboxylase inhibitor may be related to the loss of clinical efficacy of combination therapy in some patients and how it is related to end-of-dose deterioration and on-off phenomena.

  8. Tratamento do parkinsonismo com L-Dopa

    Nilton Luís Latuf


    Full Text Available São apresentados os resultados obtidos no tratamento de 54 pacientes parkinsonianos com L-Dopa. A intensidade dos sintomas foi avaliada mediante a escala NUDS (Northwestern University Disability Scales antes e após o tratamento. Os autores ressaltam a obtenção de melhores resultados no que respeita à rigidez e à discinesia, em comparação com o tremor. São apontados os efeitos colaterais, quase sempre de pequena intensidade e corrigíveis com a diminuição ou suspensão da administração medicamentosa.

  9. Design, synthesis, and preliminary pharmacological evaluation of new imidazolinones as L-DOPA prodrugs.

    Giorgioni, Gianfabio; Claudi, Francesco; Ruggieri, Sabrina; Ricciutelli, Massimo; Palmieri, Giovanni F; Di Stefano, Antonio; Sozio, Piera; Cerasa, Laura S; Chiavaroli, Annalisa; Ferrante, Claudio; Orlando, Giustino; Glennon, Richard A


    L-DOPA, the immediate biological precursor of dopamine, is still considered the drug of choice in the treatment of Parkinson's disease. However, therapy with L-DOPA is associated with a number of acute problems. With the aim to increase the bioavailability after oral administration, we designed a multi-protected L-DOPA prodrugs able to release the drug by both spontaneous chemical or enzyme catalyzed hydrolysis. The new compounds have been synthesized and preliminarily evaluated for their water solubility, log P, chemical stability, and enzymatic stability. The results indicate that the incorporation of the amino acidic moiety of L-DOPA into an imidazoline-4-one ring provides prodrugs sufficiently stable to potentially cross unchanged the acidic environment of the stomach, and to be absorbed from the intestine. They also might be able to release L-DOPA in human plasma after enzymatic hydrolysis. The ability of prodrugs 6a-b to increase basal levels of striatal DA, and influence brain neurochemistry associated with dopaminergic activity following oral administration, as well as the radical-scavenging activity against DPPH for compounds 6a-b and 15a are also reported.

  10. Renal effects of L-DOPA in heart failure.

    Grossman, E; Shenkar, A; Peleg, E; Thaler, M; Goldstein, D S


    We examined whether low-dose L-DOPA treatment induces natriuresis and diuresis in patients with congestive heart failure who have cardiac decompensation despite treatment with digoxin, a diuretic, and an angiotensin-converting enzyme inhibitor and who respond acutely to intravenously infused dopamine. In a randomized, double-blind, placebo-controlled crossover study, 11 patients with severe congestive heart failure received L-DOPA (0.10 g, p.o., t.i.d., for 1 day and then 0.25 g, p.o., t.i.d., for 2 days after a washout period of > or = 1 day), with assessments of plasma and urinary levels of catechols, urinary volume, and sodium content, and clinical and laboratory measures of improvement of congestive heart failure. L-DOPA elicited short-term, dose-related increases in urinary volume and sodium excretion. At the 0.10-g dose, L-DOPA increased plasma L-DOPA levels and urinary L-DOPA excretion by about fivefold, whereas at the 0.25-g dose, L-DOPA increased plasma and urinary L-DOPA by >50-fold. Twenty-four-hour urinary dopamine excretion increased by about fivefold after the low dose of L-DOPA and approximately 50-fold after the high dose. The results demonstrate that oral L-DOPA treatment can produce beneficial natriuretic and diuretic effects in selected patients with congestive heart failure. The bioavailability of oral L-DOPA appears to vary with the dose. These results support findings from previous studies about beneficial cardiac functional effects of L-DOPA in patients with refractory heart failure.

  11. Inhibitory kinetics of paeonol on the activity of mushroom tyrosinase oxidizing L-Dopa%丹皮酚抑制蘑菇酪氨酸酶催化氧化L-Dopa的动力学

    龚盛昭; 程江; 杨卓如


    Aim To evaluate the effect of paeonol on the activity of tyrosinase and provide experimental evidence for the treatment of hyperpigmentation disorders. Methods Tyrosinase activity was estimated by measuring the oxidation rate of L-3,4-dihydroxyphenylalanine (L-Dopa). The inhibitory effects of paeonol on the activity of mushroom tyrosinase and Michaelis-Menten kinetics were deduced from the Lineweaver-Burk plots. Results The inhibitory concentration of paeonol leading to 50% enzyme Paeonol is a potential mixed inhibitor of mushroom tyrosinase. The mixed inhibition function may originate from its ability to form a Schiff base with a primary amino group and to chelate copper at the active site of tyrosinase.%目的研究丹皮酚对酪氨酸酶活性的影响,为色素增加性皮肤病的治疗提供实验依据.方法选用酪氨酸酶催化氧化3,4-二羟基苯丙氨酸(L-Dopa)速率法体外测定酪氨酸酶活性.应用Lineweaver-Burk曲线推导丹皮酚对酪氨酸酶活性的抑制效应及Michaelis-Menten动力学.结果导致酪氨酸酶活力下降50%的丹皮酚浓度为0.60mmol·L-1.丹皮酚对游离酶的抑制常数(KI)和对酶底物络合物的抑制常数(KIS)分别为0.084和0.12 mmol·L-1.结论丹皮酚是酪氨酸酶的混合型抑制剂,该抑制作用源于其能与酶中氨基结合生成席夫碱及能与活性中心的铜生成络合物.

  12. Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells.

    Carranza, Andrea; Musolino, Patricia L; Villar, Marcelo; Nowicki, Susana


    The inward l-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of l-dopa transport into isolated rat PTCs. Insulin (50-500 microU/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 microU/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 microM). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 microM) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 microM), but not maximal (5 microM), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 microM LY-294002) and protein kinase C (PKC; 1 microM RO-318220). Insulin-induced activation of PKC-zeta was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-zeta, and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs.

  13. L-DOPA inhibits excitatory synaptic transmission in the rat nucleus tractus solitarius through release of dopamine.

    Ohi, Y; Kodama, D; Haji, A


    The mode of action of L-DOPA on excitatory synaptic transmission in second-order neurons of the nucleus tractus solitarius (NTS) was studied using the rat brainstem slices. Superfusion of L-DOPA (10μM) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without any effect on the amplitude. A low concentration (1μM) was ineffective on the mEPSCs, and the highest concentration (100μM) exerted a stronger inhibitory effect. L-DOPA (10μM) decreased the amplitude of EPSCs (eEPSCs) evoked by electrical stimulation of the tractus solitarius and increased the paired-pulse ratio. The inhibitory effects of L-DOPA on mEPSCs and eEPSCs were similar to those of dopamine (100μM). The effects of L-DOPA were blocked by a competitive antagonist, L-DOPA methyl ester (100μM) and also by a D2 receptor antagonist, sulpiride (10μM), while those of dopamine were blocked by the latter but not by the former. In reserpine (5mg/kg, s.c.)-treated rats, the effects of L-DOPA on both mEPSCs and eEPSCs were completely abolished, but those of dopamine remained unchanged. The present results suggest a possibility that L-DOPA may induce the release of dopamine from the axon terminals in the NTS and the released dopamine suppresses the glutamatergic transmission through activation of the presynaptic D2 receptors. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Aspergillus niger PA2: a novel strain for extracellular biotransformation of L-tyrosine into L-DOPA.

    Agarwal, Pragati; Pareek, Nidhi; Dubey, Swati; Singh, Jyoti; Singh, R P


    L-DOPA (3,4-dihydroxyphenyl-L-alanine), an amino acid derivative is the most widely used drug of choice for the treatment of Parkinson's disease and other neurologic injuries. The present study deals with the elevated biochemical transformation of L-tyrosine to L-DOPA by Aspergillus niger PA2, a potent tyrosinase producer, isolated from decomposed food wastes. This appears to be the first report on A. niger as a notable extracellular tyrosinase producer. The extracellular tyrosinase activity produced remarkably higher levels of L-DOPA, i.e. 2.44 mg mL(-1) when the media was supplemented with 5 mg mL(-1) L-tyrosine. The optimum pH for tyrosinase production was 6.0, with the maximal L-DOPA production at the same pH. The product thus produced was analyzed by thin-layer chromatography, UV spectroscopy, high-performance liquid chromatography and Fourier transform infrared spectroscopy, that had denoted this to be L-DOPA. Kinetic parameters viz. Y p/s, Q s and Q p had further indicated the notable levels of production. Thus, Aspergillus niger PA2 could be a promising resource and may be further exploited for large-scale production of L-DOPA.

  15. Simultaneous MAO-B and COMT inhibition in L-Dopa-treated patients with Parkinson's disease.

    Lyytinen, J; Kaakkola, S; Ahtila, S; Tuomainen, P; Teräväinen, H


    The effect of selegiline (L-deprenyl) on plasma catecholamines, clinical response, and drug tolerability was studied in 13 patients with Parkinson's disease (PD) treated with L-Dopa/benserazide and entacapone, a peripheral catechol-O-methyltransferase (COMT) inhibitor, in a placebo-controlled double-blind study. An L-Dopa test was performed on 3 study days. The first study day was with L-Dopa/benserazide only (control), the second after 14 days of treatment with 200 mg entacapone taken concomitantly with L-Dopa/benserazide in combination with either selegiline (10 mg daily) or placebo. After a 2-week washout period, selegiline and placebo treatments were switched, and the third study day was after 14 days of treatment. During the study days, clinical response was evaluated at 30-min intervals for 6 h, by using the motor score of the Unified Parkinson's Disease Rating Scale (UPDRS). In addition, repeated blood pressure measurements were made, and plasma samples were taken for analysis of L-Dopa, 3-O-methyldopa (3-OMD), dihydroxyphenyl acetic acid (DOPAC), homovanillic acid (HVA), dopamine, noradrenaline, and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG). Monoamine oxidase B (MAO-B) and COMT enzyme activities were measured from platelets and erythrocytes, respectively. Entacapone improved the clinical response to L-Dopa during both selegiline and placebo (p DOPAC levels and decreased plasma 3-OMD and MHPG levels both with selegiline and placebo. Selegiline partially inhibited the entacapone-induced increase of plasma DOPAC. Plasma dopamine and noradrenaline levels did not change. Entacapone decreased erythrocyte COMT activity by > 35% (p patient withdrew because of diarrhea, dizziness, and loss of sleep when receiving selegiline treatment. Otherwise no differences in adverse events, mean daily blood pressures, or other safety parameters were observed between selegiline and placebo treatments. Our results suggest that entacapone can be safely administered together

  16. Corticostriatal Plastic Changes in Experimental L-DOPA-Induced Dyskinesia

    Veronica Ghiglieri


    Full Text Available In Parkinson’s disease (PD, alteration of dopamine- (DA- dependent striatal functions and pulsatile stimulation of DA receptors caused by the discontinuous administration of levodopa (L-DOPA lead to a complex cascade of events affecting the postsynaptic striatal neurons that might account for the appearance of L-DOPA-induced dyskinesia (LID. Experimental models of LID have been widely used and extensively characterized in rodents and electrophysiological studies provided remarkable insights into the inner mechanisms underlying L-DOPA-induced corticostriatal plastic changes. Here we provide an overview of recent findings that represent a further step into the comprehension of mechanisms underlying maladaptive changes of basal ganglia functions in response to L-DOPA and associated to development of LID.

  17. Pro-oxidant DNA breakage induced by the interaction of L-DOPA with Cu(II): a putative mechanism of neurotoxicity.

    Perveen, Asma; Khan, Husain Yar; Hadi, S M; Damanhouri, Ghazi A; Alharrasi, Ahmed; Tabrez, Shams; Ashraf, Ghulam Md


    There are reports in scientific literature that the concentration of copper ions in Parkinsonian brain is at a level that could promote oxidative DNA damage. The possibility of copper chelation by antioxidants excited us to explore the generation of reactive oxygen species (ROS) and DNA damage by the interaction of L-DOPA with Cu(II) ions. In the present manuscript, L-DOPA was tested for its ability to bind with Cu(II) and reduce it to Cu(I). The generation of ROS, such as superoxide anion (O(2)(-)) and hydroxyl radical (OH(•)), was also ascertained. As a result of L-DOPA and Cu(II) interaction, the generation of O(2)(-) was found to be increased in a time-dependent manner. Moreover, the formation of OH(•) was also found to be enhanced with increasing concentrations of L-DOPA. Furthermore, Comet assay results clearly showed significantly higher cellular DNA breakage in lymphocytes treated with L-DOPA and Cu(II) as compared to those that were treated with L-DOPA alone. However, such DNA degradation was inhibited to a significant extent by scavengers of ROS and neocuproine, a membrane permeable Cu(I)-specific sequestering agent. These findings demonstrate that L-DOPA exhibits a pro-oxidant activity in the presence of copper ions.

  18. The CB1 cannabinoid receptor agonist reduces L-DOPA-induced motor fluctuation and ERK1/2 phosphorylation in 6-OHDA-lesioned rats.

    Song, Lu; Yang, Xinxin; Ma, Yaping; Wu, Na; Liu, Zhenguo


    The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) has been used as an effective drug for treating dopamine depletion-induced Parkinson's disease (PD). However, long-term administration of L-DOPA produces motor complications. L-DOPA has also been found to modify the two key signaling cascades, protein kinase A/dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), in striatal neurons, which are thought to play a pivotal role in forming motor complications. In the present study, we tested the possible effect of a CB1 cannabinoid receptor agonist on L-DOPA-stimulated abnormal behavioral and signaling responses in vivo. Intermittent L-DOPA administration for 3 weeks induced motor fluctuation in a rat model of PD induced by intrastriatal infusion of dopamine-depleting neurotoxin 6-hydroxydopamine (6-OHDA). A single injection of a CB1 cannabinoid receptor agonist WIN-55,212-2 had no effect on L-DOPA-induced motor fluctuation. However, chronic injections of WIN-55,212-2 significantly attenuated abnormal behavioral responses to L-DOPA in 6-OHDA-lesioned rats. Similarly, chronic injections of WIN-55,212-2 influence the L-DOPA-induced alteration of DARPP-32 and ERK1/2 phosphorylation status in striatal neurons. These data provide evidence for the active involvement of CB1 cannabinoid receptors in the regulation of L-DOPA action during PD therapy.

  19. Modulatory effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells.

    Zhang, Min; Lee, Hak Ju; Park, Keun Hong; Park, Hyun Jin; Choi, Hyun Sook; Lim, Sung Cil; Lee, Myung Koo


    The effects of sesamin on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Sesamin at concentration ranges of 20-75 μM exhibited a significant increase in intracellular dopamine levels at 24 h: 50 μM sesamin increased dopamine levels to 133% and tyrosine hydroxylase (TH) activity to 128.2% of control levels. Sesamin at 20-100 μM rapidly increased the intracellular levels of cyclic AMP (cAMP) to 158.3%-270.3% of control levels at 30 min. At 50 μM, sesamin combined with L-DOPA (50, 100 and 200 μM) further increased the intracellular dopamine levels for 24 h compared to L-DOPA alone. In the absence or presence of L-DOPA (100 and 200 μM), sesamin (50 μM) increased the phosphorylation of TH, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), as well as the mRNA levels of TH and CREB for 24 h, an effect which was reduced by L-DOPA (100 and 200 μM). In addition, 50 μM sesamin exhibited a protective effect against L-DOPA (100 and 200 μM)-induced cytotoxicity via the inhibition of reactive oxygen species (ROS) production and superoxide dismutase reduction, induction of extracellular signal-regulated kinase (ERK)1/2 and BadSer112 phosphorylation and Bcl-2 expression, and inhibition of cleaved-caspase-3 formation. These results suggested that sesamin enhanced dopamine biosynthesis and L-DOPA-induced increase in dopamine levels by inducing TH activity and TH gene expression, which was mediated by cAMP-PKA-CREB systems. Sesamin also protected against L-DOPA (100-200 μM)-induced cytotoxicity through the suppression of ROS activity via the modulation of ERK1/2, BadSer112, Bcl-2, and caspase-3 pathways in PC12 cells. Therefore, sesamin might serve as an adjuvant phytonutrient for neurodegenerative diseases.

  20. The role of the subthalamic nucleus in L-DOPA induced dyskinesia in 6-hydroxydopamine lesioned rats.

    Asier Aristieta

    Full Text Available L-DOPA is the most effective treatment for Parkinson's disease (PD, but prolonged use leads to disabling motor complications including dyskinesia. Strong evidence supports a role of the subthalamic nucleus (STN in the pathophysiology of PD whereas its role in dyskinesia is a matter of controversy. Here, we investigated the involvement of STN in dyskinesia, using single-unit extracellular recording, behavioural and molecular approaches in hemi-parkinsonian rats rendered dyskinetic by chronic L-DOPA administration. Our results show that chronic L-DOPA treatment does not modify the abnormal STN activity induced by the 6-hydroxydopamine lesion of the nigrostriatal pathway in this model. Likewise, we observed a loss of STN responsiveness to a single L-DOPA dose both in lesioned and sham animals that received daily L-DOPA treatment. We did not find any correlation between the abnormal involuntary movement (AIM scores and the electrophysiological parameters of STN neurons recorded 24 h or 20-120 min after the last L-DOPA injection, except for the axial subscores. Nonetheless, unilateral chemical ablation of the STN with ibotenic acid resulted in a reduction in global AIM scores and peak-severity of dyskinesia. In addition, STN lesion decreased the anti-dyskinetogenic effect of buspirone in a reciprocal manner. Striatal protein expression was altered in dyskinetic animals with increases in ΔFosB, phosphoDARPP-32, dopamine receptor (DR D3 and DRD2/DRD1 ratio. The STN lesion attenuated the striatal molecular changes and normalized the DRD2/DRD1 ratio. Taken together, our results show that the STN plays a role, if modest, in the physiopathology of dyskinesias.

  1. Gastrin stimulates renal dopamine production by increasing the renal tubular uptake of l-DOPA.

    Jiang, Xiaoliang; Zhang, Yanrong; Yang, Yu; Yang, Jian; Asico, Laureano D; Chen, Wei; Felder, Robin A; Armando, Ines; Jose, Pedro A; Yang, Zhiwei


    Gastrin is a peptide hormone that is involved in the regulation of sodium balance and blood pressure. Dopamine, which is also involved in the regulation of sodium balance and blood pressure, directly or indirectly interacts with other blood pressure-regulating hormones, including gastrin. This study aimed to determine the mechanisms of the interaction between gastrin and dopamine and tested the hypothesis that gastrin produced in the kidney increases renal dopamine production to keep blood pressure within the normal range. We show that in human and mouse renal proximal tubule cells (hRPTCs and mRPTCs, respectively), gastrin stimulates renal dopamine production by increasing the cellular uptake of l-DOPA via the l-type amino acid transporter (LAT) at the plasma membrane. The uptake of l-DOPA in RPTCs from C57Bl/6J mice is lower than in RPTCs from normotensive humans. l-DOPA uptake in renal cortical slices is also lower in salt-sensitive C57Bl/6J than in salt-resistant BALB/c mice. The deficient renal cortical uptake of l-DOPA in C57Bl/6J mice may be due to decreased LAT-1 activity that is related to its decreased expression at the plasma membrane, relative to BALB/c mice. We also show that renal-selective silencing of Gast by the renal subcapsular injection of Gast siRNA in BALB/c mice decreases renal dopamine production and increases blood pressure. These results highlight the importance of renal gastrin in stimulating renal dopamine production, which may give a new perspective in the prevention and treatment of hypertension. Copyright © 2017 the American Physiological Society.

  2. Synthesis of a Dopamimetic Thionated Dipeptide Prodrug of L-DOPA

    Ye WANG; Zhan Zhu LIU; Shi Zhi CHEN


    L-DOPA has gained widespread credit over the past decades as being the mainstay of the pharmacological treatment of Parkinson's disease. However, there are many adverse effects associated with the use of L-DOPA. The prodrug approach is the most promising way to solve the problem. In this article, a thionated dipeptide prodrug of L-DOPA 11 was synthesized via 10steps in a total yield of 26.5% from L-DOPA.

  3. 谷酰多巴对清醒兔的交感抑制作用不被多巴胺D2受体的激活所介导%Sympatho-inhibitory effects of γ-l-glutamyl-l-dopa are not mediated by activation of dopamine-2 receptors in conscious rabbits

    王志勤; Way D; Secombe J; McGrath BP


    AIM: To define the role of dopamine-2 receptors in the sympatho-inhibitory effects of γ-l-glutamyl-l-dopa in conscious rabbits. METHOD: γ-l-glutamyl-l-dopa ( gludopa ) was infused ivat 25 and 100 μg·kg-1·min-1 with and without prior dopamine-2 receptor blockade by YM-09151-2 (50 μg·kg-1 iv) in conscious rabbits. RESULTS: Mean arterial pressure and heart rate remained unchanged while renal plasma flow increased. Arterial norepinephrine (NE) concentration, total and renal NE spillover rate were markedly decreased in a dose-related manner, which were not affected by prior dopamine-2 receptor blockade . Gludopa was detected in the whole brain (92±112 ng/g wet brain tissue) at the end of experiment although brain tissue levodopa, NE, and dopamine contents were not much different from those in the control group. CONCLUSION: Gludopa decreased dose-dependently plasma NE concentration,and total and renal NE overflow to plasma,which were not mediated by activation of dopamine D2 receptors.

  4. Dopamine release from serotonergic nerve fibers is reduced in L-DOPA-induced dyskinesia

    Nevalainen, Nina; af Bjerkén, Sara; Lundblad, Martin; Gerhardt, Greg A.; Strömberg, Ingrid


    L-DOPA (3,4-dihydroxyphenyl-L-alanine) is the most commonly used treatment for symptomatic control in patients with Parkinson’s disease. Unfortunately, most patients develop severe side effects, such as dyskinesia, upon chronic L-DOPA treatment. The patophysiology of dyskinesia is unclear, however, involvement of serotonergic nerve fibers in converting L-DOPA to dopamine has been suggested. Therefore, potassium-evoked dopamine release was studied after local application of L-DOPA in the striata of normal, dopamine- and dopamine/serotonin-lesioned L-DOPA naïve, and dopamine-denervated chronically L-DOPA-treated dyskinetic rats using in vivo chronoamperometry. The results revealed that local L-DOPA administration into normal and intact hemisphere of dopamine-lesioned L-DOPA naïve animals significantly increased the potassium-evoked dopamine release. L-DOPA application also increased the dopamine peak amplitude in the dopamine-depleted L-DOPA naïve striatum, although these dopamine levels were several-folds lower than in the normal striatum, while no increased dopamine release was found in the dopamine/serotonin-denervated striatum. In dyskinetic animals, local L-DOPA application did not affect the dopamine release, resulting in significantly attenuated dopamine levels compared to those measured in L-DOPA naïve dopamine-denervated striatum. To conclude, L-DOPA is most likely converted to dopamine in serotonergic nerve fibers in the dopamine-depleted striatum, but the dopamine release is several-fold lower than in normal striatum. Furthermore, L-DOPA loading does not increase the dopamine release in dyskinetic animals as found in L-DOPA naïve animals, despite similar density of serotonergic innervation. Thus, the dopamine overflow produced from the serotonergic nerve fibers appears not to be the major cause of dyskinetic behavior. PMID:21534956

  5. Maladaptive synaptic plasticity in L-DOPA-induced dyskinesia

    Qiang Wang


    Full Text Available The emergence of L-DOPA-induced dyskinesia (LID in patients with Parkinson disease (PD could be due to maladaptive plasticity of corticostriatal synapses in response to L-DOPA treatment. A series of recent studies has revealed that LID is associated with marked morphological plasticity of striatal dendritic spines, particularly cell type-specific structural plasticity of medium spiny neurons (MSNs in the striatum. In addition, evidence demonstrating the occurrence of plastic adaptations, including aberrant morphological and functional features, in multiple components of cortico-basal ganglionic circuitry, such as primary motor cortex (M1 and basal ganglia (BG output nuclei. These adaptations have been implicated in the pathophysiology of LID. Here, we briefly review recent studies that have addressed maladaptive plastic changes within the cortico-BG loop in dyskinetic animal models of PD and patients with PD.

  6. Striatal overexpression of DeltaJunD resets L-DOPA-induced dyskinesia in a primate model of Parkinson disease.

    Berton, Olivier; Guigoni, Céline; Li, Qin; Bioulac, Bernard H; Aubert, Incarnation; Gross, Christian E; Dileone, Ralph J; Nestler, Eric J; Bezard, Erwan


    Involuntary movements, or dyskinesia, represent a debilitating complication of dopamine replacement therapy for Parkinson disease (PD). The transcription factor DeltaFosB accumulates in the denervated striatum and dimerizes primarily with JunD upon repeated L-3,4-dihydroxyphenylalanine (L-DOPA) administration. Previous studies in rodents have shown that striatal DeltaFosB levels accurately predict dyskinesia severity and indicate that this transcription factor may play a causal role in the dyskinesia sensitization process. We asked whether the correlation previously established in rodents extends to the best nonhuman primate model of PD, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned macaque. We used western blotting and quantitative polymerase chain reaction (PCR) to compare DeltaFosB protein and messenger RNA (mRNA) levels across two subpopulations of macaques with differential dyskinesia severity. Second, we tested the causal implication of DeltaFosB in this primate model. Serotype 2 adeno-associated virus (AAV2) vectors were used to overexpress, within the motor striatum, either DeltaFosB or DeltaJunD, a truncated variant of JunD lacking a transactivation domain and therefore acting as a dominant negative inhibitor of DeltaFosB. A linear relationship was observed between endogenous striatal levels of DeltaFosB and the severity of dyskinesia in Parkinsonian macaques treated with L-DOPA. Viral overexpression of DeltaFosB did not alter dyskinesia severity in animals previously rendered dyskinetic, whereas the overexpression of DeltaJunD dramatically dropped the severity of this side effect of L-DOPA without altering the antiparkinsonian activity of the treatment. These results establish a mechanism of dyskinesia induction and maintenance by L-DOPA and validate a strategy, with strong translational potential, to deprime the L-DOPA-treated brain.

  7. Tratamento da síndrome parkinsoniana pelo L-dopa Parkinsonism's treatment by L-Dopa

    Roberto Melaragno


    Full Text Available O trabalho refere o tratamento com L-Dopa de 33 pacientes internados, sendo 19 homens e 14 mulheres. Os casos foram agrupados, segundo uma classificação esquemática, em leves, moderados, intensos e severos. As idades dos pacientes, previamente operados ou não, variaram de 42 a 78 anos. O esquema de medicação constou de administração de L-Dopa na dose inicial de 500 mg associado a um inibidor da MAO. A dose de L-Dopa era elevada de 500 mg cada 3 ou 4 dias, segundo a tolerância de cada paciente. Todos os sintomas dependentes, direta ou indiretamente, da rigidez tiveram melhoras mais nítidas em relação aos tremores. Durante a administração do medicamento os teores sangüíneos de ácido úrico e uréia, mostraram tendência para se elevar; a reserva alcalina, pelo contrário, tendeu para a diminuição; o hemograma revelou muitas vezes eosinofilia por vezes intensa. Os efeitos colaterais foram catalogados em dois tipos — efeitos adrenérgicos e efeitos dopaminérgicos — correspondentes a duas fases: uma primeira, de pequena duração, na qual foram usados concomitantemente o L-Dopa e um inibidor da MAO, surgindo cefaléia hipertensão arterial paroxística, rubor facial, sudorese, tenesmo vesical e angina pectoris; numa segunda fase surgiram os efeitos colaterais ditos dopaminérgicos, proporcionais à gravidade clínica do caso, cujas manifestações principais foram hipotensão arterial, sudorese fria, arritmia cardíaca e lipotimia. As complicações clínicas gerais eram representados habitualmente por anorexia, obstipação intestinal, náuseas, vômitos, discretas alterações do estado psíquico com depressão e euforia. Dentre as complicações neurológicas foram assinalados o estado parkinsonóide, discinesias e acatisia.The results of the treatment with L-Dopa of 33 patients (19 males and 14 females are reported. The cases were separated, acording to their symptoms and signs, in slight, moderate, intense and severe ones

  8. Changes in kynurenine pathway metabolism in Parkinson patients with L-DOPA-induced dyskinesia

    Havelund, Jesper F; Dammann Andersen, Andreas; Binzer, Michael


    L-DOPA is the most effective drug in the symptomatic treatment of Parkinson's disease, but chronic use is associated with L-DOPA-induced dyskinesia in more than half the patients after 10 years of treatment. L-DOPA treatment may affect tryptophan metabolism via the kynurenine pathway. Altered...... levels of kynurenine metabolites can affect glutamatergic transmission and may play a role in the development of L-DOPA-induced dyskinesia. In this study we assessed kynurenine metabolites in plasma and cerebrospinal fluid of Parkinson's disease patients and controls. Parkinson patients (n=26) were...... clinically assessed for severity of motor symptoms (UPDRS) and L-DOPA-induced dyskinesia (UDysRS). Plasma and cerebrospinal fluid samples were collected after overnight fasting and 1-2 hours after intake of L-DOPA or other anti-Parkinson medication. Metabolites were analyzed in plasma and cerebrospinal fluid...

  9. L-DOPA neurotoxicity is mediated by up-regulation of DMT1-IRE expression.

    Fang Du

    Full Text Available BACKGROUND: The mechanisms underlying neurotoxicity caused by L-DOPA are not yet completely known. Based on recent findings, we speculated that the increased expression of divalent metal transporter 1 without iron-response element (DMT1-IRE induced by L-DOPA might play a critical role in the development of L-DOPA neurotoxicity. To test this hypothesis, we investigated the effects of astrocyte-conditioned medium (ACM and siRNA DMT-IRE on L-DOPA neurotoxicity in cortical neurons. METHODS AND FINDINGS: We demonstrated that neurons treated with L-DOPA have a significant dose-dependent decrease in neuronal viability (MTT Assay and increase in iron content (using a graphite furnace atomic absorption spectrophotometer, DMT1-IRE expression (Western blot analysis and ferrous iron (55Fe(II uptake. Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability. Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1-IRE expression. In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1-IRE expression as well as L-DOPA neurotoxicity. CONCLUSION: The up-regulation of DMT1-IRE and the increase in DMT1-IRE-mediated iron influx play a key role in L-DOPA neurotoxicity in cortical neurons.

  10. The Tyrosinase Produced by Lentinula boryana (Berk. & Mont. Pegler Suffers Substrate Inhibition by L-DOPA

    Rodrigo Otávio de Faria


    Full Text Available We undertook a preliminary characterization of the tyrosinase produced by a strain of Lentinula boryana from Brazil, with a view to evaluate its potential for biotechnological applications. The enzyme was similar to other fungal tyrosinases in many respects. When the crude extract was characterized, the tyrosinase activity was optimal at pH=6 and was not particularly thermostable, with half-lives of about 10 min and 1 min at 50 and 60 °C, respectively. We purified the enzyme with ammonium sulfate precipitation followed by ion exchange chromatography on a DEAE Sepharose column, obtaining a yield of 33 % and a 5.3-fold enrichment. The purified preparation gave three bands on SDS-PAGE, with molecular masses of 20, 27 and 47 kDa. This preparation showed substrate inhibition kinetics with L-DOPA (3,4-dihydroxy-L-phenylalanine, with a KM of 1.9 mM and a KI of 72 mM. Under the same reaction conditions, a commercial mushroom tyrosinase followed Michaelis-Menten kinetics, with a KM of 0.51 mM. Although the present study did not identify properties that would make the tyrosinase of L. boryana more suitable in biotechnological applications than tyrosinases from other mushrooms, it has made a contribution by showing that the enzyme suffers substrate inhibition by L-DOPA, something that has not previously been reported for mushroom tyrosinases.

  11. Association of L-DOPA with recovery following Ayurveda medication in Parkinson's disease.

    Nagashayana, N; Sankarankutty, P; Nampoothiri, M R; Mohan, P K; Mohanakumar, K P


    Ayurveda, the Indian system of traditional medicine, uses a concoction of several spices, herbs and minerals for the treatment of diseases. In a clinical prospective study we have evaluated the efficacy of Ayurveda treatment (a concoction in cow's milk of powdered Mucuna pruriens and Hyoscyamus reticulatus seeds and Withania somnifera and Sida cordifolia roots) in 18 clinically diagnosed (with a mean Hoen and Yahr value of 2.22) parkinsonian patients. As per Ayurveda principles, 13 patients underwent both cleansing (for 28 days) and palliative therapy (56 days), 5 patients underwent palliative therapy alone (84 days). Only the former group showed significant improvement in activities of daily living (ADL) and on motor examination as per UPDRS rating. Symptomatically, they exhibited better response in tremor, bradykinesia, stiffness and cramps as compared to the latter group. Excessive salivation worsened in both the groups. Analyses of powdered samples in milk, as administered in patients, revealed about 200 mg of L-DOPA per dose. The study establishes the necessity of cleansing therapy in Ayurveda medication prior to palliative therapy. It also reveals contribution of L-DOPA in the recovery as observed in Parkinson' disease following Ayurveda medication.

  12. Mediated exodus of L-dopa from human epidermal Langerhans cells.

    Falck, B; Bendsoe, N; Ronquist, G


    L-3,4-dihydroxyphenylalanine (L-dopa) is not metabolized within human epidermal Langerhans cells (LC); yet they can take up substantial amounts of this amino acid which subsequently can be released into the extracellular space. We recently reported that human epidermal energy metabolism is predominantly anaerobic and that the influx mechanism is a unidirectional L-dopa/proton counter-transport system and now we describe conditions for the mediated transport of L-dopa out of the LC. It is demonstrated that certain amino acids and one dipeptide can effectively trigger the efflux of L-dopa taken up by the LC.Thus, alpha-methyl-dopa (alpha-m-dopa), D-dopa and the dipeptide, met-ala at the outside of the plasma membrane stimulated the efflux of L-dopa from L-dopa loaded LC. Similar effects were achieved by a variety of other amino acids in the extracellular fluid while some other amino acids were inactive. The time required for 50% D-methionine-induced exodus of L-dopa from L-dopa loaded LC was in the range of 5-7 min and a complete exodus of L-dopa was attained at about 20 min of incubation. This dislocation of L-dopa to the extracellular fluid is interpreted as an expression of trans-stimulation. In the case of alpha-m-dopa, D-dopa and met-ala, which admittedly were not able to penetrate the plasma membrane of LC, the concept of trans-stimulation was given a new purport, since none of them were able to participate in an exchange reaction. Finally, it could be concluded that L-dopa escaped by a route different from the one responsible for L-dopa uptake in LC.Thus, while the influx of L-dopa supports extrusion of protons deriving from anaerobic glycolysis in the LC, L-dopa efflux can provide the cells with useful amino acids in an energy-saving way, altogether a remarkable biological process. From this follows that L-dopa has a biological function of its own, besides being a precursor in the catecholamine and pigment syntheses.

  13. Ceftriaxone reduces L-dopa-induced dyskinesia severity in 6-hydroxydopamine parkinson's disease model.

    Chotibut, Tanya; Meadows, Samantha; Kasanga, Ella A; McInnis, Tamara; Cantu, Mark A; Bishop, Christopher; Salvatore, Michael F


    Increased extracellular glutamate may contribute to l-dopa induced dyskinesia, a debilitating side effect faced by Parkinson's disease patients 5 to 10 years after l-dopa treatment. Therapeutic strategies targeting postsynaptic glutamate receptors to mitigate dyskinesia may have limited success because of significant side effects. Increasing glutamate uptake may be another approach to attenuate excess glutamatergic neurotransmission to mitigate dyskinesia severity or prolong the time prior to onset. Initiation of a ceftriaxone regimen at the time of nigrostriatal lesion can attenuate tyrosine hydroxylase loss in conjunction with increased glutamate uptake and glutamate transporter GLT-1 expression in a rat 6-hydroxydopamine model. In this article, we examined if a ceftriaxone regimen initiated 1 week after nigrostriatal lesion, but prior to l-dopa, could reduce l-dopa-induced dyskinesia in an established dyskinesia model. Ceftriaxone (200 mg/kg, intraperitoneal, once daily, 7 consecutive days) was initiated 7 days post-6-hydroxydopamine lesion (days 7-13) and continued every other week (days 21-27, 35-39) until the end of the study (day 39 postlesion, 20 days of l-dopa). Ceftriaxone significantly reduced abnormal involuntary movements at 5 time points examined during chronic l-dopa treatment. Partial recovery of motor impairment from nigrostriatal lesion by l-dopa was unaffected by ceftriaxone. The ceftriaxone-treated l-dopa group had significantly increased striatal GLT-1 expression and glutamate uptake. Striatal tyrosine hydroxylase loss in this group was not significantly different when compared with the l-dopa alone group. Initiation of ceftriaxone after nigrostriatal lesion, but prior to and during l-dopa, may reduce dyskinesia severity without affecting l-dopa efficacy or the reduction of striatal tyrosine hydroxylase loss. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

  14. Growth Hormone Response to L-Dopa and Clonidine in Autistic Children.

    Realmuto, George M.; And Others


    Seven medication-free autistic subjects (ages 6-19) were administered clonidine and L-Dopa to investigate neuroendocrine responses through changes in growth hormone levels. Findings showed that, compared to normal controls, the L-Dopa-stimulated growth hormone peak was delayed and the clonidine growth hormone peak was premature. (Author/JDD)

  15. Lotus hairy roots expressing inducible arginine decarboxylase activity.

    Chiesa, María A; Ruiz, Oscar A; Sánchez, Diego H


    Biotechnological uses of plant cell-tissue culture usually rely on constitutive transgene expression. However, such expression of transgenes may not always be desirable. In those cases, the use of an inducible promoter could be an alternative approach. To test this hypothesis, we developed two binary vectors harboring a stress-inducible promoter from Arabidopsis thaliana, driving the beta-glucuronidase reporter gene and the oat arginine decarboxylase. Transgenic hairy roots of Lotus corniculatus were obtained with osmotic- and cold-inducible beta-glucuronidase and arginine decarboxylase activities. The increase in the activity of the latter was accompanied by a significant rise in total free polyamines level. Through an organogenesis process, we obtained L. corniculatus transgenic plants avoiding deleterious phenotypes frequently associated with the constitutive over-expression of arginine decarboxylation and putrescine accumulation.

  16. Preventive effect of rikkunshito on gastric motor function inhibited by L-dopa in rats.

    Wang, Lixin; Mogami, Sachiko; Karasawa, Hiroshi; Yamada, Chihiro; Yakabi, Seiichi; Yakabi, Koji; Hattori, Tomohisa; Taché, Yvette


    We previously reported that ghrelin prevented l-dopa (LD)-induced inhibition of gastric emptying (GE) of a non-nutrient solution in rats. Parkinson's disease treatment involves the combined administration of l-dopa with the enzyme l-amino acid decarboxylase inhibitor, carbidopa (CD) to reduce peripheral formation of dopamine. We investigated the effect LD/CD given orogastrically (og) on GE of a non-nutrient or nutrient meal and whether og pretreatment with rikkunshito, a kampo medicine clinically used to treat gastroparesis, influenced LD/CD effect on GE and postprandial antral and duodenal motility in conscious rats. LD/CD (20/2 mgkg(-1)) decreased significantly GE to 26.3 ± 6.0% compared to 61.2 ± 3.2% in og vehicle monitored 20-min after a non-nutrient meal and to 41.9 ± 5.8% compared to 72.9 ± 5.2% in og vehicle monitored 60 min after a nutrient meal. Rikkunshito (0.5 or 1.0 g kg(-1)) reduced the LD/CD (20/2 mg kg(-1)) inhibition of GE of non-nutrient meal (36.9 ± 7.4% and 46.6 ± 4.8% respectively vs. 12.1 ± 7.4% in og vehicle plus LD/CD) while having no effect alone (56.6 ± 8.5%). The ghrelin antagonist, [d-Lys(3)]-GHRP-6 (1 mg kg(-1)) injected intraperitoneally partially reversed rikkunshito preventive effect on LD/CD-inhibited GE. Rikkunshito (1.0 g kg(-1)) blocked LD/CD (20/2 mg kg(-1))-induced delayed GE of a nutrient meal and the reduction of postprandial antral motility. In 6-hydroxydopamine-induced Parkinson's disease rat model, rikkunshito (1.0 g kg(-1), og) also prevented LD/CD-inhibited gastric emptying of a nutrient meal and enhanced fasting plasma levels of acylated ghrelin. These data indicate that oral rikkunshito alleviates the delayed GE induced by LD/CD in naïve and PD rat model in part through ghrelin-related mechanisms.

  17. Differential dopamine receptor occupancy underlies L-DOPA-induced dyskinesia in a rat model of Parkinson's disease.

    Gurdal Sahin

    Full Text Available Dyskinesia is a major side effect of an otherwise effective L-DOPA treatment in Parkinson's patients. The prevailing view for the underlying presynaptic mechanism of L-DOPA-induced dyskinesia (LID suggests that surges in dopamine (DA via uncontrolled release from serotonergic terminals results in abnormally high level of extracellular striatal dopamine. Here we used high-sensitivity online microdialysis and PET imaging techniques to directly investigate DA release properties from serotonergic terminals both in the parkinsonian striatum and after neuronal transplantation in 6-OHDA lesioned rats. Although L-DOPA administration resulted in a drift in extracellular DA levels, we found no evidence for abnormally high striatal DA release from serotonin neurons. The extracellular concentration of DA remained at or below levels detected in the intact striatum. Instead, our results showed that an inefficient release pool of DA associated with low D2 receptor binding remained unchanged. Taken together, these findings suggest that differential DA receptor activation rather than excessive release could be the underlying mechanism explaining LID seen in this model. Our data have important implications for development of drugs targeting the serotonergic system to reduce DA release to manage dyskinesia in patients with Parkinson's disease.

  18. Cell type-specific plasticity of striatal projection neurons in parkinsonism and L-DOPA-induced dyskinesia

    Fieblinger, Tim; Graves, Steven M.; Sebel, Luke E.; Alcacer, Cristina; Plotkin, Joshua L.; Gertler, Tracy S.; Chan, C. Savio; Heiman, Myriam; Greengard, Paul; Cenci, M. Angela; Surmeier, D. James


    Summary The striatum is widely viewed as the fulcrum of pathophysiology in Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID). In these disease states, the balance in activity of striatal direct pathway spiny projection neurons (dSPNs) and indirect pathway spiny projection neurons (iSPNs) is disrupted, leading to aberrant action selection. However, it is unclear whether countervailing mechanisms are engaged in these states. Here we report that iSPN intrinsic excitability and excitatory corticostriatal synaptic connectivity were lower in PD models than normal; L-DOPA treatment restored these properties. Conversely, dSPN intrinsic excitability was elevated in tissue from PD models and suppressed in LID models. Although the synaptic connectivity of dSPNs did not change in PD models, it fell with L-DOPA treatment. In neither case, however, was the strength of corticostriatal connections globally scaled. Thus, SPNs manifested homeostatic adaptations in intrinsic excitability and in the number but not strength of excitatory corticostriatal synapses. PMID:25360704

  19. Management of L-dopa overdose in the competitive inhibition state.

    Hinz, Marty; Stein, Alvin; Cole, Ted


    The amino acid L-3,4-dihydroxyphenylalanine (L-dopa) is prescribed for conditions where increased central and/or peripheral dopamine synthesis is desired. Its administration can establish dopamine concentrations higher than can be achieved from an optimal diet. Specific indications include Parkinson's disease and restless leg syndrome. The interaction between serotonin and dopamine exists in one of two distinctly different physiologic states: the endogenous state or the competitive inhibition state. Management with L-dopa in the competitive inhibition state is the focus of this paper. In the past, control of the competitive inhibition state was thought to be so difficult and complex that it was described in the literature as functionally "meaningless". When administering L-dopa without simultaneous administration of serotonin precursors, the patient is in the endogenous state. Experience gained with patient outcomes during endogenous L-dopa administration does not allow predictability of L-dopa outcomes in the competitive inhibition state. The endogenous approach typically increases the daily L-dopa dosing value in a linear fashion until symptoms of Parkinson's disease are under control. It is the novel observations made during treatment with the competitive inhibition state approach that L-dopa dosing values above or below the optimal therapeutic range are generally associated with the presence of the exact same Parkinson's disease symptoms with identical intensity. This recognition requires a novel approach to optimization of daily L-dopa dosing values from that used in the endogenous state. This paper outlines that novel approach through utilization of a pill stop. This approach enhances patient safety through its ability to prevent L-dopa overdose, while assisting in the establishment of the optimal therapeutic L-dopa daily dosing value.

  20. IRC-082451, a novel multitargeting molecule, reduces L-DOPA-induced dyskinesias in MPTP Parkinsonian primates.

    Romina Aron Badin

    Full Text Available The development of dyskinesias following chronic L-DOPA replacement therapy remains a major problem in the long-term treatment of Parkinson's disease. This study aimed at evaluating the effect of IRC-082451 (base of BN82451, a novel multitargeting hybrid molecule, on L-DOPA-induced dyskinesias (LIDs and hypolocomotor activity in a non-human primate model of PD. IRC-082451 displays multiple properties: it inhibits neuronal excitotoxicity (sodium channel blocker, oxidative stress (antioxidant and neuroinflammation (cyclooxygenase inhibitor and is endowed with mitochondrial protective properties. Animals received daily MPTP injections until stably parkinsonian. A daily treatment with increasing doses of L-DOPA was administered to parkinsonian primates until the appearance of dyskinesias. Then, different treatment regimens and doses of IRC-082451 were tested and compared to the benchmark molecule amantadine. Primates were regularly filmed and videos were analyzed with specialized software. A novel approach combining the analysis of dyskinesias and locomotor activity was used to determine efficacy. This analysis yielded the quantification of the total distance travelled and the incidence of dyskinesias in 7 different body parts. A dose-dependent efficacy of IRC-082451 against dyskinesias was observed. The 5 mg/kg dose was best at attenuating the severity of fully established LIDs. Its effect was significantly different from that of amantadine since it increased spontaneous locomotor activity while reducing LIDs. This dose was effective both acutely and in a 5-day sub-chronic treatment. Moreover, positron emission tomography scans using radiolabelled dopamine demonstrated that there was no direct interference between treatment with IRC-082451 and dopamine metabolism in the brain. Finally, post-mortem analysis indicated that this reduction in dyskinesias was associated with changes in cFOS, FosB and ARC mRNA expression levels in the putamen. The data

  1. Striatal proteomic analysis suggests that first L-dopa dose equates to chronic exposure.

    Birger Scholz

    Full Text Available L-3,4-dihydroxypheylalanine (L-dopa-induced dyskinesia represent a debilitating complication of therapy for Parkinson's disease (PD that result from a progressive sensitization through repeated L-dopa exposures. The MPTP macaque model was used to study the proteome in dopamine-depleted striatum with and without subsequent acute and chronic L-dopa treatment using two-dimensional difference in-gel electrophoresis (2D-DIGE and mass spectrometry. The present data suggest that the dopamine-depleted striatum is so sensitive to de novo L-dopa treatment that the first ever administration alone would be able (i to induce rapid post-translational modification-based proteomic changes that are specific to this first exposure and (ii, possibly, lead to irreversible protein level changes that would be not further modified by chronic L-dopa treatment. The apparent equivalence between first and chronic L-dopa administration suggests that priming would be the direct consequence of dopamine loss, the first L-dopa administrations only exacerbating the sensitization process but not inducing it.

  2. Imbalanced Dopaminergic Transmission Mediated by Serotonergic Neurons in L-DOPA-Induced Dyskinesia

    Sylvia Navailles


    Full Text Available L-DOPA-induced dyskinesias (LIDs are one of the main motor side effects of L-DOPA therapy in Parkinson's disease. The review will consider the biochemical evidence indicating that the serotonergic neurons are involved in the dopaminergic effects of L-DOPA in the brain. The consequences are an ectopic and aberrant release of dopamine that follows the serotonergic innervation of the brain. After mid- to long-term treatment with L-DOPA, the pattern of L-DOPA-induced dopamine release is modified. In several brain regions, its effect is dramatically reduced while, in the striatum, its effect is quite preserved. LIDs could appear when the dopaminergic effects of L-DOPA fall in brain areas such as the cortex, enhancing the subcortical impact of dopamine and promoting aberrant motor responses. The consideration of the serotonergic system in the core mechanism of action of L-DOPA opens an important reserve of possible strategies to limit LIDs.

  3. Motivational disturbances and effects of L-dopa administration in neurofibromatosis-1 model mice.

    David F Wozniak

    Full Text Available Children with neurofibromatosis type 1 (NF1 frequently have cognitive and behavioral deficits. Some of these deficits have been successfully modeled in Nf1 genetically-engineered mice that develop optic gliomas (Nf1 OPG mice. In the current study, we show that abnormal motivational influences affect the behavior of Nf1 OPG mice, particularly with regard to their response to novel environmental stimuli. For example, Nf1 OPG mice made fewer spontaneous alternations in a Y-maze and fewer arm entries relative to WT controls. However, analysis of normalized alternation data demonstrated that these differences were not due to a spatial working memory deficit. Other reported behavioral results (e.g., open-field test, below suggest that differential responses to novelty and/or other motivational influences may be more important determinants of these kinds of behavior than simple differences in locomotor activity/spontaneous movements. Importantly, normal long-term depression was observed in hippocampal slices from Nf1 OPG mice. Results from elevated plus maze testing showed that differences in exploratory activity between Nf1 OPG and WT control mice may be dependent on the environmental context (e.g., threatening or non-threatening under which exploration is being measured. Nf1 OPG mice also exhibited decreased exploratory hole poking in a novel holeboard and showed abnormal olfactory preferences, although L-dopa (50 mg/kg administration resolved the abnormal olfactory preference behaviors. Nf1 OPG mice displayed an attenuated response to a novel open field in terms of decreased ambulatory activity and rearing but only during the first 10 min of the session. Importantly, Nf1 OPG mice demonstrated investigative rearing deficits with regard to a novel hanging object suspended on one side of the field which were not rescued by L-dopa administration. Collectively, our results provide new data important for evaluating therapeutic treatments aimed at


    Gordana Glavan


    Full Text Available Motor complications after chronic l-DOPA treatment in patients with Parkinson’s diseasemay be caused by the fluctuations of l-DOPA availability in the brain that provokes thesensitization of striatal output neurons of dopamine-depleted striatum. The aim of thisstudy was to analyze the effects of intermittent l-DOPA/carbidopa treatment schedule(injection of l-DOPA/carbidopa every fourth day, 6-treatments on the development oflocomotor sensitization of hemiparkinsonian rats to l-DOPA, and on the development ofdopaminergic sensitization of striatal output neurons of the indirect and direct pathways.The development of locomotor sensitization was verified by the increased intensity ofcontralateral turning behavior after the last l-DOPA injection. It is well known that PPTmRNA is expressed predominantly by the neurons of the direct pathway, PENK mRNAby the neurons of the indirect pathway, while GAD67 mRNA is expressed in the neuronsof both pathways. Dopaminergic sensitization of striatal output neurons of dopaminedepleted striatum was thus assessed by the analysis of changes of striatal preprotachykinin(PPT, proenkephalin (PENK and GAD 67 mRNA levels 4 and 12 hours after the lastl-DOPA injection. We found, that chronic dopamine depletion by itself down-regulates theexpression of striatal PPT mRNA and up-regulates GAD67 and PENK mRNAs. These changesof basal expression were not reversed by the intermittent l-DOPA/carbidopa treatment.However, in dopamine-depleted striatum, the intermittent treatment with l-DOPA inducedincreased responsiveness of striatal PPT and GAD67, but not PENK mRNA expression, tol-DOPA. Our results are in agreement with the hypothesis, that intermittent l-DOPA treatment induces locomotor sensitization that may be linked to the increased dopaminergicresponsiveness of striatonigral neurons of the direct pathway, within dopamine-depletedstriatum.

  5. Chronopharmacology of L-DOPA: implications for orthochronal therapy in the prevention of circadian dyschronism

    Ehret, C.F.; Meinert, J.C.; Groh, K.R.


    Two experimental results showed a significant influence of the circadian phase time-of-administration of L-DOPA upon the circadian rhythm of the rat, as judged by measures of core temperature. It is concluded that, in order to minimize the deleterious side effects and maximize the beneficial effects of a drug such as L-DOPA, orthochronal therapy is indicated. How orthochronal is to be defined when extrapolated to organisms apparently deficient in either tyrosine hydroxylase or in L-DOPA itself (as in the case of Parkinson's disease) will require experimental trial with appropriate subjects.

  6. Disturbed intracortical excitability in early Parkinson's disease is l-DOPA dose related: a prospective 12-month paired TMS study.

    Bares, Martin; Kanovský, Petr; Rektor, Ivan


    We were interested to know if chronic l-DOPA treatment in Parkinson's disease (PD) patients could restore impairment of the intracortical excitability, when this difference could occur, and if it was related to the total daily dose of l-DOPA. Twelve patients with early PD were studied using paired transcranial magnetic stimulation before the administration of l-DOPA, and then after 3, 6, and 12 months of l-DOPA treatment. The level of disturbed intracortical excitability strongly correlated with the total daily dose of l-DOPA. The level of cortical excitability in PD patients seems to be indirectly related to the nigro-striatal functioning.

  7. Effect of L-dopa on plasma homocysteine in elderly patients with Parkinson’ s disease



    Objective To explore the effect of L-dopa on plasma homocysteine and folic acid in patients with Parkinsons’s disease (PD). Methods Twenty eight elderly PD patients and thirty normal subjects were enrolled in this group. The homocysteine, cobalamin and folate were examined in normal group and in PD group before treatment and after being treated with L-dopa for six months respec-

  8. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    Lietzan, Adam D; St Maurice, Martin


    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  9. Effects of (-)-sesamin on motor and memory deficits in an MPTP-lesioned mouse model of Parkinson's disease treated with l-DOPA.

    Zhao, T T; Shin, K S; Kim, K S; Park, H J; Kim, H J; Lee, K E; Lee, M K


    The present study investigated the effects of (-)-sesamin on motor and memory deficits in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) with l-3,4-dihydroxyphenylalanine (l-DOPA). MPTP-lesioned (30mg/kg/day, 5days) mice showed deficits in memory including habit learning memory and spatial memory, which were further aggravated by daily treatment with 25mg/kg l-DOPA for 21days. However, daily treatment with (-)-sesamin (25 and 50mg/kg) for 21days ameliorated memory deficits in an MPTP-lesioned mouse model of PD treated with l-DOPA (25mg/kg). Both (-)-sesamin doses reduced decreases in the retention latency time in the passive avoidance test, latency to fall of rotarod test and distance traveled in the open field test, and attenuated decreases in tyrosine hydroxylase (TH)-immunopositive cells, dopamine, and its metabolites in the substantia nigra-striatum. (-)-Sesamin reduced increases in the retention transfer latency time in the elevated plus-maze test and N-methyl-d-aspartate receptor (NMDAR) expression and reduced decreases in the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus. In contrast, daily treatment with 10mg/kg l-DOPA for 21days ameliorated memory deficits in MPTP-lesioned mice, and this effect was further improved by treatment with (-)-sesamin (25 and 50mg/kg). These results suggest that (-)-sesamin protects against habit learning memory deficits by activating the dopamine neuronal system, while spatial memory deficits are decreased by its modulatory effects on the NMDAR-ERK1/2-CREB system. Accordingly, (-)-sesamin may act as an adjuvant phytonutrient for motor and memory deficits in patients with PD receiving l-DOPA.

  10. Boronophenylalanine uptake in C6 glioma model is dramatically increased by L-DOPA preloading

    Capuani, S. [CNR-INFM SOFT, Department of Physics, Sapienza University of Rome, Piazzale Aldo Moro 2, Rome (Italy); Enrico Fermi Center, Compendio Viminale, Rome (Italy)], E-mail:; Gili, T. [CNR-INFM SOFT, Department of Physics, Sapienza University of Rome, Piazzale Aldo Moro 2, Rome (Italy); Enrico Fermi Center, Compendio Viminale, Rome (Italy); Bozzali, M. [Neuroimaging Laboratory, Santa Lucia Foundation, Via Ardeatina 306, Rome (Italy); Russo, S. [Victor Horsley Department of Neurosurgery, National Hospital for Neurology and Neurosurgery, Queen Square, London (United Kingdom); Porcari, P. [CNR-INFM SOFT, Department of Physics, Sapienza University of Rome, Piazzale Aldo Moro 2, Rome (Italy); Cametti, C. [CNR-INFM SOFT, Department of Physics, Sapienza University of Rome, Piazzale Aldo Moro 2, Rome (Italy); Department of Physics, Sapienza University of Rome, Piazzale Aldo Moro 2, Rome (Italy); Muolo, M. [Department of Biological Science, University ' Rome III' , Viale G. Marconi 446, Rome (Italy); D' Amore, E. [Serv. Qual./Sicurezza Sperim. Anim., Istituto Superiore di Sanita, Rome (Italy); Maraviglia, B. [Enrico Fermi Center, Compendio Viminale, Rome (Italy); Neuroimaging Laboratory, Santa Lucia Foundation, Via Ardeatina 306, Rome (Italy); Lazzarino, G. [Laboratory of Biochemistry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, Catania (Italy); Pastore, F.S. [Department of Neuroscience, Institute of Neurosurgery, University ' Tor Vergata' , Via Montpellier 1, Rome (Italy)


    One of the main limitations for BNCT effectiveness is the insufficient intake of {sup 10}B nuclei within tumour cells. This work was aimed at investigating the use of L-DOPA as enhancer for boronophenylalanine (BPA) uptake in the C6 glioma model. The investigation was first performed in vitro, and then extended in vivo to the animal model. BPA accumulation in C6 glioma cells was assessed, using radiowave dielectric spectroscopy (RDS), with and without L-DOPA preloading. C6 glioma cells were also implanted in the brain of 25 rats, randomly assigned to two experimental branches: (1) intra-carotid BPA infusion; (2) intra-carotid BPA infusion after pre-treatment with L-DOPA, administrated 24 h before BPA infusion. All animals were sacrificed, and assessment of BPA concentrations in tumour tissue, normal brain, and blood samples was performed using high performance liquid chromatography (HPLC). L-DOPA preloading induced a massive increase of BPA concentration either in vitro on C6 glioma cells or in vivo in the animal model tumour. Moreover, no significant difference was found in the normal brain and blood samples between the two animal groups. This study suggests the potential use of L-DOPA as enhancer for BPA accumulation in malignant gliomas eligible for BNCT.

  11. Serotonergic modulation of receptor occupancy in rats treated with L-DOPA after unilateral 6-OHDA lesioning

    Nahimi, Adjmal; Høltzermann, Mette; Landau, Anne M.


    Recent studies suggest that l-3,4 dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID), a severe complication of conventional L-DOPA therapy of Parkinson's disease, may be caused by dopamine (DA) release originating in serotonergic neurons. To evaluate the in vivo effect of a 5-HT(1A) agonist...... [(±)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide, 8-OHDPAT] on the L-DOPA-induced increase in extracellular DA and decrease in [(11) C]raclopride binding in an animal model of advanced Parkinson's disease and LID, we measured extracellular DA in response to L-DOPA or a combination of L...

  12. Modification of Fe3O4 Magnetic Nanoparticles by L-dopa or Dopamine as an Enzyme Support

    PENG Hong; ZHANG Xiao; HUANG Kaixun; XU Huibi


    Fe3O4 magnetic nanoparticles were prepared by co-precipitation of Fe2+ and Fe3+ in an ammonia solution,and its size was about 36 am measured by an atomic force microscope.Fe3O4 magnetic nanopanicles were modified by L-dopa or dopamine using sonication method.The analysis of FTIR clearly indicated the formation of Fe-O-C bond.Direct immobilization of trypsin(EC: Fe3O4 magnetic nanoparticles with L-dopa and dopamine spacer was investigated using glutaraldehyde as a coupling agent.No significant changes in the size and magnetic property of the three kinds of magnetic nanoparticles linked with or without trypsin were observed.The existence of the spacer molecule on magnetic nanoparticles could greatly improve the activity and the storage stability of bound trypsin through increasing the flexibility of enzyme and changing the microenvironment on nanoparticles surface compared to the naked magnetic nanoparticles.

  13. Palmitoyl Serotonin Inhibits L-dopa-induced Abnormal Involuntary Movements in the Mouse Parkinson Model.

    Park, Hye-Yeon; Ryu, Young-Kyoung; Go, Jun; Son, Eunjung; Kim, Kyoung-Shim; Kim, Mee Ree


    L-3,4-dihydroxyphenylalanine (L-DOPA) is the most common treatment for patients with Parkinson's disease (PD). However, long term use of L-DOPA for PD therapy lead to abnormal involuntary movements (AIMs) known as dyskinesia. Fatty acid amide hydrolase (FAAH) is enriched protein in basal ganglia, and inhibition of the protein reduces dyskinetic behavior of mice. Palmitoyl serotonin (PA-5HT) is a hybrid molecule patterned after arachidonoyl serotonin, antagonist of FAAH. However, the effect of PA-5HT on L-DOPA-induced dyskinesia (LID) in PD have not yet been elucidated. To investigate whether PA-5HT relieve LID in PD and decrease hyperactivation of dopamine D1 receptors, we used the 6-hydroxydopomine (6-OHDA)-lesioned mouse model of PD and treated the L-DOPA (20 mg/kg) for 10 days with PA-5HT (0.3 mg/kg/day). The number of wall contacts with the forelimb in the cylinder test was significantly decreased by 6-OHDA lesion in mice and the pharmacotherapeutic effect of L-DOPA was also revealed in PA-5HT-treated mice. Moreover, in AIMs test, PA-5HT-treated mice showed significant reduction of locomotive, axial, limb, and orofacial AIMs score compared to the vehicle-treated mice. LID-induced hyper-phosphorylation of ERK1/2 and overexpression of FosB/ΔFosB was markedly decreased in 6-OHDA-lesioned striatum of PA-5HT-treated mice, indicating that PA-5HT decreased the dopamine D1 receptor-hyperactivation induced by chronic treatment of L-DOPA in dopamine-denervated striatum. These results suggest that PA-5HT effectively attenuates the development of LID and enhance of ERK1/2 phosphorylation and FosB/ΔFosB expression in the hemi-parkinsonian mouse model. PA-5HT may have beneficial effect on the LID in PD.

  14. Effects of dopamine depletion on LFP oscillations in striatum are task- and learning-dependent and selectively reversed by L-DOPA.

    Lemaire, Nuné; Hernandez, Ledia F; Hu, Dan; Kubota, Yasuo; Howe, Mark W; Graybiel, Ann M


    A major physiologic sign in Parkinson disease is the occurrence of abnormal oscillations in cortico-basal ganglia circuits, which can be normalized by L-DOPA therapy. Under normal circumstances, oscillatory activity in these circuits is modulated as behaviors are learned and performed, but how dopamine depletion affects such modulation is not yet known. We here induced unilateral dopamine depletion in the sensorimotor striatum of rats and then recorded local field potential (LFP) activity in the dopamine-depleted region and its contralateral correspondent as we trained the rats on a conditional T-maze task. Unexpectedly, the dopamine depletion had little effect on oscillations recorded in the pretask baseline period. Instead, the depletion amplified oscillations across delta (~3 Hz), theta (~8 Hz), beta (~13 Hz), and low-gamma (~48 Hz) ranges selectively during task performance times when each frequency band was most strongly modulated, and only after extensive training had occurred. High-gamma activity (65-100 Hz), in contrast, was weakened independent of task time or learning stage. The depletion also increased spike-field coupling of fast-spiking interneurons to low-gamma oscillations. L-DOPA therapy normalized all of these effects except those at low gamma. Our findings suggest that the task-related and learning-related dynamics of LFP oscillations are the primary targets of dopamine depletion, resulting in overexpression of behaviorally relevant oscillations. L-DOPA normalizes these dynamics except at low-gamma, linked by spike-field coupling to fast-spiking interneurons, now known to undergo structural changes after dopamine depletion and to lack normalization of spike activity following l-DOPA therapy.

  15. Mutation of Aspergillus oryzae for improved production of 3, 4-dihydroxy phenyl-L-alanine (L-DOPA from L-tyrosine Mutação de Aspergillus orizae para produção melhorada de 3,4-dihidroxi fenil-L-alanina (L-DOPA a partir de L-tirosina

    Ikram-ul Haq


    Full Text Available Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-DOPA from L-tyrosine using chemical mutation. Different putative mutant strains of organism were tested for the production of L-DOPA in submerged fermentation. Among these putative mutant strains, mutant designated SI-12 gave maximum production of L-DOPA (300 mg L-DOPA.g-1 cells. The production of L-DOPA from different carbon source solutions (So= 30 g.l-1 by mutant culture was investigated at different nitrogen sources, initial pH and temperature values. At optimum pH (pHo= 5.0, and temperature (t=30ºC, 100% sugars were utilized for production and cell mass formation, corresponding to final L-DOPA product yield of 150 mg.g-1 substrate utilized, and maximum volumetric and specific productivities of 125 mg.l-1.h-1, and 150 mg.g-1 cells. h-1, respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is the highest reported in literature. To explain the kinetic mechanism of L-DOPA formation and thermal inactivation of tyrosinase, the thermodynamic parameters were determined with the application of Arrhenius model: activation enthalpy and entropy for product formation, in case of mutant derivative, were 40 k j/mol and 0.076 k j/mol. K for L-DOPA production and 116 k j/mol and 0.590 k j/mol. K for thermal inactivation, respectively. The respective values for product formation were lower while those for product deactivation were higher than the respective values for the parental culture. Therefore, the mutant strain was thermodynamically more resistant to thermal denaturation.A produção de L-DOPA a partir de tirosina pela cepa mutante de Aspergillus orizae UV-7 foi melhorada através de mutação química. Diferentes cepas foram testadas quanto a produção de L-DOPA por fermentação submersa, observando-se que a cepa denominada SI-12 foi a melhor produtora (300 mg de L-DOPA por g de células. A produção de L-DOPA pela cepa

  16. L-DOPA treatment in MPTP-mouse model of Parkinson's disease potentiates homocysteine accumulation in substantia nigra.

    Bhattacharjee, Nivedita; Mazumder, Muhammed Khairujjaman; Paul, Rajib; Choudhury, Amarendranath; Choudhury, Sabanum; Borah, Anupom


    One of the intermediates of methionine cycle, the homocysteine (Hcy), elevates in plasma of Parkinson's disease (PD) patients undergoing L-DOPA (3,4-dihydroxyphenylalanine) therapy and has been regarded as a risk factor of the disease. Several evidences pointed out that Hcy causes degeneration of dopaminergic neurons. In rodent, elevated level of Hcy in brain or infusion of the same directly into the substantia nigra (SN) potentiates dopaminergic neurodegeneration. However, the influence of L-DOPA therapy on the levels of Hcy in dopamine-rich regions of the brain (striatum and SN) of experimental models of PD is not known. The present study, for the first time, tested the hypothesis that L-DOPA treatment in experimental mouse model of PD potentiates Hcy accumulation in the dopamine-rich regions of the brain. We found a significant elevation of Hcy level in nigrostriatum in naïve as well as parkinsonian mice as a result of chronic L-DOPA treatment. Interestingly, L-DOPA treatment significantly elevates Hcy level in nigra but not in striatum of parkinsonian mice, when compared with L-DOPA naïve group. However, there is no significant decrease in the number of dopaminergic neurons in SN region in the parkinsonian mice given L-DOPA treatment. Thus, the present study demonstrates that L-DOPA treatment potentiates the level of Hcy in the SN without causing aggravated neurodegeneration in parkinsonian mice model.

  17. Fully automated synthesis module for the high yield one-pot preparation of 6-[F-18]fluoro-L-DOPA

    de Vries, EFJ; Luurtsema, G; Brussermann, M; Elsinga, PH; Vaalburg, W


    A fully automated one-pot synthesis of 6-[F-18]fluoro-L-DOPA, an important radiopharmaceutical for studies on the presynaptic dopamine metabolism with positron emission tomography,is described. 6-[F-18]Fluoro-L-DOPA was prepared in high radiochemical yield (33 +/- 4%, c.f.d.) and radiochemical

  18. Gynostemma pentaphyllum Ethanolic Extract Protects Against Memory Deficits in an MPTP-Lesioned Mouse Model of Parkinson's Disease Treated with L-DOPA.

    Kim, Kyung Sook; Zhao, Ting Ting; Shin, Keon Sung; Park, Hyun Jin; Cho, Yoon Jeong; Lee, Kyung Eun; Kim, Seung Hwan; Lee, Myung Koo


    This study investigated the effects of ethanol extract from Gynostemma pentaphyllum (GP-EX) on memory deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) (MPTP-lesioned mice). MPTP (30 mg/kg/day, 5 days)-lesioned mice showed deficits of habit learning memory and spatial memory, which were further aggravated by treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) (25 mg/kg, 21 days). However, treatment with GP-EX (50 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice treated with L-DOPA (25 mg/kg): GP-EX prevented the decreases in retention latency time in the passive avoidance test and tyrosine hydroxylase-immunopositive cells and dopamine levels in the nigrostriatum. GP-EX also reduced increases in retention transfer latency time of the elevated plus-maze test and expression of N-methyl-D-aspartate (NMDA) receptor and improved decreases in phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus in the same models. By contrast, L-DOPA treatment (10 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice, which were further improved by GP-EX treatment. These results suggest that GP-EX ameliorates habit learning memory deficits by activating dopaminergic neurons and spatial memory deficits by modulating NMDA receptor-ERK1/2-CREB system in MPTP-lesioned mice treated with L-DOPA. GP-EX may serve as an adjuvant phytonutrient for memory deficits in PD.

  19. Enzymatic Synthesis of Agmatine by Immobilized Escherichia coli Cells with Arginine Decarboxylase Activity

    ZHANG Wei-guo; ZHAO Gen-hai; LIU Jun-zhong; LIU Qian; JIAO Qing-cai


    A new method for the enzymatic synthesis of agmatine by immobilized Escherichia coli cells with arginine decarboxylase(ADC)activity was established and a series of optimal reaction conditions was set down.The arginine decarboxylase showed the maximum activity when the pyridoxal phosphate(PLP)concentration was 50 mmol/L,pH=7 and 45 ℃.The arginine decarboxylase exhibited the maximum production efficiency when the substrate concentration was 100 mmol/L and the reaction time was 15 h.It was also observed that the appropriate concentration of Mg2+,especially at 0.5 mmol/L promoted the arginine decarboxylase activity; Mn2+ had little effect on the arginine decarboxylase activity.The inhibition of Cu2+ and Zn2+ to the arginine decarboxylase activity was significant.The immobilized cells were continuously used 6 times and the average conversion rate during the six-time usage was 55.6%.The immobilized cells exhibited favourable operational stability.After optimization,the maximally cumulative amount of agmatine could be up to 20 g/L.In addition,this method can also catalyze D,L-arginine to agmatine,leaving the pure optically D-arginine simultaneously.The method has a very important guiding significance to the enzymatic preparation of agmatine.

  20. Cerebrospinal fluid levels of catecholamine metabolites in Parkinson’s disease and L-DOPA-induced dyskinesia

    Dammann Andersen, Andreas; Binzer, Michael; Stenager, Egon;

    Levodopa (L-DOPA) is effective in the symptomatic treatment of Parkinson’s disease (PD), but chronic use is associated with L-DOPA-induced dyskinesia (LID). In the 6-hydroxydopamine rat model of PD, L-DOPA treatment increases dopamine, its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC...... fasting and 1 hour after intake of medication. Results and conclusion: PD patients receiving levodopa had 10-20 fold higher L-DOPA levels and about 100 fold higher levels of 3-OMD than age-matched controls or PD not receiving L-DOPA. DOPAC levels were not different among controls and subgroups of PD. HVA...... levels were significantly lower in non-DOPA-treated PD. Ratios of DOPAC/DOPA or HVA/DOPA were much lower in levodopa-treated patients, not distinguishing dyskinetic (N=6) from non-dyskinetic PD patients (N=5). More patients need to be included. Tryptophan/kynurenine metabolites will be analysed using LC...

  1. Four Hour Temporal Relation of 5-HTP and L-DOPA Induces Inhibitory Responses in Recrudescing Gonad of Indian Palm Squirrel (Funambulus pennantii).

    Jaiwal, Ranjana; Chaturvedi, C M


    Daily injections of L-dihydroxyphenylalanine (L-DOPA, dopamine precursor) given 4 h after 5-hydroxytryptophan (5-HTP, serotonin precursor) induced inhibitory responses in recrudescing gonad (in the first week of December) of Indian palm squirrel, a seasonally breeding subtropical animal. Other temporal relations (L-DOPA given at 0, 8, 12, 16, and 20 h after 5-HTP administration) did not show any effect on the recrudescing gonad. This inhibitory effect of 4 h was evident under short day length (6 : 18) group but was masked by the increasing day length of nature (NDL, late December onwards) and increased photoperiod of long day group (16 : 8). It is apparent that seasonal testicular recrudescence of Indian palm squirrel during short day length by 4 h relation of 5-HTP and L-DOPA is not a pharmacological effect but actually is an alteration of seasonality in this annually breeding mammal. It seems that endogenous mechanism controlling seasonal testicular recrudescence of Indian palm squirrel is reset by timed daily injections of these neurotransmitter drugs. It is suggested that in spite of different environmental factors (photoperiod, humidity, etc.) used by different species to time their annual reproduction, basic mechanism of seasonality appears to be the same, that is, the temporal synergism of neurotransmitter activity.

  2. Optimization of PLGA nanoparticles formulation containing L-DOPA by applying the central composite design.

    Zhou, Yong Zhi; Alany, Raid G; Chuang, Victor; Wen, Jingyuan


    The aim of this work was to prepare L-DOPA loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles by a modified water-in-oil-in-water (W(1)/O/W(2)) emulsification solvent evaporation method. A central composite design was applied for optimization of the formulation parameters and for studying the effects of three independent variables: PLGA concentration, polyvinyl alcohol (PVA) concentration and organic solvent removal rate on the particle size and the entrapment efficiency (response variables). Second-order models were obtained to adequately describe the influence of the independent variables on the selected responses. The analysis of variance showed that the three independent variables had significant effects (p overlay contour plots, the optimal preparation area can be highlighted. It was found that the optimum values of the responses could be obtained at higher concentration of PLGA (5%, w/v) and PVA (6%, w/v); and faster organic solvent removal rate (700 rpm). The corresponding particle size was 256.2 nm and the entrapment efficiency was 62.19%. FTIR investigation confirmed that the L-DOPA and PLGA polymer maintained its backbone structure in the fabrication of nanoparticles. The scanning electron microscopic images of nanoparticles showed that all particles had spherical shape with porous outer skin. The results suggested that PLGA nanoparticles might represent a promising formulation for brain delivery of L-DOPA. The preparation of L-DOPA loaded PLGA nanoparticles can be optimized by the central composite design.

  3. Presynaptic Mechanisms of l-DOPA-Induced Dyskinesia: The Findings, the Debate, and the Therapeutic Implications.

    Cenci, M Angela


    The dopamine (DA) precursor l-DOPA has been the most effective treatment for Parkinson's disease (PD) for over 40 years. However, the response to this treatment changes with disease progression, and most patients develop dyskinesias (abnormal involuntary movements) and motor fluctuations within a few years of l-DOPA therapy. There is wide consensus that these motor complications depend on both pre- and post-synaptic disturbances of nigrostriatal DA transmission. Several presynaptic mechanisms converge to generate large DA swings in the brain concomitant with the peaks-and-troughs of plasma l-DOPA levels, while post-synaptic changes engender abnormal functional responses in dopaminoceptive neurons. While this general picture is well-accepted, the relative contribution of different factors remains a matter of debate. A particularly animated debate has been growing around putative players on the presynaptic side of the cascade. To what extent do presynaptic disturbances in DA transmission depend on deficiency/dysfunction of the DA transporter, aberrant release of DA from serotonin neurons, or gliovascular mechanisms? And does noradrenaline (which is synthetized from DA) play a role? This review article will summarize key findings, controversies, and pending questions regarding the presynaptic mechanisms of l-DOPA-induced dyskinesia. Intriguingly, the debate around these mechanisms has spurred research into previously unexplored facets of brain plasticity that have far-reaching implications to the treatment of neuropsychiatric disease.

  4. l-Dopa responsiveness is associated with distinctive connectivity patterns in advanced Parkinson's disease.

    Akram, Harith; Wu, Chengyuan; Hyam, Jonathan; Foltynie, Thomas; Limousin, Patricia; De Vita, Enrico; Yousry, Tarek; Jahanshahi, Marjan; Hariz, Marwan; Behrens, Timothy; Ashburner, John; Zrinzo, Ludvic


    Neuronal loss and dopamine depletion alter motor signal processing between cortical motor areas, basal ganglia, and the thalamus, resulting in the motor manifestations of Parkinson's disease. Dopamine replacement therapy can reverse these manifestations with varying degrees of improvement. To evaluate functional connectivity in patients with advanced Parkinson's disease and changes in functional connectivity in relation to the degree of response to l-dopa, 19 patients with advanced Parkinson's disease underwent resting-state functional magnetic resonance imaging in the on-medication state. Scans were obtained on a 3-Tesla scanner in 3 × 3 × 2.5 mm(3) voxels. Seed-based bivariate regression analyses were carried out with atlas-defined basal ganglia regions as seeds, to explore relationships between functional connectivity and improvement in the motor section of the UPDRS-III following an l-dopa challenge. False discovery rate-corrected P was set at motor area, mesial primary motor, and primary sensory areas). Our findings show characteristic basal ganglia resting-state functional connectivity patterns associated with different degrees of l-dopa responsiveness in patients with advanced Parkinson's disease. l-Dopa exerts a graduated influence on remapping connectivity in distinct motor control networks, potentially explaining some of the variance in treatment response. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

  5. Imaging mass spectrometry reveals elevated nigral levels of dynorphin neuropeptides in L-DOPA-induced dyskinesia in rat model of Parkinson's disease.

    Anna Ljungdahl

    Full Text Available L-DOPA-induced dyskinesia is a troublesome complication of L-DOPA pharmacotherapy of Parkinson's disease and has been associated with disturbed brain opioid transmission. However, so far the results of clinical and preclinical studies on the effects of opioids agonists and antagonists have been contradictory at best. Prodynorphin mRNA levels correlate well with the severity of dyskinesia in animal models of Parkinson's disease; however the identities of the actual neuroactive opioid effectors in their target basal ganglia output structures have not yet been determined. For the first time MALDI-TOF imaging mass spectrometry (IMS was used for unbiased assessment and topographical elucidation of prodynorphin-derived peptides in the substantia nigra of a unilateral rat model of Parkinson's disease and L-DOPA induced dyskinesia. Nigral levels of dynorphin B and alpha-neoendorphin strongly correlated with the severity of dyskinesia. Even if dynorphin peptide levels were elevated in both the medial and lateral part of the substantia nigra, MALDI IMS analysis revealed that the most prominent changes were localized to the lateral part of the substantia nigra. MALDI IMS is advantageous compared with traditional molecular methods, such as radioimmunoassay, in that neither the molecular identity analyzed, nor the specific localization needs to be predetermined. Indeed, MALDI IMS revealed that the bioconverted metabolite leu-enkephalin-arg also correlated positively with severity of dyskinesia. Multiplexing DynB and leu-enkephalin-arg ion images revealed small (0.25 by 0.5 mm nigral subregions with complementing ion intensities, indicating localized peptide release followed by bioconversion. The nigral dynorphins associated with L-DOPA-induced dyskinesia were not those with high affinity to kappa opioid receptors, but consisted of shorter peptides, mainly dynorphin B and alpha-neoendorphin that are known to bind and activate mu and delta opioid receptors

  6. High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143

    Shultz Jeffry L


    Full Text Available Abstract Background The 3,4-dihydroxy phenyl L-alanine (L-dopa is a drug of choice for Parkinson's disease, controlling changes in energy metabolism enzymes of the myocardium following neurogenic injury. Aspergillus oryzae is commonly used for L-dopa production; however, potential improvements in ease of handling, growth rate and environmental impact have led to an interest in exploiting alternative yeasts. The two important elements required for L-dopa production are intracellular tyrosinases (thus pre-grown yeast cells are required for the transformation of L-tyrosine to L-dopa and L-ascorbate, which acts as a reducing agent. Results Pre-grown cells of Yarrowia lipolytica NRRL-143 were used for the microbiological transformation of L-tyrosine to L-dopa. Different diatomite concentrations (0.5–3.0 mg/ml were added to the acidic (pH 3.5 reaction mixture. Maximum L-dopa biosynthesis (2.96 mg/ml L-dopa from 2.68 mg/ml L-tyrosine was obtained when 2.0 mg/ml diatomite was added 15 min after the start of the reaction. After optimizing reaction time (30 min, and yeast cell concentration (2.5 mg/ml, an overall 12.5 fold higher L-dopa production rate was observed when compared to the control. Significant enhancements in Yp/s, Qs and qs over the control were observed. Conclusion Diatomite (2.0 mg/ml addition 15 min after reaction commencement improved microbiological transformation of L-tyrosine to L-dopa (3.48 mg/ml; p ≤ 0.05 by Y. lipolytica NRRL-143. A 35% higher substrate conversion rate was achieved when compared to the control.

  7. Influence of L-dopa and of thymus fraction on the survival rate of whole-body irradiated mice

    Busse, E.; Helmholz, M. (Humboldt-Universitaet, Berlin (German Democratic Republic). Bereich Medizin (Charite))


    The survival rate of mice with exposure of the whole body (7 Gy) was hardly changed by one dose as well as several doses of the phosphodiesterase inhibitor amantadine and the interferon inductor measles vaccine. However, the survival rates were increased by one administration of L-dopa or by the long-term therapy using L-dopa at 7 and 9 Gy, resp. The survival rates were also increased at 7 and 9 Gy, resp. if the thymus factor was three times applied to the animals after irradiation. The increased survival rates gained by using L-dopa and thymus factor are correlated with the leukocyte values determined.

  8. Genetic, temporal and diurnal influences on L-dopa-induced dyskinesia in the 6-OHDA model.

    Monville, Christelle; Torres, Eduardo M; Pekarik, Vladimir; Lane, Emma L; Dunnett, Stephen B


    Current treatments for Parkinson's disease rely on a dopamine replacement strategy and are reasonably effective, particularly in the early stages of the disease. However, chronic dopaminergic therapy is limited by the development of a range of side effects, including the onset of abnormal movements ('dyskinesia'). The neural mechanisms that underlie dyskinesia are far from clear but they have been associated with pulsatile stimulation of dopamine receptors, downstream changes in proteins and genes, and abnormalities in non-dopamine transmitter systems. However, there has been no pathophysiological explanation for the worsening motor symptoms in the afternoon and evening reported by Parkinsonian patients in long-term L-dopa therapy, and no direct relationship has been found with the pharmacokinetics of the drug. Moreover, there continues to be a debate about whether the development of dyskinesias in patients is dependent upon the duration of L-dopa treatment or on the degree of denervation/advanced stage of the disease, both factors that are difficult to resolve experimentally in the human disease. The objective of this study was to characterise, in an animal model, factors that predispose some individuals to develop dyskinesia after a prolonged treatment with L-dopa, whereas others continue to exhibit symptom alleviation without the side effects. We report that none of the parameters studied--genetic variation within and between strains, delay of treatment onset after lesion, or time of day of the drug treatment--were found to influence directly the formation of dyskinesias after L-dopa treatment. We conclude that a complex combination of individual factors are likely to interact to regulate the onset and development of abnormal movements in some animals but not others.

  9. Asymmetric Pictet-Spengler Reactions:Synthesis of Tetrahydroisoquinoline Derivatives from L-DOPA

    Ye WANG; Zhan Zhu LIU; Shi Zhi CHEN; Xiao Tian LIANG


    The cis-1-substituted-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid esters 3 can be obtained in a highly diastereoselective fashion through 1,3-induction Pictet- Spengler (P-S) cyclization of the L-DOPA (3,4-dihydroxyphenylalanine) methyl ester with aromatic or aliphatic aldehydes under acidic conditions. Their epimers 4 are also obtained as minor products.


    A. Rabbani.


    Full Text Available Growth hormone stimulation tests have been used to assess the growth hormone reserve of the pituitary gland in both children and adults. We have assessed the effect of clonidine, insulin, L-Dopa and exercise on growth hormone secretion in 261 short children. The" results found in this study revealed that there are no significant differences in these stimulation tests (P=0.28 .

  11. The role of L-DOPA on melanization and mycelial production in Malassezia furfur.

    Sirida Youngchim

    Full Text Available Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.

  12. The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur

    Youngchim, Sirida; Nosanchuk, Joshua D.; Pornsuwan, Soraya; Kajiwara, Susumu; Vanittanakom, Nongnuch


    Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization. PMID:23762233

  13. Rapid Electrochemical Method for the Determination of L-DOPA in Extract From the Seeds of Mucuna Prurita.

    Stankovič, Dalibor M; Samphao, Anchalee; Dojcinović, Biljana; Kalcher, Kurt


    This work presents the electrochemical behavior of levodopa (L-DOPA), at boron-doped diamond (BDD) electrodes, using cycling voltammetry (CV), in Britton-Robinson (BR) buffer solution, and application of the proposed electrode for the determination of L-DOPA in extracts from the seeds of velvet bean (Mucuna prurita Hook or Mucuna pruriens (L.) DC.). L-DOPA provides a well-defined and single oval-shape oxidation peak at +0.8 V vs. Ag/AgCl (3 M KCl) reference electrode in BR buffer solution at pH 3.0. Experimental parameters, such as pH of supporting electrolyte and square wave voltammetry (SWV) operating parameters (frequency and modulation amplitude) were optimized. The effect of possible interferences was evaluated. Under optimal conditions the detection limit of the developed method was 0.8 μM and the calibration curve for L-DOPA was linear in the range from 2 to 100 μM. The proposed method was successfully applied to the determination of L-DOPA in an extract from the seeds of Mucuna prurita. The obtained result was in good agreement with obtained by photometry with 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The developed approach can be beneficial for the quantification of L-DOPA using a BDD electrode as up-to-date potential alternative sensor for electroanalytical applications.


    M. Zarabian


    Full Text Available Avulsion is one of the most serious emergencies in dental office. In the event of any problem, the tooth should be stored in a medium that supports the periodontal ligament cell viability. In other clinical situations, preserving media, growth factors and mitogenic products may be useful in repairing the traumatized tissues. It has been previously reported that levodopa (L-dopa accelerates healing by increasing the growth hormone level. In this study, the local effect of L-dopa, as a mitogen, on human periodontal ligament fibroblast (HPLF cells was evaluated. Samples from impacted or semiimpacted wisdom or canine teeth, which were devoid of inflammation, were taken. The cells obtained from this tissue were cultured in an appropriate medium. The passage numbers between 3-6 were taken for further experiments. The viability of HPLF cells, which were treated by L-dopa, was evaluated by trypan blue dye exclusion and neutral red assay. Results indicated that low concentration of L-dopa produces significant increase of these cells compared to control group. These results confirmed previous studies about direct action of L- dopa on the viability of HPLF cells. On the basis of this study and previous reports, presence of L-dopa in preserving media may be useful in increasing the self-life transferring HPLF cells.

  15. Ornithine Decarboxylase Activity Is Required for Prostatic Budding in the Developing Mouse Prostate.

    Melissa Gamat

    Full Text Available The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their

  16. Fast-scan cyclic voltammetry reveals that L-Dopa produces regionally selective, bimodal influences on striatal dopamine kinetics in vivo

    Harun, R; Munoz, M; Grassi, C; Hare, K; Brough, E; Torres, GE; Grace, AA; Wagner, AK


    Parkinson’s disease (PD) is a debilitating condition that is caused by a relatively specific degeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta. Levodopa (L-Dopa) was introduced as a viable treatment option for PD over 40 years ago and still remains the most common and effective therapy for PD. Though the effects of L-Dopa to augment striatal DA production are well known, little is actually known about how L-Dopa alters the kinetics of DA neurotransmission that contribute to its beneficial and adverse effects. In this study, we examined the effects of L-Dopa administration (100mg/kg carbidopa/250mg/kg L-Dopa) on regional electrically stimulated DA response kinetics using fast-scan cyclic voltammetry (FSCV) in anesthetized rats. We demonstrate that L-Dopa enhances DA release in both the dorsal striatum (D-STR) and nucleus accumbens (NAc), but surprisingly causes a delayed inhibition of release in the D-STR, a finding that may be related to high-dose L-Dopa effects. In both regions, L-Dopa progressively attenuated reuptake kinetics through a decrease in Vmax and an increase in Km. This finding is consistent with recent clinical studies suggesting that L-Dopa chronically down-regulates the DA transporter (DAT), which may relate to the common development of L-Dopa induced dyskinesias (LID) in PD subjects. PMID:26611352

  17. Articulation disorders and duration, severity and L-dopa dosage in idiopathic Parkinson's disease.

    Pawlukowska, Wioletta; Gołąb-Janowska, Monika; Safranow, Krzysztof; Rotter, Iwona; Amernik, Katarzyna; Honczarenko, Krystyna; Nowacki, Przemysław


    Parkinson's disease (PD) is one of the most common diseases of the central nervous system (CNS). It is frequently heralded by speech disturbances, which are one of its first symptoms. The aim of this paper is to share our own experience concerning the correlation between the severity of speech disorders and the PD duration, its severity and the intake of L-dopa. The research included 93 patients with idiopathic PD, aged 26-86 years (mean age 65.1 years). Participants were examined neurologically according to the Unified Parkinson's Disease Rating Scale (UPDRS) and the Hoehn and Yahr Scale. They were also assessed by Frenchay Dysarthria Assessment. Considerable and severe disorders were concurrent with impairments in the mobility of the tongue, lips, the jaw as well as the pitch and loudness of the voice. The strongest correlation but at a moderate level was found to exist between the severity of labial impairment, voice loudness and the length of the disease. There was also a positive correlation between lip movement while the motions were being diversified, lip arrangement while speaking and the intake of L-dopa. As PD progresses a significant decline in vocal articulation can be observed, which is due to reduced mobility within the lips and the jaw. Exacerbation of articulation disorders resulting from progression of the disease does not materially influence the UPDRSS scores. L-dopa has been found to positively affect the mobility of the lips while the patient is speaking and their arrangement at rest. Copyright © 2015 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  18. Tratamento da síndrome parkinsoniana pelo L-dopa

    Roberto Melaragno


    Full Text Available O trabalho refere o tratamento com L-Dopa de 33 pacientes internados, sendo 19 homens e 14 mulheres. Os casos foram agrupados, segundo uma classificação esquemática, em leves, moderados, intensos e severos. As idades dos pacientes, previamente operados ou não, variaram de 42 a 78 anos. O esquema de medicação constou de administração de L-Dopa na dose inicial de 500 mg associado a um inibidor da MAO. A dose de L-Dopa era elevada de 500 mg cada 3 ou 4 dias, segundo a tolerância de cada paciente. Todos os sintomas dependentes, direta ou indiretamente, da rigidez tiveram melhoras mais nítidas em relação aos tremores. Durante a administração do medicamento os teores sangüíneos de ácido úrico e uréia, mostraram tendência para se elevar; a reserva alcalina, pelo contrário, tendeu para a diminuição; o hemograma revelou muitas vezes eosinofilia por vezes intensa. Os efeitos colaterais foram catalogados em dois tipos — efeitos adrenérgicos e efeitos dopaminérgicos — correspondentes a duas fases: uma primeira, de pequena duração, na qual foram usados concomitantemente o L-Dopa e um inibidor da MAO, surgindo cefaléia hipertensão arterial paroxística, rubor facial, sudorese, tenesmo vesical e angina pectoris; numa segunda fase surgiram os efeitos colaterais ditos dopaminérgicos, proporcionais à gravidade clínica do caso, cujas manifestações principais foram hipotensão arterial, sudorese fria, arritmia cardíaca e lipotimia. As complicações clínicas gerais eram representados habitualmente por anorexia, obstipação intestinal, náuseas, vômitos, discretas alterações do estado psíquico com depressão e euforia. Dentre as complicações neurológicas foram assinalados o estado parkinsonóide, discinesias e acatisia.

  19. A single dose of L-DOPA changes perceptual experiences and decreases latent inhibition in Parkinson's disease.

    Györfi, Orsolya; Nagy, Helga; Bokor, Magdolna; Kelemen, Oguz; Kéri, Szabolcs


    Despite the well-known neuropsychiatric side effects of dopaminergic medications, the possible subjective psychotomimetic effects of a single dose of L-DOPA in newly diagnosed, drug-naïve patients with Parkinson's disease (PD) are not known. To investigate this question, we used a visual search task for latent inhibition (LI), the Community Assessment of Psychic Experiences (CAPE) scale, and visual analog scales for psychotomimetic effects (perception, relaxation, and dysphoria) in 28 de novo PD patients before (off) and after (on) the adminstration of L-DOPA and in 25 matched healthy control individuals. Results revealed increased LI in PD-off and decreased LI in PD-on relative to the control subjects. After the administration of L-DOPA, we observed a significant decline in LI in PD. L-DOPA also enhanced perceptual experiences (changes in subjective feelings in thinking, time perception, and mental "highness"). Greater reduction in LI was associated with enhanced perceptual experiences. These results suggest that a single dose of L-DOPA has a significant psychotomimetic effect, which is associated with decreased LI, a behavioral marker of psychosis-like experiences.

  20. Hypertrophy of IMC of carotid artery in Parkinson's disease is associated with L-DOPA, homocysteine, and MTHFR genotype.

    Nakaso, Kazuhiro; Yasui, Kenichi; Kowa, Hisanori; Kusumi, Masayoshi; Ueda, Keigo; Yoshimoto, Yuko; Takeshima, Takao; Sasaki, Kiyohiro; Nakashima, Kenji


    In recent years, an intense interest has developed in the association between Parkinson's disease (PD) and hyperhomocysteinemia. Homocysteine (Hcy) is a neuronal excitotoxic amino acid, and is well known as a risk factor for vascular diseases. Some reports suggest that the administration of L-DOPA may promote hyperhomocysteinemia and idiopathic atherosclerosis. In this study, we report that a mild hypertrophy of the intima-media complex (IMC) of the carotid artery, which has been established as a marker for systemic atherosclerosis, is observed in PD patients compared with normal subjects. PD patients that were treated with L-DOPA for long durations showed a hypertrophic IMC, while the patients that were not treated with L-DOPA did not show any hypertrophic changes in the IMC. These hypertrophic changes were observed primarily in patients with a Hoehn-Yahr stage of 3-5. PD patients with hypertrophic IMC of the carotid artery also exhibited elevated plasma levels of Hcy associated with the C677T genotype of 5,10-methylenetetrahydrofolate reductase (MTHFR). Moreover, a prolonged duration of treatment with L-DOPA in patients with MTHFR T/T genotype enhanced the hypertrophy of IMC, compared with patients with the C/C or C/T genotype. These results suggest that hyperhomocysteinemia promoted by the C677T genotype of MTHFR and prolonged treatment with L-DOPA enhances atherosclerosis in PD patients and affects their general condition.

  1. Increase of histidine decarboxylase activity in mice hypothalamus after intracerebroventricular administration of lipopolysaccharide.

    Niimi, M; Mochizuki, T; Cacabelos, R; Yamatodani, A


    The effect of intracerebroventricular (icv) administration of lipopolysaccharide on histidine decarboxylase activity and histamine content in the hypothalamus were investigated in male mice of ddY strain in vivo. Two-fold increase in histidine decarboxylase activity (HDC) was observed 4 h after administration of 50 mcg lipopolysaccharide, and HDC activity returned to the basal level within 12 h after injection. Furthermore, histamine contents showed a slight decrease at 1 and 2 h and a mild increase at 12 h after administration. However, changes in histamine content were not statistically significant. These results suggest that the increase of HDC activity in the hypothalamus by lipopolysaccharide may be involved in the central neuroimmune responses.

  2. Fast-scan cyclic voltammetry demonstrates that L-DOPA produces dose-dependent regionally selective, bimodal effects on striatal dopamine kinetics in vivo.

    Harun, R; Hare, K M; Brough, M E; Munoz, M J; Grassi, C M; Torres, G E; Grace, A A; Wagner, A K


    Parkinson's disease (PD) is a debilitating condition that is caused by a relatively specific degeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta. Levodopa (L-DOPA) was introduced as a viable treatment option for PD over 40 years ago and still remains the most common and effective therapy for PD. Though the effects of L-DOPA to augment striatal DA production are well known, little is actually known about how L-DOPA alters the kinetics of DA neurotransmission that contribute to its beneficial and adverse effects. In this study, we examined the effects of L-DOPA administration (50mg/kg carbidopa + 0, 100, and 250mg/kg L-DOPA) on regional electrically stimulated DA response kinetics using fast-scan cyclic voltammetry (FSCV) in anesthetized rats. We demonstrate that L-DOPA enhances DA release in both the dorsal striatum (D-STR) and nucleus accumbens (NAc), but surprisingly causes a delayed inhibition of release in the D-STR. In both regions, L-DOPA progressively attenuated reuptake kinetics, predominantly through a decrease in Vmax. These findings have important implications on understanding the pharmacodynamics of L-DOPA, which can be informative for understand its therapeutic effects and also common side effects like L-DOPA induced dyskinesias (LID). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  3. DAT1 polymorphism determines L-DOPA effects on learning about others' prosociality.

    Christoph Eisenegger

    Full Text Available Despite that a wealth of evidence links striatal dopamine to individualś reward learning performance in non-social environments, the neurochemical underpinnings of such learning during social interaction are unknown. Here, we show that the administration of 300 mg of the dopamine precursor L-DOPA to 200 healthy male subjects influences learning about a partners' prosocial preferences in a novel social interaction task, which is akin to a repeated trust game. We found learning to be modulated by a well-established genetic marker of striatal dopamine levels, the 40-bp variable number tandem repeats polymorphism of the dopamine transporter (DAT1 polymorphism. In particular, we found that L-DOPA improves learning in 10/10R genoype subjects, who are assumed to have lower endogenous striatal dopamine levels and impairs learning in 9/10R genotype subjects, who are assumed to have higher endogenous dopamine levels. These findings provide first evidence for a critical role of dopamine in learning whether an interaction partner has a prosocial or a selfish personality. The applied pharmacogenetic approach may open doors to new ways of studying psychiatric disorders such as psychosis, which is characterized by distorted perceptions of others' prosocial attitudes.

  4. Gastroretentive drug delivery systems with L-dopa based on carrageenans and hydroxypropylmethylcellulose.

    Dorożyński, P; Kulinowski, P; Mendyk, A; Jachowicz, R


    A comprehensive study was conducted to investigate the effects of carrageenans, and hydroxypropylmethylcellulose (HPMC) on the properties of hydrodynamically balanced systems (HBS) containing L-dopa as a model drug. The novel integrated approach included measurements of: solvent uptake, erosion, apparent density and changes in the internal structure of dosage forms during dissolution test by means of a USP4 compatible MRI. Differences in water ingress into the matrices with pure carrageenans (ι, κ, λ) or low viscous HPMC, were detected by non-invasive magnetic resonance imaging. Matrices based on carrageenans subjected to rapid hydration and erosion, were not able to maintain satisfactory floating properties for a sufficiently long period of time. The application of carrageenans in mixtures with HMC promoted water uptake by HBS formulations. The effect produced by varying the polymer blend's composition on release of the L-dopa was also studied. Dissolution data was fitted to Korsmeyer-Peppas equation. For matrices containing mixtures of carrageenan and HPMC, the linear increase in the releasing rate constant, K, with the carrageenan content in the matrix was observed.

  5. Phosphorylation of Ser-204 and Tyr-405 in human malonyl-CoA decarboxylase expressed in silkworm Bombyx mori regulates catalytic decarboxylase activity.

    Hwang, In-Wook; Makishima, Yu; Suzuki, Tomohiro; Kato, Tatsuya; Park, Sungjo; Terzic, Andre; Chung, Shin-Kyo; Park, Enoch Y


    Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC is a vital catalytic reaction of lipid metabolism. While it is established that phosphorylation of MCD modulates the enzymatic activity, the specific phosphorylation sites associated with the catalytic function have not been documented due to lack of sufficient production of MCD with proper post-translational modifications. Here, we used the silkworm-based Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system to express human MCD (hMCD) and mapped phosphorylation effects on enzymatic function. Purified MCD from silkworm displayed post-translational phosphorylation and demonstrated coherent enzymatic activity with high yield (-200 μg/silkworm). Point mutations in putative phosphorylation sites, Ser-204 or Tyr-405 of hMCD, identified by bioinformatics and proteomics analyses reduced the catalytic activity, underscoring the functional significance of phosphorylation in modulating decarboxylase-based catalysis. Identified phosphorylated residues are distinct from the decarboxylation catalytic site, implicating a phosphorylation-induced global conformational change of MCD as responsible in altering catalytic function. We conclude that phosphorylation of Ser-204 and Tyr-405 regulates the decarboxylase function of hMCD leveraging the silkworm-based BmNPV bacmid expression system that offers a fail-safe eukaryotic production platform implementing proper post-translational modification such as phosphorylation.

  6. Inhibitory Effect of Ferulic Acid on Oxidation of L-DOPA Catalyzed by Mushroom Tyrosinase%阿魏酸对蘑菇酪氨酸酶的抑制效应

    龚盛昭; 程江; 杨卓如


    The inhibitory effect of ferulic acid on the diphenolase activity of mushroom tyrosinase and the kinetic behavior were studied with L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate. The inhibitor concentration from Lineweaver-Burk plots shows that ferulic acid is a competitive inhibitor and the inhibition of tyrosinase by ferulic acid is a reversible reaction. The equilibrium constant for ferulic acid binding with the tyrosinase was determined to be 0.25 mmol· L-1 for diphenolase.

  7. On the synthesis of radiofluorinated amino acids by isotope exchange based on the example of 6-[{sup 18}F]Fluor-L-DOPA; Zur Synthese radiofluorierter aromatischer Aminosaeuren mittels Isotopenaustausch am Beispiel von 6-[{sup 18}F]Fluor-L-DOPA

    Wagner, F.M.


    In nuclear medical diagnosis, 6-[{sup 18}F]fluoro-L-3,4-dihydroxyphenylalanine (6-[{sup 18}F]fluoro-LDOPA), an analogue of L-DOPA, is one of the few established radiopharmaceuticals used for the in vivo investigation of the presynaptic dopaminergic metabolism and of some kind of tumours via Positron Emission Tomography (PET). The presently used method of preparation of the radiotracer by electrophilic labelling is limited to low amounts of activity at high costs. Known nucleophilic syntheses, however, result either in insufficient enantiomeric purity or the known multi-step syntheses are hard to automate, due to their complexity. During this work a novel, easy to automate alternative for the preparation of 6-[{sup 18}F]fluoro-L-DOPA, was developed and evaluated, using a direct nucleophilic {sup 18}F-fluorination of a protected amino acid derivative. The resulting product has a very high enantiomeric purity. At first, the general suitability of the (S)-BOC-BMI-derivatives for the synthesis of {sup 18}F-labelled amino acids, used in this work, was investigated using a less complex precursor, which resulted in the amino acid 6-[{sup 18}F]fluoro-L-m-tyrosin via acidic hydrolysis. The preparation of a useful precursor for the nucleophilic {sup 18}F-isotope substitution, namely the (2S,5S)-tert.-butyl- 5-(2-fluoro-5-formylbenzyl)-2-tert.-butyl-3-methyl-4-oxoimidazolidine-1-carboxylate, was investigated in three general different ways. At first it was tried to obtain this product via formylation after coupling with the BOC-BMI, secondly via {alpha},{beta}-dehydro amino acid derivatives and finally via a systematic multi-step synthesis. Only the last mentioned way resulted in a precursor with sufficient purity that could be labelled. The radiochemical yield of the isotopic exchange was about 60 %. In the next step, the presented concept was modified to synthesize a precursor for the preparation of 6-[{sup 18}F]fluoro-L-DOPA. Only a combination of the protecting groups

  8. Strong preferences of dopamine and l-dopa towards lipid head group: importance of lipid composition and implication for neurotransmitter metabolism

    Orlowski, A.; Grzybek, M.; Bunker, A.


    J. Neurochem. (2012) 122, 681690. Abstract The interactions of the neurotransmitter dopamine, and its precursor l-dopa, with membrane lipids were investigated through a set of molecular dynamic simulations with all atom resolution. The results obtained indicate that both dopamine and l-dopa have...

  9. Dopamine-Induced Changes in Gαolf Protein Levels in Striatonigral and Striatopallidal Medium Spiny Neurons Underlie the Genesis of l-DOPA-Induced Dyskinesia in Parkinsonian Mice

    Morigaki, Ryoma; Okita, Shinya; Goto, Satoshi


    The dopamine precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), exerts powerful therapeutic effects but eventually generates l-DOPA-induced dyskinesia (LID) in patients with Parkinson’s disease (PD). LID has a close link with deregulation of striatal dopamine/cAMP signaling, which is integrated by medium spiny neurons (MSNs). Olfactory type G-protein α subunit (Gαolf), a stimulatory GTP-binding protein encoded by the GNAL gene, is highly concentrated in the striatum, where it positively couples with dopamine D1 (D1R) receptor and adenosine A2A receptor (A2AR) to increase intracellular cAMP levels in MSNs. In the striatum, D1Rs are mainly expressed in the MSNs that form the striatonigral pathway, while D2Rs and A2ARs are expressed in the MSNs that form the striatopallidal pathway. Here, we examined the association between striatal Gαolf protein levels and the development of LID. We used a hemi-parkinsonian mouse model with nigrostriatal lesions induced by 6-hydroxydopamine (6-OHDA). Using quantitative immunohistochemistry (IHC) and a dual-antigen recognition in situ proximity ligation assay (PLA), we here found that in the dopamine-depleted striatum, there appeared increased and decreased levels of Gαolf protein in striatonigral and striatopallidal MSNs, respectively, after a daily pulsatile administration of l-DOPA. This leads to increased responsiveness to dopamine stimulation in both striatonigral and striatopallidal MSNs. Because Gαolf protein levels serve as a determinant of cAMP signal-dependent activity in striatal MSNs, we suggest that l-DOPA-induced changes in striatal Gαolf levels in the dopamine-depleted striatum could be a key event in generating LID.

  10. Growth Hormone Response after Administration of L-dopa, Clonidine, and Growth Hormone Releasing Hormone in Children with Down Syndrome.

    Pueschel, Seigfried M.


    This study of eight growth-retarded children with Down's syndrome (aged 1 to 6.5 years) found that administration of growth hormone was more effective than either L-dopa or clonidine. Results suggest that children with Down's syndrome have both anatomical and biochemical hypothalamic derangements resulting in decreased growth hormone secretion and…

  11. Alterations in cerebellar glutamic acid decarboxylase (GAD) activity in a genetic model of torsion dystonia (rat).

    Oltmans, G A; Beales, M; Lorden, J F; Gordon, J H


    Glutamic acid decarboxylase (GAD) activity was studied in specific brain regions of a newly identified genetic (rat) model of human torsion dystonia. GAD activity was found to be significantly increased in the deep cerebellar nuclei of dystonic rats at 16, 20, and 24 days of age. GAD activity in the other regions examined (vermis, cerebellar hemispheres, caudate nucleus, and globus pallidus) did not differ from that of age-matched normal littermate controls. Diazepam treatment significantly reduced the frequency of dystonic movements in the mutant.

  12. Combined treatment with acupuncture reduces effective dose and alleviates adverse effect of L-dopa by normalizing Parkinson's disease-induced neurochemical imbalance.

    Kim, Seung-Nam; Doo, Ah-Reum; Park, Ji-Yeun; Choo, Hyunwoo J; Shim, Insop; Park, Jongbae J; Chae, Younbyoung; Lee, Bena; Lee, Hyejung; Park, Hi-Joon


    This study first showed the behavioural benefits of novel combination therapy of L-dopa with acupuncture on Parkinson's disease, and its underlying mechanisms within basal ganglia. The previous study reported that acupuncture may improve the motor function of a Parkinson's disease (PD) mouse model by increasing the dopamine efflux and turnover ratio of dopamine. Hence, we hypothesised that combining L-dopa with acupuncture would have a behavioural benefit for those with PD. We performed unilateral injections of 6-OHDA into the striatum of C57Bl/6 mice to model hemi-Parkinsonian attributes. To test motor function and dyskinetic anomalies, we examined cylinder behaviour and abnormal involuntary movement (AIM), respectively. We found that (1) a 50% reduced dose of L-dopa (7.5 mg/kg) combined with acupuncture showed an improvement in motor function that was comparable to mice given the standard dose of L-dopa treatment (15 mg/kg) only, and that (2) the combination treatment (L-dopa +acupuncture) was significantly superior in reducing AIM scores when equivalent doses of L-dopa were used. The combination treatment also significantly reduces the abnormal increase of GABA contents in the substantia nigra compared to the standard L-dopa treatment. Furthermore, abnormal expression of FosB, the immediate early gene of L-dopa induced dyskinesia (LID), was mitigated in the striatum by the combination treatment. All of these results indicate that acupuncture enhances the benefits of L-dopa on motor function with reduced dose of L-dopa and alleviating LID by normalising neurochemical imbalance within the basal ganglia.

  13. Gypenosides ameliorate memory deficits in MPTP-lesioned mouse model of Parkinson's disease treated with L-DOPA.

    Zhao, Ting Ting; Kim, Kyung Sook; Shin, Keon Sung; Park, Hyun Jin; Kim, Hyun Jeong; Lee, Kyung Eun; Lee, Myung Koo


    Previous studies have revealed that gypenosides (GPS) improve the symptoms of anxiety disorders in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rat model of Parkinson's disease (PD). The present study aimed to investigate the effects of GPS on memory deficits in an MPTP-lesioned mouse model of PD treated with L-3,4-dihydroxyphenylalanine (L-DOPA). MPTP (30 mg/kg/day, 5 days)-lesioned mice were treated with GPS (50 mg/kg) and/or L-DOPA (10 and 25 mg/kg) for 21 days. After the final treatments, behavioral changes were assessed in all mice using passive avoidance and elevated plus-maze tests. We then evaluated the biochemical influences of GPS treatment on levels of tyrosine hydroxylase (TH), dopamine, N-methyl-D-aspartate (NMDA) receptors, extracellular signal-regulated kinase (ERK1/2), and cyclic AMP-response element binding protein (CREB) phosphorylation. MPTP-lesioned mice exhibited deficits associated with habit learning and spatial memory, which were further aggravated by treatment with L-DOPA (25 mg/kg). However, treatment with GPS (50 mg/kg) ameliorated memory deficits. Treatment with GPS (50 mg/kg) also improved L-DOPA (25 mg/kg)-treated MPTP lesion-induced decreases in retention latency on the passive avoidance test, as well as levels of TH-immunopositive cells and dopamine in the substantia nigra and striatum. GPS treatment also attenuated increases in retention transfer latency on the elevated plus-maze test and in NMDA receptor expression, as well as decreases in the phosphorylation of ERK1/2 and CREB in the hippocampus. Treatment with L-DOPA (10 mg/kg) also ameliorated deficits in habit learning and spatial memory in MPTP-lesioned mice, and this effect was further enhanced by treatment with GPS (50 mg/kg). GPS ameliorate deficits in habit learning and spatial memory by modulating the dopaminergic neuronal and N-methyl-D-aspartate receptor-mediated signaling systems in MPTP-lesioned mice treated with L-DOPA. GPS may serve as an adjuvant

  14. NADH: ubiquinone oxidoreductase inhibitors block induction of ornithine decarboxylase activity in MCF-7 human breast cancer cells.

    Rowlands, J C; Casida, J E


    Rotenone is the classical inhibitor of NADH: ubiquinone oxidoreductase and its analogue deguelin is a potent inhibitor of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase mRNA steady state level and enzyme activity in mouse 308 cells (Gerhäuser et al. 1995). In MCF-7 human breast cancer cells, rotenone, deguelin and two structurally-unrelated miticides (pyridaben and fenazaquin) inhibit not only NADH: ubiquinone oxidoreductase but also induced ornithine decarboxylase activity with IC50 values of < 1 to 70 nM. Rotenone inhibits ornithine decarboxylase activity equally well as induced by TPA, insulin-like growth factor I and 17 beta-oestradiol. Pyridaben is the most potent of the four inhibitors not only for NADH: ubiquinone oxidoreductase activity (bovine heart enzyme) and TPA-induced ornithine decarboxylase activity and mRNA steady state level but also for TPA-induced reactive oxygen species. It is therefore proposed that NADH: ubiquinone oxidoreductase inhibitors block multiple and possibly reactive oxygen species-modulated pathways which regulate ornithine decarboxylase activity.

  15. The ornithine decarboxylase, NO-synthase activities and phospho-c-Jun content under experimental gastric mucosa malignancy

    Mariia Tymoshenko


    Full Text Available Ornithine decarboxylase is the first and key regulatory enzyme in synthesis of polyamines, which are essential for cell proliferation and differentiation, so its aberrant regulation is reported to play a role in neoplastic transformation and tumours growth. That's why, there were analysed some major links of metabolic pathways that are closely related to tumorigenesis: ornithine decarboxylase, and the NADPH-dependent enzyme nitric oxide synthase, the nuclear phosphoprotein c-Jun, that could play an important role in the development of gastric cancer malignancy.The gastric carcinogenesis was initiated in rats by 10-week replacement of drinking water by 0.01% N-methyl-N-nitro-N-nitrosoguanidine solution, at the same time they were redefined on the diet containing 5% NaCl. After this period expiry the animals were fed with standard diet till the end of the 24th week. The gastric mucosa cells were extracted at the end of the 4th, 6th, 8th, 10th, 12th, 18th and 24th week and underwent biochemical examinations. It was established the elevated phospho-c-Jun content, ornithine decarboxylase and inducible nitric oxide synthase activities from 6th to 24th week of gastric cancer development compared to the control references. The increasing of ornithine decarboxylase activity could probably be caused by the growth of phospho-c-Jun, it is also belonging to an ornithine decarboxylase transactivation effects. Thus, it was shown that the increase of ornithine decarboxylase and inducible nitric oxide synthase activities, phospho-c-Jun and nitrite-ions accumulation in gastric mucosa epithelial cells were associated with the gastric malignant progression. The complex relationships between the examined enzymes and transcription activator that pointed to an aggravation of pathological disturbances due to reciprocal action between ornithine decarboxylase and c-Jun and nitric oxide synthase participation. [Biomed Res Ther 2016; 3(4.000: 596-604

  16. Enhancing the Activity of Glutamate Decarboxylase from Lactobacillus brevis by Directed Evolution☆

    Ling Lin; Sheng Hu; Kai Yu; Jun Huang; Shanjing Yao; Yinlin Lei; Guixiang Hu; Lehe Mei


    Glutamate decarboxylase (GAD, EC4.1.1.15) can catalyze the decarboxylation of L-glutamate to form γ-aminobutyrate (GABA), which is in great demand in some foods and pharmaceuticals. In our previous study, gad, the gene coding glutamate decarboxylase from Lactobacil us brevis CGMCC 1306, was cloned and its soluble expression was realized. In this study, error-prone PCR was conducted to improve its activity, followed by a screening. Mutant Q51H with high activity [55.4 mmol·L−1·min−1·(mg protein)−1, 120%higher than that of the wild type at pH 4.8] was screened out from the mutant library. In order to investigate the potential role of this site in the regulation of enzymatic activity, site-directed saturation mutagenesis at site 51 was carried out, and three specific mutants, N-terminal truncated GAD, Q51P, and Q51L, were identified. The kinetic parameters of the three mutants and Q51H were characterized. The results reveal that aspartic acid at site 88 and N-terminal domain are essential to the activity as well as correct folding of GAD. This study not only improves the activity of GAD, but also sheds new light on the structure–function relationship of GAD.

  17. Catalytic irreversible inhibition of bacterial and plant arginine decarboxylase activities by novel substrate and product analogues.

    Bitonti, A J; Casara, P J; McCann, P P; Bey, P


    Arginine decarboxylase (ADC) activity from Escherichia coli and two plant species (oats and barley) was inhibited by five new substrate (arginine) and product (agmatine) analogues. The five compounds, (E)-alpha-monofluoromethyldehydroarginine (delta-MFMA), alpha-monofluoromethylarginine (MFMA), alpha-monofluoromethylagatine (FMA), alpha-ethynylagmatine (EA) and alpha-allenylagmatine (AA), were all more potent inhibitors of ADC activity than was alpha-difluoromethylarginine (DFMA), the only irreversible inhibitor of this enzyme described previously. The inhibition caused by the five compounds was apparently enzyme-activated and irreversible, since the loss of enzyme activity followed pseudo-first-order kinetics, was time-dependent, the natural substrate of ADC (arginine) blocked the effects of the inhibitors, and the inhibition remained after chromatography of inhibited ADC on Sephadex G-25 or on overnight dialysis of the enzyme. DFMA, FMA, delta-MFMA and MFMA were effective at very low concentrations (10 nM-10 microM) at inhibiting ADC activity in growing E. coli. FMA was also shown to deplete putrescine effectively in E. coli, particularly when combined with an inhibitor of ornithine decarboxylase, alpha-monofluoromethyl-putrescine. The potential uses of the compounds for the study of the role of polyamine biosynthesis in bacteria and plants is discussed.

  18. NMDA receptor antagonism potentiates the L-DOPA-induced extracellular dopamine release in the subthalamic nucleus of hemi-parkinson rats.

    El Arfani, Anissa; Bentea, Eduard; Aourz, Najat; Ampe, Ben; De Deurwaerdère, Philippe; Van Eeckhaut, Ann; Massie, Ann; Sarre, Sophie; Smolders, Ilse; Michotte, Yvette


    Long term treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) is associated with several motor complications. Clinical improvement of this treatment is therefore needed. Lesions or high frequency stimulation of the hyperactive subthalamic nucleus (STN) in Parkinson's disease (PD), alleviate the motor symptoms and reduce dyskinesia, either directly and/or by allowing the reduction of the L-DOPA dose. N-methyl-D-aspartate (NMDA) receptor antagonists might have similar actions. However it remains elusive how the neurochemistry changes in the STN after a separate or combined administration of L-DOPA and a NMDA receptor antagonist. By means of in vivo microdialysis, the effect of L-DOPA and/or MK 801, on the extracellular dopamine (DA) and glutamate (GLU) levels was investigated for the first time in the STN of sham and 6-hydroxydopamine-lesioned rats. The L-DOPA-induced DA increase in the STN was significantly higher in DA-depleted rats compared to shams. MK 801 did not influence the L-DOPA-induced DA release in shams. However, MK 801 enhanced the L-DOPA-induced DA release in hemi-parkinson rats. Interestingly, the extracellular STN GLU levels remained unchanged after nigral degeneration. Furthermore, administration of MK 801 alone or combined with L-DOPA did not alter the STN GLU levels in both sham and DA-depleted rats. The present study does not support the hypothesis that DA-ergic degeneration influences the STN GLU levels neither that MK 801 alters the GLU levels in lesioned and non-lesioned rats. However, NMDA receptor antagonists could be used as a beneficial adjuvant treatment for PD by enhancing the therapeutic efficacy of l-DOPA at least in part in the STN.

  19. Unmasking the Effects of L-DOPA on Rapid Dopamine Signaling with an Improved Approach for Nafion Coating Carbon-Fiber Microelectrodes.

    Qi, Lingjiao; Thomas, Elina; White, Stephanie H; Smith, Samantha K; Lee, Christie A; Wilson, Leslie R; Sombers, Leslie A


    L-DOPA has been the gold standard for symptomatic treatment of Parkinson's disease. However, its efficacy wanes over time as motor complications develop. Very little is known about how L-DOPA therapy affects the dynamics of fluctuating dopamine concentrations in the striatum on a rapid time scale (seconds). Electrochemical studies investigating the effects of L-DOPA treatment on electrically evoked dopamine release have reported conflicting results with significant variability. We hypothesize that the uncertainty in the electrochemical data is largely due to electrode fouling caused by polymerization of L-DOPA and endogenous catecholamines on the electrode surface. Thus, we have systematically optimized the procedure for fabricating cylindrical, Nafion-coated, carbon-fiber microelectrodes. This has enabled rapid and reliable detection of L-DOPA's effects on striatal dopamine signaling in intact rat brain using fast-scan cyclic voltammetry. An acute dose of 5 mg/kg L-DOPA had no significant effect on dopamine dynamics, demonstrating the highly efficient regulatory mechanisms at work in the intact brain. In contrast, administration of 200 mg/kg L-DOPA significantly increased the amplitude of evoked dopamine release by ∼200%. Overall, this work describes a reliable tool that allows a better measure of L-DOPA augmented dopamine release in vivo, measured using fast-scan cyclic voltammetry. It provides a methodology that improves the stability and performance of the carbon-fiber microelectrode when studying the molecular mechanisms underlying L-DOPA therapy and also promises to benefit a wide variety of studies because Nafion is so commonly used in electroanalytical chemistry.

  20. Cerebrospinal fluid levels of catecholamines and its metabolites in Parkinson's disease: Effect of L-DOPA treatment and changes in levodopa-induced dyskinesia.

    Andersen, A D; Blaabjerg, M; Binzer, M; Kamal, A; Thagesen, H; Kjaer, T W; Stenager, E; Gramsbergen, J B


    Levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) is the most effective drug in the symptomatic treatment of Parkinson's disease (PD), but chronic use initiates a maladaptive process leading to L-DOPA-induced dyskinesia (LID). Risk factors for early onset LID include younger age, more severe disease at baseline and higher daily L-DOPA dose, but biomarkers to predict the risk of motor complications are not yet available. Here we investigated whether CSF levels of catecholamines and its metabolites are altered in PD patients with LID (PD-LID, n=8)) as compared to non-dyskinetic PD patients receiving L-DOPA (PD-L, n=6), or not receiving L-DOPA (PD-N, n=7) as well as non-PD controls (n=16). PD patients were clinically assessed using the Unified Parkinson's Disease Rating Scale and Unified Dyskinesia Rating Scale and CSF was collected after overnight fasting and 1-2 hours after oral intake of L-DOPA or other anti-Parkinson medication. CSF catecholamines and its metabolites were analyzed by HPLC with electrochemical detection. We observed (1) decreased levels of dihydroxyphenylacetic acid and homovanillic acid in PD patients not receiving L-DOPA (2) higher DA levels in LID as compared to controls (3) higher DA/L-DOPA and lower DOPAC/DA ratio's in LID as compared to PDL and (4) an age-dependent increase of DA and decrease of DOPAC/DA ratio in controls. These results suggest increased DA release from non-DA cells and deficient DA re-uptake in PD-LID. Monitoring DA and DOPAC in CSF of L-DOPA-treated PD patients may help identify patients at risk of developing LID. This article is protected by copyright. All rights reserved.

  1. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase.

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan


    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  2. Ornithine decarboxylase, mitogen-activated protein kinase and matrix metalloproteinase-2 expressions in human colon tumors

    Takahiro Nemoto; Shunichiro Kubota; Hideyuki Ishida; Nobuo Murata; Daijo Hashimoto


    AIM: To investigate the expressions of omithine decarboxylase (ODC), MMP-2, and Erk, and their relationship in human colon tumors.METHODS: ODC activity, MMP-2 expression, and mitogenactivated protein (MAP) kinase activity (Erk phosphorylation) were determined in 58 surgically removed human colon tumors and their adjacent normal tissues, using [1-14C]-ornithine as a substrate, ELISA assay, and Western blotting, respectively.RESULTS: ODC activity, MMP-2 expression, and Erk phosphorylation were significantly elevated in colon tumors, compared to those in adjacent normal tissues. A significant correlation was observed between ODC activities and MMP-2 levels.CONCLUSION: This is the first report showing a significant correlation between ODC activities and MMP-2 levels in human colon tumors. As MMP-2 is involved in cancer invasion and metastasis, and colon cancer overexpresses ODC, suppression of ODC expression may be a rational approach to treat colon cancer which overexpresses ODC.

  3. Impact of selenium, iron, copper and zinc in on/off Parkinson's patients on L-dopa therapy.

    Qureshi, G A; Qureshi, A A; Memon, S A; Parvez, S H


    We have quantitated CSF and serum levels of Selenium, iron, copper and zinc by Atomic absorption spectrophotometer in 36 patients with parkinson's disease all on L-dopa therapy. Out of these 19 showed on or positive response to L-dopa where as 21 patients showed on and off response. These data were compared with 21 healthy controls. The results showed that serum levels of iron, copper and zinc remained unchanged where as in CSF, significant decrease in zinc was found in both on and on/off PD patients indicating the deficiency of zinc which continues in the worsening clinical condition of off patients. The level of copper remained unchanged in both on and on/off PD patients. Iron and selenium increase in CSF of both patients which is a clear evidence of relationship between increased iron and selenium level in brain which could be correlated with decrease in dopamine levels and oxidative stress in PD Patients.

  4. A dual-tracer study of extrastriatal 6-[18F]fluoro-m-tyrosine and 6-[18F]-Fluoro-L-dopa uptake in Parkinson's disease

    Li, Clarence; Palotti, Matthew; Holden, James E.; Oh, Jen; Okonkwo, Ozioma; Christian, Bradley T.; Bendlin, Barbara B.; Buyan-Dent, Laura; Harding, Sandra J.; Stone, Charles K.; Dejesus, Onofre T.; Nickles, Robert J.; Gallagher, Catherine L


    6-[18F]-Fluoro-L-dopa (FDOPA) has been widely used as a biomarker for catecholamine synthesis, storage, and metabolism—its intense uptake in the striatum, and fainter uptake in other brain regions, is correlated with the symptoms and pathophysiology of Parkinson's disease (PD). 6-[18F]fluoro-m-tyrosine (FMT), which also targets L-amino acid decarboxylase, has potential advantages over FDOPA as a radiotracer because it does not form catechol-O-methyltransferase (COMT) metabolites. The purpose of the present study was to compare the regional distribution of these radiotracers in the brains of PD patients. 15 Parkinson's patients were studied with FMT and FDOPA positron emission tomography (PET) as well as high-resolution structural magnetic resonance imaging (MRI). MRI's were automatically parcellated into neuroanatomical regions of interest (ROIs) in Freesurfer (; region-specific uptake rate constants (Kocc) were generated from coregistered PET using a Patlak graphical approach. The essential findings were as follows: (1) regional Kocc were highly correlated between the radiotracers and in agreement with a previous FDOPA studies that used different ROI selection techniques; (2) FMT Kocc were higher in extrastriatal regions of relatively large uptake such as amygdala, pallidum, brainstem, hippocampus, entorhinal cortex, and thalamus, whereas cortical Kocc were similar between radiotracers; (3) while subcortical uptake of both radiotracers was related to disease duration and severity, cortical uptake was not. These results suggest that FMT may have advantages for examining pathologic changes within allocortical loop structures, which may contribute to cognitive and emotional symptoms of PD. PMID:24710997

  5. Rational design of ornithine decarboxylase with high catalytic activity for the production of putrescine.

    Choi, Hyang; Kyeong, Hyun-Ho; Choi, Jung Min; Kim, Hak-Sung


    Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes.

  6. Combined treatment with acupuncture reduces effective dose and alleviates adverse effect of l-dopa by normalizing Parkinson’s disease-induced neurochemical imbalance

    Kim, Seung-Nam; Doo, Ah-Reum; Park, Ji-Yeun; Choo, Hyunwoo J.; Shim, Insop; Park, Jongbae J.; Chae, Younbyoung; Lee, Bena; Lee, Hyejung; Park, Hi-Joon


    This study first showed the behavioural benefits of novel combination therapy of l-dopa with acupuncture on Parkinson’s disease, and its underlying mechanisms within basal ganglia. The previous study reported that acupuncture may improve the motor function of a Parkinson’s disease (PD) mouse model by increasing the dopamine efflux and turnover ratio of dopamine. Hence, we hypothesised that combining l-dopa with acupuncture would have a behavioural benefit for those with PD. We performed unila...

  7. Apical and basal uptake of L-dopa and L-5-HTP and their corresponding amines, dopamine and 5-HT, in OK cells.

    Vieira-Coelho, M A; Soares-da-Silva, P


    The present study defined the kinetic characteristics of L-3,4-dihydroxyphenylalanine (L-dopa) and L-5-hydroxytryptophan (L-5-HTP) transport and their decarboxylation products, dopamine and 5-hydroxytryptamine (5-HT), respectively, in OK cells. The apical uptake of both L-dopa and 5-HTP was temperature dependent and stereoselective. Nonlinear analysis of the saturation curves revealed for L-dopa and L-5-HTP Michaelis constants of 13.8 and 29.1 microM, respectively, and maximal velocities of 21 and 29 protein-1.6 min-1, respectively, L-5-HTP inhibited the apical uptake of L-dopa with a half-maximal inhibitory constant of 29.1 microM. The accumulation of L-dopa and L-5-HTP from the basolateral cell border was dependent on the concentration used and saturable at nearly 50 microM. The accumulation of dopamine and 5-HT was found to be saturable only when the substrates were applied from the basolateral cell border. These results demonstrate that the L-dopa and L-5-HTP transporters in OK cells are located in both the apical and basolateral cell borders, whereas the uptake of dopamine and 5-HT is a saturable process only when the substrates are applied from the basolateral cell border.

  8. Augmentation by L-Dopa of growth inhibition and melanin formation of X-irradiated Harding-Passey melanoma cells in culture

    Schachtschabel, D.O.; Pfab, R.; Hess, F.; Paul, N.


    Treatment of exponentially proliferating melanogenic Harding-Passey melanoma cells in monolayer culture (HPM-73 line) with a single dose of X-irradiation (up to 8 Gy) or continuously (for several weeks) with L-3,4-dihydroxyphenylalanine (L-Dopa) up to 5x10/sup -4/ M resulted in a dose-dependent inhibition of cell proliferation, but not in death of all cells. Actually, 8 Gy-irradiated or L-Dopa (2x10/sup -4/ M)-treated cultures finally reached the cell number and cell density of controls. However, a combination of a single dose of radiation (8 Gy) followed by L-Dopa (2x10/sup -4/ M)-treatment resulted in destruction of all cells. Melanin formation was stimulated by L-dopa-treatment or X-irradiation, and was further elevated by the combined application of radiation and L-Dopa-exposure. Whether the effects of exogenously applied L-Dopa, an intermediary metabolite of melanin synthesis, are due to the conversion to growth-inhibitory metabolites (quinones, radicals, etc.) inside or outside the cell, was discussed. The latter might result from release (due to membrane damage or cell desintegration) of tyrosinase or/and melanosomes into the culture medium with the consequence of extracellular synthesis of potentially cytotoxic metabolites from medium substrates. Further, endocytosis of exogenous melanosomes and tyrosinase with potentially harmful effects is feasible. An application of such a combination therapy of melanoma to clinical medicine should be considered.

  9. Structural characterization of the mechanism through which human glutamic acid decarboxylase auto-activates

    Langendorf, Christopher G.; Tuck, Kellie L.; Key, Trevor L. G.; Fenalti, Gustavo; Pike, Robert N.; Rosado, Carlos J.; Wong, Anders S. M.; Buckle, Ashley M.; Law, Ruby H. P.; Whisstock, James C.


    Imbalances in GABA (γ-aminobutyric acid) homoeostasis underlie psychiatric and movement disorders. The ability of the 65 kDa isoform of GAD (glutamic acid decarboxylase), GAD65, to control synaptic GABA levels is influenced through its capacity to auto-inactivate. In contrast, the GAD67 isoform is constitutively active. Previous structural insights suggest that flexibility in the GAD65 catalytic loop drives enzyme inactivation. To test this idea, we constructed a panel of GAD65/67 chimaeras and compared the ability of these molecules to auto-inactivate. Together, our data reveal the important finding that the C-terminal domain of GAD plays a key role in controlling GAD65 auto-inactivation. In support of these findings, we determined the X-ray crystal structure of a GAD65/67 chimaera that reveals that the conformation of the catalytic loop is intimately linked to the C-terminal domain. PMID:23126365

  10. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C


    Uroporphyrinogen decarboxylase (URO-D; EC, the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  11. Influence of chronic L-DOPA treatment on immune response following allogeneic and xenogeneic graft in a rat model of Parkinson's disease.

    Breger, Ludivine S; Kienle, Korbinian; Smith, Gaynor A; Dunnett, Stephen B; Lane, Emma L


    Although intrastriatal transplantation of fetal cells for the treatment of Parkinson's disease had shown encouraging results in initial open-label clinical trials, subsequent double-blind studies reported more debatable outcomes. These studies highlighted the need for greater preclinical analysis of the parameters that may influence the success of cell therapy. While much of this has focused on the cells and location of the transplants, few have attempted to replicate potentially critical patient centered factors. Of particular relevance is that patients will be under continued L-DOPA treatment prior to and following transplantation, and that typically the grafts will not be immunologically compatible with the host. The aim of this study was therefore to determine the effect of chronic L-DOPA administered during different phases of the transplantation process on the survival and function of grafts with differing degrees of immunological compatibility. To that end, unilaterally 6-OHDA lesioned rats received sham surgery, allogeneic or xenogeneic transplants, while being treated with L-DOPA before and/or after transplantation. Irrespective of the L-DOPA treatment, dopaminergic grafts improved function and reduced the onset of L-DOPA induced dyskinesia. Importantly, although L-DOPA administered post transplantation was found to have no detrimental effect on graft survival, it did significantly promote the immune response around xenogeneic transplants, despite the administration of immunosuppressive treatment (cyclosporine). This study is the first to systematically examine the effect of L-DOPA on graft tolerance, which is dependent on the donor-host compatibility. These findings emphasize the importance of using animal models that adequately represent the patient paradigm.

  12. Characterization of the activity and expression of arginine decarboxylase in human and animal Chlamydia pathogens.

    Bliven, Kimberly A; Fisher, Derek J; Maurelli, Anthony T


    Chlamydia pneumoniae encodes a functional arginine decarboxylase (ArgDC), AaxB, that activates upon self-cleavage and converts l-arginine to agmatine. In contrast, most Chlamydia trachomatis serovars carry a missense or nonsense mutation in aaxB abrogating activity. The G115R missense mutation was not predicted to impact AaxB functionality, making it unclear whether AaxB variations in other Chlamydia species also result in enzyme inactivation. To address the impact of gene polymorphism on functionality, we investigated the activity and production of the Chlamydia AaxB variants. Because ArgDC plays a critical role in the Escherichia coli acid stress response, we studied the ability of these Chlamydia variants to complement an E. coli ArgDC mutant in an acid shock assay. Active AaxB was detected in four additional species: Chlamydia caviae, Chlamydia pecorum, Chlamydia psittaci, and Chlamydia muridarum. Of the C. trachomatis serovars, only E appears to encode active enzyme. To determine when functional enzyme is present during the chlamydial developmental cycle, we utilized an anti-AaxB antibody to detect both uncleaved and cleaved enzyme throughout infection. Uncleaved enzyme production peaked around 20 h postinfection, with optimal cleavage around 44 h. While the role ArgDC plays in Chlamydia survival or virulence is unclear, our data suggest a niche-specific function.


    Vijayambika C; Jegadeesan M; Ganthi A. Saravana


    Mucuna pruriens (L.) DC. (Papilionaceae) is used in male impotency, as aphrodisiac, in sexual debility, and as nervine tonic. It also possesses anti-parkinson property, possibly due to the presence of L-DOPA. Market samples of ‘Atmagupta’ (Mucuna pruriens (L.) DC) an Indian Ayurvedic and Siddha drug contain seeds of seven taxa. The dried powders of commercial and genuine samples were analysed for anti-nutritional factors and L–DOPA content. Raw M. deeringiana seeds were rich in Tannin, Hydro...

  14. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe


    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

  15. LC-MSdetermination of L-DOPA concentration in the leaf and flower tissues of six faba bean (Vicia fabaL. lines with common and rare flowercolors

    Jinguo Hu


    Full Text Available Background: Parkinson’s disease (PD is the second most common neurodegenerative disorder characterized by the loss of muscle control, which causes trembling of the limbs and head as well as impaired balance. L-DOPA (L-3,4-dihydroxy phenylalanine is the major ingredient of several prescription drugs used to treat PD. Faba bean (Vicia faba L. is one of the few plant species that is known to produce L-DOPA and has the potential to be developed as a functional food crop for people suffering with PD. Objective: Aimed to provide needed information for people who want to use faba bean as a natural remedy or functional food to relieve PD symptoms, this study analyzed the variation of L-DOPA concentration in the leaf and flower tissues of six faba bean lines with common and rare flower colors. Methods: Leaf and flower samples were taken from field grown plants with different flower colors, namely, pink with purple lines and black dots, pure white, brown, and crimson. Samples were freeze-dried and L-DOPA was quantified by a LC-MS system consisting of an ACQUITY UPLC in line with a Synapt G2 HDMS quadrupole time-of-flight mass spectrometer. This experiment was carried out in two consecutive years (2012 and 2013 and the plants used in the second year were grown from the seeds harvested from the plants used in the first year. Results and Discussion: Our two-year study revealed a high level of variation in L-DOPA concentration for leaf and flower tissues among the six faba bean lines studied. The average L-DOPA concentration based on dry weight (DW in flowers ranged from 27.8 to 63.5 mg/g and 18.2 to 48.7 mg/g for leaf tissues. There was no significant correlation between L-DOPA concentrations in flowers and leaves. The L-DOPA concentration in flowers and in leaves of the same line varied but were not statistically significant between the two years. Ideally, the genotype with the highest average L-DOPA concentration in both flowers and leaves would be grown

  16. Autoradiographic studies on distribution of L-3,4-dihydroxyphenylalanine (L-DOPA)-14C and L-5-hydroxytryptophan (L-5-HTP)-14C in the cat brain.

    Miyakoshi, N; Tanaka, M; Shindo, H


    The distribution and metabolism of L-DOPA-14C and L-5-HTP-14C in the cat brain were examined by means of autoradiography and chromatography. The results revealed that an appreciable amount of radioactivity in the gray matter, but not the white, and that the localization of radioactivity of L-DOPA and L-5-HTP significantly differed. After L-DOPA-14C administration, a high accumulation was found in the caudate nucleus, putamen and pallidum. With L-5-HTP-14C administration, high radioactivity was observed in the hypothalamus, raphe nucleus, substantia nigra, inferior olivalis and caudate nucleus. An analysis of the main metabolites of both amino acids in various regions of the brain was also made. When L-DOPA was given, a high concentration of dopamine was detected in the caudate nucleus, followed by the hypothalamus. In the case of L-5-HTP, a high concentration of serotonin was detected in the hypothalamus and the medulla oblongata. These results suggest that amines derived from exogenously administered L-DOPA and L-5-HTP accumulate in the brain regions known as the corresponding amine rich regions, under physiological conditions.

  17. The H3 receptor agonist immepip does not affect l-dopa-induced abnormal involuntary movements in 6-OHDA-lesioned rats.

    Papathanou, Maria; Jenner, Peter; Iravani, Mahmoud; Jackson, Michael; Stockwell, Kim; Strang, Isabel; Zeng, Bai-Yun; McCreary, Andrew C; Rose, Sarah


    The treatment of dyskinesia in Parkinson׳s disease remains poor but H3 receptor agonists have been suggested as a novel pharmacological approach. We examined the effects of the H3 agonist, immepip, in 6-OHDA-lesioned rats exhibiting AIMs (abnormal involuntary movements), a rat analogue of dyskinesia, in response to l-dopa compared to the known anti-dyskinetic agents amantadine, MK-801 and 8-OHDPAT. We then attempted to extend these studies in to dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated common marmosets. Amantadine, MK-801 and 8-OHDPAT all dose-dependently reduced l-dopa-induced axial, lingual and oral (ALO) AIMs in 6-OHDA-lesioned animals accompanied by a reduction in contralateral rotation with higher doses of amantadine and MK-801. By contrast, immepip had no effect on AIMs expression or contralateral rotation. In the MPTP-treated common marmoset exhibiting dyskinesia to l-dopa, immepip alone induced retching and in combination with l-dopa administered subcutaneously or orally induced the rapid onset of retching and vomiting which was not controlled by pretreatment with domperidone. Administration of the unrelated H3 agonist, imetit had the same effect. Despite causing negative side-effects, it appears that both agonists reduced the antiparkinsonian response to l-dopa resulting in reduced dyskinesia. H3 agonists appear unlikely candidates for the treatment of dyskinesia in PD based on lack of evidence of efficacy and potential adverse effects.

  18. Enzyme Architecture: The Activating Oxydianion Binding Domain for Orotidine 5′-Monophophate Decarboxylase

    Spong, Krisztina; Amyes, Tina L.; Richard, John P.


    Orotidine 5′-monophosphate decarboxylase catalyzes the decarboxylation of truncated substrate (1-β-D-erythrofuranosyl)orotic acid (EO) to form (1-β-D-erythrofuranosyl)uracil (EU). This enzymecatalyzed reaction is activated by tetrahedral oxydianions, which bind weakly to unliganded OMPDC and tightly to the enzyme-transition state complex, with the following intrinsic oxydianion binding energies (kcal/mole): SO32−, −8.3; HPO32−, −7.7; S2O32−, −4.6; SO42−, −4.5; HOPO32−, −3.0; HOAsO32−, no activation detected. We propose that oxydianion and orotate binding domains perform complementary functions in catalysis of decarboxylation reactions. (1) The orotate binding domain carries out decarboxylation of the orotate ring. (2) The activating oxydianion binding domain has the cryptic function of utilizing binding interactions with tetrahedral inorganic oxydianions to drive an enzyme conformational change that results in the stabilization of transition states at the distant orotate domain. PMID:24274746

  19. Enzyme architecture: the activating oxydianion binding domain for orotidine 5'-monophophate decarboxylase.

    Spong, Krisztina; Amyes, Tina L; Richard, John P


    Orotidine 5'-monophosphate decarboxylase catalyzes the decarboxylation of truncated substrate (1-β-D-erythrofuranosyl)orotic acid to form (1-β-D-erythrofuranosyl)uracil. This enzyme-catalyzed reaction is activated by tetrahedral oxydianions, which bind weakly to unliganded OMPDC and tightly to the enzyme-transition state complex, with the following intrinsic oxydianion binding energies (kcal/mol): SO3(2-), -8.3; HPO3(2-), -7.7; S2O3(2-), -4.6; SO4(2-), -4.5; HOPO3(2-), -3.0; HOAsO3(2-), no activation detected. We propose that the oxydianion and orotate binding domains of OMPDC perform complementary functions in catalysis of decarboxylation reactions: (1) The orotate binding domain carries out decarboxylation of the orotate ring. (2) The activating oxydianion binding domain has the cryptic function of utilizing binding interactions with tetrahedral inorganic oxydianions to drive an enzyme conformational change that results in the stabilization of transition states at the distant orotate domain.

  20. Measurement of activity for S-adenosylmethionine decarboxylase using radioisotope {sup 14}C

    Ko, Kyong Cheol; Park, Sang Hyun [Radiation Research Center for Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Kamio, Yoshiyuku [Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University (Japan)


    Polyamines are essential for normal cell growth and have important physiological function. They are polycationic compounds that are present in all biological materials. Also, they have been implicated in a wide variety of biological reactions. Generally, putrescine and spermidine are contained high amount in prokaryote, but spermidine and spermine are in eukaryote, respectively. However, S. ruminantium cells contain the polyamins such as spermidine and spermine. Addition of an aminopropyl group to putrescine conducts to the synthesis of spermidine. Aminopropyl group is derived from the dcSAM, a decarboxylation of S-adenosylmethionine, through action of S-adenosylmethionine decarboxylase (SAMDC). We suggested that S. ruminantium has a different pathway compare with prokaryote for polyamine synthesis. Assay for SAMDC activity was used {sup 14}C labeled substrate. Key enzyme in the biosynthesis of polyamines, SAMDC, was purified from S. ruminantium and characterized. The enzyme was purified about 1,259-fold to electrophoretic homogeneity with a specific activity of 1.89×10{sup -5} kat kg'-{sup 1} of protein.

  1. Singlet oxygen generation during the oxidation of L-tyrosine and L-dopa with mushroom tyrosinase

    Miyaji, Akimitsu [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan); Kohno, Masahiro [Department of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-G1-25 Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan); Inoue, Yoshihiro [Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida, Tokyo 194-8543 (Japan); Baba, Toshihide, E-mail: [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan)


    The generation of singlet oxygen during the oxidation of tyrosine and L-dopa using mushroom tyrosinase in a phosphate buffer (pH 7.4), the model of melanin synthesis in melanocytes, was examined. The reaction was performed in the presence of 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen and the electron spin resonance (ESR) of the spin adduct, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), was measured. An increase in the ESR signal attributable to 4-oxo-TEMPO was observed during the oxidation of tyrosine and L-dopa with tyrosinase, indicating the generation of singlet oxygen. The results suggest that {sup 1}O{sub 2} generation via tyrosinase-catalyzed melanin synthesis occurs in melanocyte. - Highlights: • Generation of singlet oxygen was observed during tyrosinase-catalyzed tyrosine oxidation. • The singlet oxygen generated when tyrosine was converted into dopachrome. • The amount of singlet oxygen is not sufficient for cell toxicity. • It decreased when the hydroxyl radicals and/or superoxide anions were trapped.

  2. Facile fouling resistant surface modification of microfiltration cellulose acetate membranes by using amino acid L-DOPA.

    Azari, Sara; Zou, Linda; Cornelissen, Emile; Mukai, Yasushito


    A major obstacle in the widespread application of microfiltration membranes in the wet separation processes such as wastewater treatment is the decline of permeates flux as a result of fouling. This study reports on the surface modification of cellulose acetate (CA) microfiltration membrane with amino acid L-3,4-dihydroxy-phenylalanine (L-DOPA) to improve fouling resistance of the membrane. The membrane surface was characterised using Fourier transform infrared spectroscopy (FTIR), water contact angle and zeta potential measurement. Porosity measurement showed a slight decrease in membrane porosity due to coating. Static adsorption experiments revealed an improved resistance of the modified membranes towards the adhesion of bovine serum albumin (BSA) as the model foulant. Dead end membrane filtration tests exhibited that the fouling resistance of the modified membranes was improved. However, the effect of the modification depended on the foulant solution concentration. It is concluded that L-DOPA modification is a convenient and non-destructive approach to enable low-BSA adhesion surface modification of CA microfiltration membranes. Nevertheless, the extent of fouling resistance improvement depends on the foulant concentration.

  3. Highly active and stable oxaloacetate decarboxylase Na⁺ pump complex for structural analysis.

    Inoue, Michio; Li, Xiaodan


    The oxaloacetate decarboxylase primary Na(+) pump (Oad) produces energy for the surviving of some pathogenic bacteria under anaerobic conditions. Oad composes of three subunits: Oad-α, a biotinylated soluble subunit and catalyzes the decarboxylation of oxaloacetate; Oad-β, a transmembrane subunit and functions as a Na(+) pump; and Oad-γ, a single transmembrane α-helical anchor subunit and assembles Oad-α/β/γ complex. The molecular mechanism of Oad complex coupling the exothermic decarboxylation to generate the Na(+) electrochemical gradient remains unsolved. Our biophysical and biochemical studies suggested that the stoichiometry of Oad complex from Vibrio cholerae composed of α, β, γ in 4:2:2 stoichiometry not that of 4:4:4. The high-resolution structure determination of the Oad complex would reveal the energetic transformation mechanism from the catalytical soluble α subunit to membrane β subunit. Sufficient amount stable, conformational homogenous and active Oad complex with the right stoichiometry is the prerequisite for structural analysis. Here we report an easy and reproducible protocol to obtain high quantity and quality Oad complex protein for structural analysis.

  4. Substrate activation of brewers' yeast pyruvate decarboxylase is abolished by mutation of cysteine 221 to serine.

    Baburina, I; Gao, Y; Hu, Z; Jordan, F; Hohmann, S; Furey, W


    Brewers' yeast pyruvate decarboxylase (EC, a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Structural Basis for Putrescine Activation of Human S-Adenosylmethionine Decarboxylase

    Bale, Shridhar; Lopez, Maria M.; Makhatadze, George I.; Fang, Qingming; Pegg, Anthony E.; Ealick, Steven E. (Cornell); (Penn)


    Putrescine (1,4-diaminobutane) activates the autoprocessing and decarboxylation reactions of human S-adenosylmethionine decarboxylase (AdoMetDC), a critical enzyme in the polyamine biosynthetic pathway. In human AdoMetDC, putrescine binds in a buried pocket containing acidic residues Asp174, Glu178, and Glu256. The pocket is away from the active site but near the dimer interface; however, a series of hydrophilic residues connect the putrescine binding site and the active site. Mutation of these acidic residues modulates the effects of putrescine. D174N, E178Q, and E256Q mutants were expressed and dialyzed to remove putrescine and studied biochemically using X-ray crystallography, UV-CD spectroscopy, analytical ultracentrifugation, and ITC binding studies. The results show that the binding of putrescine to the wild type dimeric protein is cooperative. The D174N mutant does not bind putrescine, and the E178Q and E256Q mutants bind putrescine weakly with no cooperativity. The crystal structure of the mutants with and without putrescine and their complexes with S-adenosylmethionine methyl ester were obtained. Binding of putrescine results in a reorganization of four aromatic residues (Phe285, Phe315, Tyr318, and Phe320) and a conformational change in the loop 312-320. The loop shields putrescine from the external solvent, enhancing its electrostatic and hydrogen bonding effects. The E256Q mutant with putrescine added shows an alternate conformation of His243, Glu11, Lys80, and Ser229, the residues that link the active site and the putrescine binding site, suggesting that putrescine activates the enzyme through electrostatic effects and acts as a switch to correctly orient key catalytic residues.

  6. The added value of [{sup 18}F]fluoro-L-DOPA PET in the diagnosis of hyperinsulinism of infancy: a retrospective study involving 49 children

    Ribeiro, Maria-Joao; Bourgeois, Sandrine; Delzescaux, Thierry [Frederic Joliot Hospital, Biomedical Imaging Institute, Life Sciences Division, CEA, Orsay (France); Boddaert, Nathalie; Brunelle, Francis [Necker-Enfants Malades Hospital, AP-HP, Department of Radiology, Paris (France); Bellanne-Chantelot, Christine [Saint-Antoine Hospital, AP-HP, Department of Cytogenetics, Paris (France); Valayannopoulos, Vassili; Lonlay, Pascale de [Necker-Enfants Malades Hospital, AP-HP, Department of Pediatrics, Paris (France); Jaubert, Francis [Necker-Enfants Malades Hospital, AP-HP, Department of Pathology, Paris (France); Nihoul-Fekete, Claire [Necker-Enfants Malades Hospital, AP-HP, Department of Surgery, Paris (France)


    Neuroendocrine diseases are a heterogeneous group of entities with the ability to take up amine precursors, such as L-DOPA, and convert them into biogenic amines, such as dopamine. Congenital hyperinsulinism of infancy (HI) is a neuroendocrine disease secondary to either focal adenomatous hyperplasia or a diffuse abnormal pancreatic insulin secretion. While focal hyperinsulinism may be reversed by selective surgical resection, diffuse forms require near-total pancreatectomy when resistant to medical treatment. Here, we report the diagnostic value of PET with [{sup 18}F]fluoro-L-DOPA in distinguishing focal from diffuse HI. Forty-nine children were studied with [{sup 18}F]fluoro-L-DOPA. A thoraco-abdominal scan was acquired 45-65 min after the injection of 4.2 {+-} 1.0 MBq/kg of [{sup 18}F]fluoro-L-DOPA. Additionally, 12 of the 49 children were submitted to pancreatic venous catheterisation for blood samples (PVS) and 31 were also investigated using MRI. We identified abnormal focal pancreatic uptake of [{sup 18}F]fluoro-L-DOPA in 15 children, whereas diffuse radiotracer uptake was observed in the pancreatic area in the other 34 patients. In children studied with both PET and PVS, the results were concordant in 11/12 cases. All patients with focal radiotracer uptake and nine of the patients with diffuse pancreatic radiotracer accumulation, unresponsive to medical treatment, were submitted to surgery. In 21 of these 24 patients, the histopathological results confirmed the PET findings. In focal forms, selective surgery was followed by clinical remission without carbohydrate intolerance. These data demonstrate that PET with [{sup 18}F]fluoro-L-DOPA is an accurate non-invasive technique allowing differential diagnosis between focal and diffuse forms of HI. (orig.)

  7. Aspergillus niger PA2 Tyrosinase Covalently Immobilized on a Novel Eco-Friendly Bio-Composite of Chitosan-Gelatin and Its Evaluation for L-DOPA Production

    Agarwal, Pragati; Dubey, Swati; Singh, Mukta; Singh, Rajesh P.


    Tyrosinase (EC a copper-containing monooxygenase, isolated from a fungal isolate Aspergillus niger PA2 was subjected for immobilization onto a composite consisting of chitosan and gelatin biopolymers. The homogeneity of the chitosan-gelatin biocomposite film was characterized by X-ray diffraction analyses. To evaluate immobilization efficiency, chitosan-gelatin-Tyr bio-composite films were analyzed by field emission scanning electron microscopy, atomic force microscopy and UV-spectroscopy. The rough morphology of the film led to a high loading of enzyme and it could retain its bioactivity for a longer period. The enzyme adsorbed onto the film exhibited 72% of its activity after 10 days and exhibited good repeatability for up to nine times, after intermittent storage. Moreover, the immobilized enzyme exhibited broader pH and temperature profile as compared to free counterpart. Immobilized enzyme was further evaluated for the synthesis of L-DOPA (2,4-dihydroxy phenylalanine) which is a precursor of dopamine and a potent drug for the treatment of Parkinson's disease and for myocardium neurogenic injury. PMID:28066399

  8. Aromatic L-amino acid decarboxylase enzyme activity in deficient patients and heterozygotes.

    Verbeek, M.M.; Geurtz, P.B.H.; Willemsen, M.A.A.P.; Wevers, R.A.


    BACKGROUND: Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disorder characterised by developmental delay, motor retardation and autonomic dysfunction. Very low concentrations in cerebrospinal fluid (CSF) of homovanillic acid (HVA) and 5-hydroxy indole acetic acid

  9. L-dopa methyl ester attenuates amblyopia-induced neuronal injury in visual cortex of amblyopic cat.

    Li, Rong; Liang, Tao; Chen, Zhaoni; Zhang, Shijun; Lin, Xing; Huang, Renbin


    In the present study, we aimed to assess the potential anti-amblyopic effects of L-dopa methyl ester (LDME) on visual cortex area 17 in an amblyopic feline model induced by monocular vision deprivation. After LDME administration, pathophysiologic and ultrastructural observations were utilized to examine the morphological changes of nerve cells in visual cortex area 17. Dopamine (DA) and its metabolite contents in visual cortex area 17 were investigated through HPLC analysis. Apoptotic cells in visual cortex area 17 were evaluated by TUNEL assay. Additionally, the c-fos expression both at gene and protein levels was assessed using RT-PCR and immunohistochemistry analyses, respectively. The contents of DA and its metabolites were elevated in visual cortex area 17. Neuronal rejuvenation which occurred in visual cortex area 17 was observed through anatomical and physiological assessments. Similarly, TUNEL results showed that neuronal apoptosis was inhibited in the visual cortex of amblyopic cats by both L-dopa and LDME therapies. Meanwhile, the c-fos expression was notably up-regulated at both the mRNA and protein levels by the treatments. These findings suggested that LDME treatment could effectively increase DA and its metabolite contents, and restrain the apoptotic process, as well as elevate the c-fos expression in nerve cells of visual cortex area 17. Taken together, LDME might ameliorate the functional cytoarchitecture in visual cortex area 17 through mechanisms that elevate DA content and increase endogenous c-fos expression, as well as inhibit neuronal lesion in visual cortex tissue.

  10. [Hemodynamic effects of one administration of l-dopa in patients with left ventricular heart failure (introductory remarks)].

    Leszek, P; Zieliński, T; Korewicki, J


    The study group consisted of 8 men with congestive heart failure. They ranged in age from 33 do 63 years (mean 48). Three patients were in class III NYHA, five in class IV. Idiopathic cardiomyopathy was diagnosed in 6 patients, one individual displayed ischemic heart disease and one patient was after mitral and aortic-valve replacement despite a normally functioning prosthetic valve. L-dopa was given orally beginning with 250 mg every six hours until a total daily dose of 4.0 g was achieved with no side effects (patients additionally received 50 mg of pyridoxine hydrochloride). Afterwards L-dopa was withheld for 24-36 hours. Having completed this washout period, patients underwent right heart catheterization, with placement of a balloon-tipped thermodilution catheter in a pulmonary artery, so that balloon inflation allowed recording of the pulmonary capillary wedge pressure. Right atrial and pulmonary arterial pressures were monitored continuously. Systemic arterial pressure was measured by mercury manometer. Cardiac output was determined by the thermodilution technique. Rest and effort hemodynamic measurements were repeated before and one, three hours after administration of 1-dopa. The base-line hemodynamic values were consistent with the clinical presentation of severe congestive heart failure. The average cardiac index (CI-1/min/m2--rest--1.93; 25 Watt-3.1) and stroke volume index (SVI-m1/m2--rest--22.2; 25 Watt-32.0) were markedly lowered. Left ventricular filling pressure and pulmonary artery pressure were elevated. The systemic vascular resistance was significantly increased (SVR -j.W.--rest--22.7; 25 Watt-14.2). Administration of 1-dopa resulted in the increase in cardiac index and stroke volume index accompanied by a substantial reduction in systemic vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. L-DOPA-treatment in primates disrupts the expression of A(2A) adenosine-CB(1) cannabinoid-D(2) dopamine receptor heteromers in the caudate nucleus.

    Bonaventura, Jordi; Rico, Alberto J; Moreno, Estefanía; Sierra, Salvador; Sánchez, Marta; Luquin, Natasha; Farré, Daniel; Müller, Christa E; Martínez-Pinilla, Eva; Cortés, Antoni; Mallol, Josefa; Armentero, Marie-Therese; Pinna, Annalisa; Canela, Enric I; Lluís, Carme; McCormick, Peter J; Lanciego, José L; Casadó, Vicent; Franco, Rafael


    The molecular basis of priming for L-DOPA-induced dyskinesias in Parkinson's disease (PD), which depends on the indirect pathway of motor control, is not known. In rodents, the indirect pathway contains striatopallidal GABAergic neurons that express heterotrimers composed of A(2A) adenosine, CB(1) cannabinoid and D(2) dopamine receptors that regulate dopaminergic neurotransmission. The present study was designed to investigate the expression of these heteromers in the striatum of a primate model of Parkinson's disease and to determine whether their expression and pharmacological properties are altered upon L-DOPA treatment. By using the recently developed in situ proximity ligation assay and by identification of a biochemical fingerprint, we discovered a regional distribution of A(2A)/CB(1) /D(2) receptor heteromers that predicts differential D(2)-mediated neurotransmission in the caudate-putamen of Macaca fascicularis. Whereas heteromers were abundant in the caudate nucleus of both naïve and MPTP-treated monkeys, L-DOPA treatment blunted the biochemical fingerprint and led to weak heteromer expression. These findings constitute the first evidence of altered receptor heteromer expression in pathological conditions and suggest that drugs targeting A(2A)-CB(1) -D(2) receptor heteromers may be successful to either normalize basal ganglia output or prevent L-DOPA-induced side effects. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Evaluation of blood-brain barrier transport and CNS drug metabolism in diseased and control brain after intravenous L-DOPA in a unilateral rat model of Parkinson's disease

    Ravenstijn Paulien GM


    Full Text Available Abstract Background Changes in blood-brain barrier (BBB functionality have been implicated in Parkinson's disease. This study aimed to investigate BBB transport of L-DOPA transport in conjunction with its intra-brain conversion, in both control and diseased cerebral hemispheres in the unilateral rat rotenone model of Parkinson's disease. Methods In Lewis rats, at 14 days after unilateral infusion of rotenone into the medial forebrain bundle, L-DOPA was administered intravenously (10, 25 or 50 mg/kg. Serial blood samples and brain striatal microdialysates were analysed for L-DOPA, and the dopamine metabolites DOPAC and HVA. Ex-vivo brain tissue was analyzed for changes in tyrosine hydroxylase staining as a biomarker for Parkinson's disease severity. Data were analysed by population pharmacokinetic analysis (NONMEM to compare BBB transport of L-DOPA in conjunction with the conversion of L-DOPA into DOPAC and HVA, in control and diseased cerebral hemisphere. Results Plasma pharmacokinetics of L-DOPA could be described by a 3-compartmental model. In rotenone responders (71%, no difference in L-DOPA BBB transport was found between diseased and control cerebral hemisphere. However, in the diseased compared with the control side, basal microdialysate levels of DOPAC and HVA were substantially lower, whereas following L-DOPA administration their elimination rates were higher. Conclusions Parkinson's disease-like pathology, indicated by a huge reduction of tyrosine hydroxylase as well as by substantially reduced levels and higher elimination rates of DOPAC and HVA, does not result in changes in BBB transport of L-DOPA. Taking the results of this study and that of previous ones, it can be concluded that changes in BBB functionality are not a specific characteristic of Parkinson's disease, and cannot account for the decreased benefit of L-DOPA at later stages of Parkinson's disease.

  13. Subcellular redistribution of the synapse-associated proteins PSD-95 and SAP97 in animal models of Parkinson's disease and L-DOPA-induced dyskinesia.

    Nash, J E; Johnston, T H; Collingridge, G L; Garner, C C; Brotchie, J M


    Abnormalities in subcellular localization and interaction between receptors and their signaling molecules occur within the striatum in Parkinson's disease (PD) and L-DOPA-induced dyskinesia (LID). Synapse-associated proteins (SAPs), for example, PSD-95 and SAP97 organize the molecular architecture of synapses and regulate interactions between receptors and downstream-signaling molecules. Here, we show that expression and subcellular distribution of PSD-95 and SAP97 are altered in the striatum of unilateral 6-OHDA-lesioned rats following repeated vehicle (a model of PD) or L-DOPA administration (a model of L-DOPA-induced dyskinesia). Furthermore, following dopamine-depletion and development of behavioral deficits in Rotorod performance, indicative of parkinsonism, we observed a dramatic decrease in total striatal levels of PSD-95 and SAP97 (to 25.6 +/- 9.9% and 19.0 +/- 5.0% of control, respectively). The remaining proteins were redistributed from the synapse into vesicular compartments. L-DOPA (6.5mg/kg twice a day, 21 days) induced a rotational response, which became markedly enhanced with repeated treatment (day 1: -15.8+/-7.3 rotations cf day 21: 758.2+/-114.0 rotations). Post L-DOPA treatment, PSD-95 and SAP97 levels increased (367.4 +/- 43.2% and 159.9 +/- 9.5% from control values, respectively), with both being redistributed toward synaptic membranes from vesicular compartments. In situ hybridization showed that changes in total levels of PSD-95, but not SAP97, were accompanied by qualitatively similar changes in mRNA. These data highlight the potential role of abnormalities in the subcellular distribution of SAPs in the pathophysiology of a neurological disease.

  14. Structural basis of enzymatic activity for the ferulic acid decarboxylase (FADase from Enterobacter sp. Px6-4.

    Wen Gu

    Full Text Available Microbial ferulic acid decarboxylase (FADase catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD superfamily. Structural analysis revealed that FADase catalyzed reactions by an "open-closed" mechanism involving a pocket of 8 × 8 × 15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer.

  15. Physiological relation between respiration activity and heterologous expression of selected benzoylformate decarboxylase variants in Escherichia coli

    Pohl Martina


    Full Text Available Abstract Background The benzoylformate decarboxylase (BFD from Pseudomonas putida is a biotechnologically interesting biocatalyst. It catalyses the formation of chiral 2-hydroxy ketones, which are important building blocks for stereoselective syntheses. To optimise the enzyme function often the amino acid composition is modified to improve the performance of the enzyme. So far it was assumed that a relatively small modification of the amino acid composition of a protein does not significantly influence the level of expression or media requirements. To determine, which effects these modifications might have on cultivation and product formation, six different BFD-variants with one or two altered amino acids and the wild type BFD were expressed in Escherichia coli SG13009 pKK233-2. The oxygen transfer rate (OTR as parameter for growth and metabolic activity of the different E. coli clones was monitored on-line in LB, TB and modified PanG mineral medium with the Respiratory Activity MOnitoring System (RAMOS. Results Although the E. coli clones were genetically nearly identical, the kinetics of their metabolic activity surprisingly differed in the standard media applied. Three different types of OTR curves could be distinguished. Whereas the first type (clones expressing Leu476Pro-Ser181Thr or Leu476Pro had typical OTR curves, the second type (clones expressing the wild type BFD, Ser181Thr or His281Ala showed an early drop of OTR in LB and TB medium and a drastically reduced maximum OTR in modified PanG mineral medium. The third type (clone expressing Leu476Gln behaved variable. Depending on the cultivation conditions, its OTR curve was similar to the first or the second type. It was shown, that the kinetics of the metabolic activity of the first type depended on the concentration of thiamine, which is a cofactor of BFD, in the medium. It was demonstrated that the cofactor binding strength of the different BFD-variants correlated with the differences

  16. Value of {sup 18}F-fluoro-L-dopa PET in the Preoperative Localization of Focal Lesions in Congenital Hyperinsulinism

    Capito, C.; Khen-Dunlop, N.; Brunelle, F.; Aigrain, Y.; Cretolle, C.; Jaubert, F.; De Lonlay, P.; Nihoul-Fekete, C. [Necker-Enfants Malades Hospital, AP-HP, 149 rue de Sevres 75015 Paris (France); Ribeiro, M.J. [Biomedical Imaging Institute, Life Sciences Division CEA, Frederic Joliot Hospital, Orsay (France)


    Purpose: To retrospectively compare fluorine 18 ({sup 18}F) fluoro-L-dopa positron emission tomography (PET) and pancreatic venous sampling (PVS) in the preoperative differentiation of diffuse from focal congenital hyperinsulinism (CHI) and localization of focal lesions. Materials and Methods: This study was approved by the institutional ethical committee, and informed consent for the research study was obtained from the parents of all subjects. Fifty-one patients evaluated for focal CHI between January 1, 1995, and January 31, 2008, were included. Thirty five underwent PVS evaluation alone, and 16 underwent a PET evaluation alone. The sensitivity values of each technique for the diagnosis and localization of focal lesions were compared in regard to results of surgery and pathologic analyses. In each patient, perioperative treatment was reviewed, and the presence of postoperative hypoglycemia was assessed as evidence of incomplete resection. Comparisons of the sensitivity values and recurrence rates were performed by using the Fisher exact test in regard to the number of patients. Comparisons of median age, weight, or number of biopsies were performed with a two-tailed unpaired Mann-Whitney U test. A difference with P {<=}.05 was considered significant. Results: For PVS and PET groups, there was no error in differentiating focal from diffuse forms. PVS was not completed in four of 35 patients. In 27 (87%) of 31 patients in whom PVS was completed and 13 (81%) of 16 patients in whom PET was completed, preoperative localization of the focal lesion was in accordance with the surgical findings (P =.7). Although not significant, the number of biopsies performed before discovering the focal lesion was higher in the PET group compared with the PVS group (P .06). Inadequate localization occurred in two (6%) patients in the PVS group and five (31%) patients in the PET group at initial preoperative imaging study; these patients underwent repeat surgery for residual CHI (P

  17. Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA

    Harris Nilangani N


    Full Text Available Abstract Background Carotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding. Results The biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA dioxygenases (DODs, and feeding of DOD substrate (L-DOPA, was sufficient to induce betalain production in cell cultures of Solanum tuberosum (potato and petals of Antirrhinum majus. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from Amanita muscaria or a plant DOD (from Portulaca grandiflora, and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed Arabidopsis thaliana (arabidopsis lines containing 35S: AmDOD produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production. Conclusions The fact that the introduction of DOD along with a supply of its substrate (L-DOPA was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to cyclo-DOPA (or dopaxanthin to betacyanin in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction

  18. Raman, IR, UV-vis and EPR characterization of two copper dioxolene complexes derived from L-dopa and dopamine.

    Barreto, Wagner J; Barreto, Sônia R G; Ando, Rômulo A; Santos, Paulo S; DiMauro, Eduardo; Jorge, Thiago


    The anionic complexes [Cu(L(1-))3](1-), L(-)=dopasemiquinone or L-dopasemiquinone, were prepared and characterized. The complexes are stable in aqueous solution showing intense absorption bands at ca. 605 nm for Cu(II)-L-dopasemiquinone and at ca. 595 nm for Cu(II)-dopasemiquinone in the UV-vis spectra, that can be assigned to intraligand transitions. Noradrenaline and adrenaline, under the same reaction conditions, did not yield Cu-complexes, despite the bands in the UV region showing that noradrenaline and adrenaline were oxidized during the process. The complexes display a resonance Raman effect, and the most enhanced bands involve ring modes and particularly the nuCC+nuCO stretching mode at ca. 1384 cm(-1). The free radical nature of the ligands and the oxidation state of the Cu(II) were confirmed by the EPR spectra that display absorptions assigned to organic radicals with g=2.0005 and g=2.0923, and for Cu(II) with g=2.008 and g=2.0897 for L-dopasemiquinone and dopasemiquinone, respectively. The possibility that dopamine and L-dopa can form stable and aqueous-soluble copper complexes at neutral pH, whereas noradrenaline and adrenaline cannot, may be important in understanding how Cu(II)-dopamine crosses the cellular membrane as proposed in the literature to explain the role of copper in Wilson disease.

  19. Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

    Frederick Daidone

    Full Text Available Dopa decarboxylase (DDC, a pyridoxal 5'-phosphate (PLP enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD. PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a to use virtual screening to identify potential human DDC inhibitors and (b to evaluate the reliability of our virtual-screening (VS protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

  20. Preloading with L-BPA, L-tyrosine and L-DOPA enhances the uptake of [(18)F]FBPA in human and mouse tumour cell lines.

    Wingelhofer, Bettina; Kreis, Katharina; Mairinger, Severin; Muchitsch, Viktoria; Stanek, Johann; Wanek, Thomas; Langer, Oliver; Kuntner, Claudia


    Aim of this study was to investigate if cellular [(18)F]FBPA uptake can be increased upon preloading with amino acids. [(18)F]FBPA uptake was assessed in HuH-7, CaCo-2 and B16-F1 cells pretreated with different concentrations or incubation times of L-BPA, L-tyrosine or L-DOPA. Without preloading, highest uptake of [(18)F]FBPA was observed in B16-F1 cells, followed by CaCo-2 cells and HuH-7 cells. In all cell lines higher [(18)F]FBPA accumulation (up to 1.65-fold) was obtained with increasing L-BPA, L-DOPA and L-tyrosine concentrations.

  1. L-DOPA Oppositely Regulates Synaptic Strength and Spine Morphology in D1 and D2 Striatal Projection Neurons in Dyskinesia

    Suarez, Luz M; Solis, Oscar; Aguado, Carolina; Lujan, Rafael; Moratalla, Rosario


    Dopamine depletion in Parkinson's disease (PD) produces dendritic spine loss in striatal medium spiny neurons (MSNs) and increases their excitability. However, the synaptic changes that occur in MSNs in PD, in particular those induced by chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, are still poorly understood. We exposed BAC-transgenic D1-tomato and D2-eGFP mice to PD and dyskinesia model paradigms, enabling cell type-specific assessment of changes in synaptic physiology and morphology. The distinct fluorescence markers allowed us to identify D1 and D2 MSNs for analysis using intracellular sharp electrode recordings, electron microscopy, and 3D reconstructions with single-cell Lucifer Yellow injections. Dopamine depletion induced spine pruning in both types of MSNs, affecting mushroom and thin spines equally. Dopamine depletion also increased firing rate in both D1- and D2-MSNs, but reduced evoked-EPSP amplitude selectively in D2-MSNs. L-DOPA treatment that produced dyskinesia differentially affected synaptic properties in D1- and D2-MSNs. In D1-MSNs, spine density remained reduced but the remaining spines were enlarged, with bigger heads and larger postsynaptic densities. These morphological changes were accompanied by facilitation of action potential firing triggered by synaptic inputs. In contrast, although L-DOPA restored the number of spines in D2-MSNs, it resulted in shortened postsynaptic densities. These changes in D2-MSNs correlated with a decrease in synaptic transmission. Our findings indicate that L-DOPA-induced dyskinesia is associated with abnormal spine morphology, modified synaptic transmission, and altered EPSP-spike coupling, with distinct effects in D1- and D2-MSNs. PMID:27613437

  2. Effects of prolonged neuronal nitric oxide synthase inhibition on the development and expression of L-DOPA-induced dyskinesia in 6-OHDA-lesioned rats.

    Padovan-Neto, Fernando Eduardo; Cavalcanti-Kiwiatkoviski, Roberta; Carolino, Ruither Oliveira Gomes; Anselmo-Franci, Janete; Del Bel, Elaine


    It is well known that nitric oxide (NO) interacts with dopamine (DA) within the striatal circuitry. The anti-dyskinetic properties of NO synthase (NOS) inhibitors demonstrate the importance of NO in L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID). Here, we investigated the ability of a daily co-treatment of the preferential neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI, 30 mg/kg), with L-DOPA (30 mg/kg) to counteract LID in unilaterally 6-OHDA-lesioned rats. We analyzed striatal nNOS-expressing interneurons, DA and 5-HT neurochemistry in the striatum and alterations of the Fos-B/ΔFosB expression in the corticostriatal, nigrostriatal and mesolimbic pathways. Prolonged administration of 7-NI inhibited the manifestation of chronic L-DOPA treatment-induced abnormal involuntary movements (AIMs). LID was associated with an up-regulation in the number of nNOS-expressing interneurons in the lateral but not medial striatum. nNOS inhibition reduced the number of nNOS-expressing interneurons. The anti-dyskinetic effects of 7-NI correlated with a reduction in DA and 5-HT turnover in the striatum. At postsynaptic striatal sites, 7-NI prevented L-DOPA-induced Fos-B/ΔFosB up-regulation in the motor cortex, nucleus accumbens and striatum. Finally, 7-NI blocked Fos-B/ΔFosB expression in nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-positive interneurons in the striatum. These results provide further evidence of the molecular mechanisms by which NOS-inhibiting compounds attenuate LID. The involvement of NO with DA and 5-HT neurochemistry may contribute to the understanding of this new, non-dopaminergic therapy for the management of LID. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. L-DOPA Oppositely Regulates Synaptic Strength and Spine Morphology in D1 and D2 Striatal Projection Neurons in Dyskinesia.

    Suarez, Luz M; Solis, Oscar; Aguado, Carolina; Lujan, Rafael; Moratalla, Rosario


    Dopamine depletion in Parkinson's disease (PD) produces dendritic spine loss in striatal medium spiny neurons (MSNs) and increases their excitability. However, the synaptic changes that occur in MSNs in PD, in particular those induced by chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, are still poorly understood. We exposed BAC-transgenic D1-tomato and D2-eGFP mice to PD and dyskinesia model paradigms, enabling cell type-specific assessment of changes in synaptic physiology and morphology. The distinct fluorescence markers allowed us to identify D1 and D2 MSNs for analysis using intracellular sharp electrode recordings, electron microscopy, and 3D reconstructions with single-cell Lucifer Yellow injections. Dopamine depletion induced spine pruning in both types of MSNs, affecting mushroom and thin spines equally. Dopamine depletion also increased firing rate in both D1- and D2-MSNs, but reduced evoked-EPSP amplitude selectively in D2-MSNs. L-DOPA treatment that produced dyskinesia differentially affected synaptic properties in D1- and D2-MSNs. In D1-MSNs, spine density remained reduced but the remaining spines were enlarged, with bigger heads and larger postsynaptic densities. These morphological changes were accompanied by facilitation of action potential firing triggered by synaptic inputs. In contrast, although L-DOPA restored the number of spines in D2-MSNs, it resulted in shortened postsynaptic densities. These changes in D2-MSNs correlated with a decrease in synaptic transmission. Our findings indicate that L-DOPA-induced dyskinesia is associated with abnormal spine morphology, modified synaptic transmission, and altered EPSP-spike coupling, with distinct effects in D1- and D2-MSNs. © The Author 2016. Published by Oxford University Press.

  4. Ultrastructural investigations about the effects of irradiation and L-3,4-dihydroxyphenylalanine (L-Dopa) - alone and in combination - on cells of Harding-Passey-melanoma. Elektronenmikroskopische Untersuchungen zur Wirkung von Roentgenstrahlen und L-3,4-Dihydroxyphenylalanin (L-Dopa) - allein und in Kombination - auf Harding-Passey-Melanomzellen in Monolayerzellkultur

    Proske, H. (Klinik fuer Strahlentherapie, Medizinisches Zentrum fuer Radiologie, Univ. Marburg/Lahn (Germany)); Schachtschabel, D.O. (Inst. fuer Physiologische Chemie, Univ. Marburg/Lahn (Germany)); Plamper, G. (Klinik fuer Strahlentherapie, Medizinisches Zentrum fuer Radiologie, Univ. Marburg/Lahn (Germany)); Paul, N. (Inst. fuer Anatomie und Zellbiologie, Univ. Marburg/Lahn (Germany)); Pfab, R. (Klinik fuer Strahlentherapie, Medizinisches Zentrum fuer Radiologie, Univ. Marburg/Lahn (Germany))


    Monolayer cultures of Harding-Passey melanoma cells in exponential growth phase were exposed to 8 or 16 Gy by X-ray treatment. The 8 Gy treated cells revealed little ultrastructural changes, while the 16 Gy exposed cells showed increased damage as segregates, swollen mitochondria and vacuoles. Sole treatment with L-3,4-dihydroxyphenylalanine (2x10[sup -4]M L-Dopa) resulted in insignificant electronmicroscopically tangible cell alterations. Combined treatment - starting with 8 Gy irradiation followed by a six-day incubation in the presence of 2x10[sup -]4M L-Dopa - revealed more pronounced cell damage with final cell desintegration; the cytoplasm contained an increased number of vacuoles and segregates, a strongly decreased endoplasmic reticulum as well as swollen mitochondria and less pinocytosis vesicles; the cell surface showed less microvilli. Melanin containing organelles increased after the combination treatment. The growth inhibitory and cell destructive influence of L-Dopa on X-ray pretreated melanogenic melanoma cells was explained with the formation of cytotoxic oxidation products of L-Dopa. (orig.)

  5. The Adhesion Mechanism of Marine Mussel Foot Protein: Adsorption of L-Dopa on α- and β-Cristobalite Silica Using Density Functional Theory

    Shabeer Ahmad Mian


    Full Text Available Marine mussels strongly adhere to various surfaces and endure their attachment under a variety of conditions. In order to understand the basic mechanism involved, we study the adsorption of L-dopa molecule on hydrophilic geminal and terminal isolated silanols of silica (001 surface. High content of modified amino acid L-dopa is found in the glue-like material secreted by the mussels through which it sticks to various surfaces under water. To understand the adsorption behavior, we have made use of periodic Density Functional Theory (DFT study. The L-dopa molecule adheres to silica surfaces terminated with geminal and terminal silanols via its catechol part. In both cases, the adhesion is achieved through the formation of 4 H-bonds. A binding energy of 29.48 and 31.67 kcal/mol has been estimated, after the inclusion of dispersion energy, for geminal and terminal silanols of silica, respectively. These results suggest a relatively stronger adhesion of dopa molecule for surface with terminal isolated silanols.

  6. Striatal kinetics of ( sup 11 C)-(+)-nomifensine and 6-( sup 18 F)fluoro-L-dopa in Parkinson's disease measured with positron emission tomography

    Tedroff, J.; Aquilonius, S.-M. (Department of Neurology, University Hospital (Sweden)); Laihinen, A.; Rinne, U. (Department of Neurology, University of Turku (Finland)); Hartvig, P. (Department of Hospital Pharmacy, University Hospital (Sweden)); Anderson, J. (Department of Radiation Sciences, University Hospital (Sweden)); Lundqvist, H. (Svenberg Laboratory, Uppsala University (Sweden)); Haaparanta, M.; Solin, O. (Medical Cyclotron Laboratory, University of Turku (Finland)); Antoni, G.; Gee, A.D.; Ulin, J.; Laangstroem, B. (Department of Organic Chemistry, University Hospital (Sweden))


    The kinetics in brain of the dopamine reuptake blocking agent ({sup 11}C)-(+)-nomifensine and the L-dopa analogue 6-({sup 18}F)fluoro-L-dopa were compared in 3 patients with idopathic Parkinson's disease and agematched healthy volunteers using positron emission tomography. Regional uptake was analyzed and quantified according to a 3-compartment model. Retention of both tracers in striatal regions of the parkinsonian patients were reduced compared with the healthy volunteers mainly in the putamen, while the caudate nuclleus was only mildly affected. The reductions were considerably less than the decrease previously reported postmortem for striatal dopamine content in the basal ganglia of patients with Parkinson's disease. A fairly constant ratio between 6-({sup 18}F)fluoro-L-dopa utilization and ({sup 11}C)-(+)-nomifensine binding in the caudate nucleus and the putamen were found in both groups unrelated to the size of the estimated parameters. This indicates that a limiting factor for the utilization of exogenous levodopa in Parkinsons's disease may be a reduced transport capacity for the amino acid into the dopaminergic terminals. (author).

  7. Correlation between the Movement Disorders Society Unified Parkinson's Disease rating scale (MDS-UPDRS) and the Unified Parkinson's Disease rating scale (UPDRS) during L-dopa acute challenge.

    Merello, Marcelo; Gerschcovich, Eliana Roldan; Ballesteros, Diego; Cerquetti, Daniel


    While Movement Disorders Society Unified Parkinson's Disease rating scale (MDS-UPDRS) validation has been exhaustive; performance evaluation to detect acute changes arising after administration of a single dose of L-dopa has yet to be explored. To determine the correlation between UPDRS and MDS-UPDRS during the acute challenge with Ldopa and the MDS-UPDRS equivalent to 30% cutoff score of UPDRS for defining responsiveness, 64 patients were assessed. Consecutive assessments were performed immediately before and after administration of a single dose of L-dopa/carbidopa 250/25 mg using the motor section of the UPDRS and the MDS-UPDRS. Good diagnostic accuracy, consistent with published findings of high correlation between scales was observed. Area under the curve (AUC) was 0.99 (CI = 0.97-1.00, P UPDRS and MDS-UPDRS and that the 30% of variation in UPDRS score used for predicting sustained long term L-dopa response was equivalent to 24% in MDS-UPDRS.

  8. Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts.

    Nitta, Yoko; Yasukata, Fumiko; Kitamoto, Noritoshi; Ito, Mikiko; Sakaue, Motoyoshi; Kikuzaki, Hiroe; Ueno, Hiroshi


    Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 μM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

  9. Interruption of the MnO2 oxidative process on dopamine and L-dopa by the action of S2O3(2-).

    Barreto, W J; Barreto, S R; Santos, M A; Schimid, R; Paschoal, F M; Mangrich, A S; deOliveira, L F


    The oxidation effects of Mn2+, Mn3+ or MnO2 on dopamine can be studied in vitro and, therefore, this offers a model of the auto-oxidation process that appears naturally in neurons causing Parkinson's disease. The use of MnO, as an oxidizer in aqueous solution at pH 7 causes the oxidation of catecholamines (L-dopa, dopamine, noradrenaline and adrenaline) to melanin. However, this work shows that, in water at pH 6-7, the oxidation of catecholamines by MnO2 in the presence of sodium thiosulphate (Na2S2O3) occurs by other mechanisms. For dopamine and L-dopa, MLCT complexes were formed with bands at 312, 350 (sh), 554 (sh) nm, and an intense band at 597 nm (epsilon approximately/= 4 x 10(3) M(-1) cm(-1)) and at ca. 336, 557 (sh) nm, and an intense band at 597 nm (epsilon approximately 6 x 10(3) M(-1) cm(-1)), respectively. The latter transitions were assigned to d(pi)-->pi*-SQ. Noradrenaline and adrenaline do not form this blue complex in solution, but generate soluble oxidized compounds. The resonance Raman spectra of these complexes in solution showed bands at 950, 1006, 1258, 1378, 1508 and 1603 cm(-1) for the complex derivation of L-dopa and at 948, 1010, 1255, 1373, 1510 and 1603 cm(-1) for the dopamine-derived compound. The most intense Raman band at ca. 1378 cm(-1) was assigned to C-O stretching with major C1-C2 characteristics and indicated that dopamine and L-dopa do not occur complexed with manganese in the catecholate or quinone form, but suggests an intermediate compound such as an anionic o-semiquinone (SQ-), forming a complex such as [Mn(II)(SQ-)3]-. All enhanced Raman frequencies are characteristic of the benzenic ring without the participation of the aminic nitrogen. A mechanism is proposed for the formation of the dopamine and L-dopa complexes and a computational simulation was performed to support it.

  10. Autonomic nervous system response to L-dopa in patients with advanced Parkinson's disease.

    Ruonala, Verneri; Tarvainen, Mika P; Karjalainen, Pasi A; Pekkonen, Eero; Rissanen, Saara M


    Levodopa is the main treatment method for reducing the symptoms of Parkinson's disease. Whereas it reduces the motor symptoms efficiently, its effect on autonomous nervous system is not clear. The information about effect of levodopa on heart rate variability is not coherent between the studies. In this study, ECG of 11 patients with Parkinson's disease was measured during levodopa challenge with pronounced dose of fast release levodopa to ensure the positive drug effect for deep brain stimulation treatment. Heart rate variability analysis was done at three time points, before administration of levodopa, 30 and 60 minutes after administration. After 30 minutes of administration, the HRV parameters show that parasympathetic nervous system activity is decreased and the sympatho-vagal balance is shifted towards sympathetic control. At 60 minutes after administration the parasympathetic nervous system activates slightly and causes a decrease in heart rate.

  11. Horseradish peroxidase-catalyzed polymerization of L-DOPA for mono-/bi-enzyme immobilization and amperometric biosensing of H2O2 and uric acid.

    Dai, Mengzhen; Huang, Ting; Chao, Long; Xie, Qingji; Tan, Yueming; Chen, Chao; Meng, Wenhua


    Horseradish peroxidase (HRP)-catalyzed polymerization of L-DOPA (vs. dopamine) in the presence of H2O2 (and uricase (UOx)) was exploited to immobilize mono-/bi-enzymes for hydroquinone-mediated amperometric biosensing of H2O2 and uric acid (UA). The relevant polymeric biocomposites (PBCs) were prepared in phosphate buffer solution containing HRP and L-DOPA (or plus UOx) after adding H2O2. The mono-/bi-enzyme amperometric biosensors were prepared simply by casting some of the PBCs on Au-plated Au (Au(plate)/Au) electrodes, followed by coating with an outer-layer chitosan (CS) film for each. UV-vis spectrophotometry, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy were used for film characterization and/or process monitoring. The HRP immobilized by enzyme catalysis well preserved its bioactivity, as confirmed by UV-vis spectrophotometry. Under optimized conditions, the monoenzyme CS/HRP-poly(L-DOPA) (PD)/Au(plate)/Au electrode potentiostated at -0.1V responded linearly to H2O2 concentration from 0.001 to 1.25mM with a sensitivity of 700μA mM(-1)cm(-2) and a limit of detection (LOD) of 0.1μM, and the bienzyme CS/UOx-HRP-PD/Au(plate)/Au electrode at -0.1V responded linearly to UA concentration from 0.001 to 0.4mM with a sensitivity of 349μA mM(-1)cm(-2) and a LOD of 0.1μM. The mono-/bi-enzyme biosensors based on biosynthesized PD performed better than many reported analogues and those based on similarly biosynthesized polydopamine.

  12. Excessive sensitivity to uncertain visual input in L-dopa induced dyskinesias in Parkinson’s disease: further implications for cerebellar involvement

    James eStevenson


    Full Text Available When faced with visual uncertainty during motor performance, humans rely more on predictive forward models and proprioception and attribute lesser importance to the ambiguous visual feedback. Though disrupted predictive control is typical of patients with cerebellar disease, sensorimotor deficits associated with the involuntary and often unconscious nature of L-dopa-induced dyskinesias in Parkinson’s disease (PD suggests dyskinetic subjects may also demonstrate impaired predictive motor control. Methods: We investigated the motor performance of 9 dyskinetic and 10 non-dyskinetic PD subjects on and off L-dopa, and of 10 age-matched control subjects, during a large-amplitude, overlearned, visually-guided tracking task. Ambiguous visual feedback was introduced by adding ‘jitter’ to a moving target that followed a Lissajous pattern. Root mean square (RMS tracking error was calculated, and ANOVA, robust multivariate linear regression and linear dynamical system analyses were used to determine the contribution of speed and ambiguity to tracking performance. Results: Increasing target ambiguity and speed contributed significantly more to the RMS error of dyskinetic subjects off medication. L-dopa improved the RMS tracking performance of both PD groups. At higher speeds, controls and PDs without dyskinesia were able to effectively de-weight ambiguous visual information. Conclusions: PDs’ visually-guided motor performance degrades with visual jitter and speed of movement to a greater degree compared to age-matched controls. However, there are fundamental differences in PDs with and without dyskinesia: subjects without dyskinesia are generally slow, and less responsive to dynamic changes in motor task requirements but, PDs with dyskinesia there was a trade-off between overall performance and inappropriate reliance on ambiguous visual feedback. This is likely associated with functional changes in posterior parietal-ponto-cerebellar pathways.

  13. The effects of dopamine on regional cerebral blood flow in patients with Parkinson`s disease before and after L-dopa. Measurement by Xe-enhanced CT

    Aiba, Ikuko [Nagoya Univ. (Japan). School of Medicine; Indo, Toshikatsu; Takahashi, Akira


    Regional cerebral blood flow (rCBF) was measured using the stable xenon enhanced CT method in previously untreated 13 patients with Parkinson`s disease to evaluate CBF abnormality related to dysfunction of the nigrostriatal dopaminergic neurons. The patients comprised 5 men and 8 women with Hoehn-Yahr stage II-III. Age at onset ranged from 51 to 73 years (mean{+-}SD, 61.8{+-}8.9) and the duration of illness ranged from 1 to 96 months (15.1{+-}24.1 months). In this series, there was no clinical evidence of hypertension, diabetes mellitus and cognitive impairment. rCBF was measured during 4-5-minutes inhalation, of 33% stable xenon gas-67% oxygen. The first measurement of rCBF was performed in all of the patients before L-dopa treatment. After initiation of L-dopa treatment (333.3{+-}47.1 mg/day), the second measurement was carried out in 6 patients (1 man and 5 women) who had shown symptomatic improvement. The interval between both measurements was 57.7{+-}16.9 days. The following results were obtained. (1) No significant CBF asymmetry was noted in any of the striatum, pallidum, thalamus, cerebrum, cerebellum and frontal lobe in untreated patients with Parkinson`s disease. (2) After L-dopa treatment, rCBF was significantly increased only in the striatum as compared with the pretreatment level (51.9{+-}9.3{yields}63.1{+-}9.9 ml/100g/min, p<0.01). (3) This increase was significantly greater on the more severely affected side (contralateral to the predominantly symptomatic limb) (p<0.05). These results suggest that the increase of rCBF in the striatum is closely related to functional improvement of the nigrostriatal dopaminergic neurons. (author).

  14. Dynamic changes in gamma-aminobutyric acid and glutamate decarboxylase activity in oats (Avena nuda L.) during steeping and germination.

    Xu, Jian Guo; Hu, Qing Ping; Duan, Jiang Lian; Tian, Cheng Rui


    Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system and provides beneficial effects for human and other animals health. To accumulate GABA, samples from two different naked oat cultivars, Baiyan II and Bayou I, were steeped and germinated in an incubator. The content of GABA and glutamic acid as well as the activity of the glutamate decarboxylase (GAD) in oats during steeping and germination were investigated with an amino acid automatic analyzer. Compared with raw groats, an increase in GABA content of oat groats during steeping and germination was continuously observed for two oat cultivars. The activity of GAD increased greatly at the end of steeping and the second stage of germination for Baiyan II and Bayou I, respectively. Glutamic acid content of treated oat groats was significantly lower than that in raw groats until the later period of germination. GABA was correlated (p<0.01) significantly and positively with the glutamic acid rather than GAD activity in the current study. The results indicates that steeping and germination process under highly controlled conditions can effectively accumulate the GABA in oat groats for Baiyan II and Bayou I, which would greatly facilitate production of nutraceuticals or food ingredients that enable consumers to gain greater access to the health benefits of oats. However, more assays need to be further performed with more oat cultivars.

  15. Trypanocidal activity of 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]-(methylamino)}adenosine (Genz-644131), an adenosylmethionine decarboxylase inhibitor.

    Bacchi, Cyrus J; Barker, Robert H; Rodriguez, Aixa; Hirth, Bradford; Rattendi, Donna; Yarlett, Nigel; Hendrick, Clifford L; Sybertz, Edmund


    Genzyme 644131, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.


    张莉; 李文; 肖正华; 王云霞; 夏仕文


    以在L-酪氨酸诱导下高效表达酪氨酸酚解酶的菌株Citrobacter freundii48003-3的休止细胞为生物催化剂,以邻苯二酚、丙酮酸钠、醋酸铵为前体,选择性合成L-DOPA.研究了反应温度、pH和前体浓度等对合成L-DOPA的影响.最优反应条件下,反应12h,L-DOPA的量可达到9.5g/L.

  17. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    Yan Huang

    Full Text Available Glutamate decarboxylase (GAD catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA. In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C. Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C, superior thermostability (2.8-fold greater than that of GAD-C, and higher kcat/Km (1.6-fold higher than that of GAD-C. Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.

  18. Levodopa activates apoptosis signaling kinase 1 (ASK1) and promotes apoptosis in a neuronal model: implications for the treatment of Parkinson's disease.

    Liedhegner, Elizabeth A Sabens; Steller, Kelly M; Mieyal, John J


    Oxidative stress is implicated in the etiology of Parkinson's disease (PD), the second most common neurodegenerative disease. PD is treated with chronic administration of l-3,4-dihydroxyphenylalanine (levodopa, L-DOPA), and typically, increasing doses are used during progression of the disease. Paradoxically, L-DOPA is a pro-oxidant and induces cell death in cellular models of PD through disruption of sulfhydryl homeostasis involving loss of the thiol-disulfide oxidoreductase functions of the glutaredoxin (Grx1) and thioredoxin (Trx1) enzyme systems [Sabens, E. A., Distler, A. M., and Mieyal, J. J. (2010) Biochemistry 49 (12), 2715-2724]. Considering this loss of both Grx1 and Trx1 activities upon L-DOPA treatment, we sought to elucidate the mechanism(s) of L-DOPA-induced apoptosis. In other contexts, both the NFκB (nuclear factor κB) pathway and the ASK1 (apoptosis signaling kinase 1) pathway have been shown to be regulated by both Grx1 and Trx1, and both pathways have been implicated in cell death signaling in model systems of PD. Moreover, mixed lineage kinase (MLK) has been considered as a potential therapeutic target for PD. Using SHSY5Y cells as model dopaminergic neurons, we found that NFκB activity was not altered by L-DOPA treatment, and the selective MLK inhibitor (CEP-1347) did not protect the cells from L-DOPA. In contrast, ASK1 was activated with L-DOPA treatment as indicated by phosphorylation of its downstream mitogen-activated protein kinases (MAPK), p38 and JNK. Chemical inhibition of either p38 or JNK provided protection from L-DOPA-induced apoptosis. Moreover, direct knockdown of ASK1 protected from L-DOPA-induced neuronal cell death. These results identify ASK1 as the main pro-apoptotic pathway activated in response to L-DOPA treatment, implicating it as a potential target for adjunct therapy in PD.

  19. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    Addo, James K; Skaff, D Andrew; Miziorko, Henry M


    Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 μM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.

  20. The Parkinsonian Basal Ganglia Network: Measures of Power, Linear and Non-Linear Synchronization and their Relationship to L-DOPA Treatment and OFF State Motor Severity

    Timothy West


    Full Text Available In this paper we investigated the dopaminergic modulation of neuronal interactions occurring in the subthalamic nucleus (STN during Parkinson’s disease (PD. We utilized linear measures of local and long range synchrony such as power and coherence, as well as Detrended Fluctuation Analysis for Phase Synchrony (DFA-PS- a recently developed non-linear method that computes the extent of long tailed autocorrelations present in the phase interactions between two coupled signals. Through analysis of local field potentials (LFPs taken from the STN we seek to determine changes in the neurodynamics that may underpin the pathophysiology of PD in a group of 12 patients who had undergone surgery for deep brain stimulation. We demonstrate up modulation of alpha-theta (5-12 Hz band power in response to L-DOPA treatment, whilst low beta band power (15-20 Hz band-power is suppressed. We also find evidence for significant local connectivity within the region surrounding STN although there was no evidence for modulation via administration of L-DOPA. Further to this we present evidence for a positive correlation between the phase ordering of bilateral STN interactions and the severity of bradykinetic and rigidity symptoms in PD. Although the ability of non-linear measures to predict clinical state did not exceed standard measures such as beta power, these measures may help identify the connections which play a role in pathological dynamics.

  1. Long-term treatment with L-DOPA or pramipexole affects adult neurogenesis and corresponding non-motor behavior in a mouse model of Parkinson's disease.

    Chiu, W-H; Depboylu, C; Hermanns, G; Maurer, L; Windolph, A; Oertel, W H; Ries, V; Höglinger, G U


    Non-motor symptoms such as hyposmia and depression are often observed in Parkinson's disease (PD) and can precede the onset of motor symptoms for years. The underlying pathological alterations in the brain are not fully understood so far. Dysregulation of adult neurogenesis in the dentate gyrus of the hippocampus and the olfactory bulb has been recently suggested to be implicated in non-motor symptoms of PD. However, there is so far no direct evidence to support the relationship of non-motor symptoms and the modulation of adult neurogenesis following dopamine depletion and/or dopamine replacement. In this study, we investigated the long-term effects of l-DOPA and pramipexole, a dopamine agonist, in a mouse model of bilateral intranigral 6-OHDA lesion, in order to assess the impact of adult neurogenesis on non-motor behavior. We found that l-DOPA and pramipexole can normalize decreased neurogenesis in the hippocampal dentate gyrus and the periglomerular layer of the olfactory bulb caused by a 6-OHDA lesion. Interestingly, pramipexole showed an antidepressant and anxiolytic effect in the forced swim test and social interaction test. However, there was no significant change in learning and memory function after dopamine depletion and dopamine replacement, respectively.

  2. [Determination of L-dopa and dopamine in rat brain microdialysate by ultra high performance liquid chromatography-tandem mass spectrometry using stable isotope-coded derivatization coupled with dispersive liquid-liquid microextraction].

    Qi, Weimei; Zhao, Xian-en; Qi, Yong; Sun, Zhiwei; Chen, Guang; You, Jinmao; Suo, Yourui


    The sensitive detection method of levodopa (L-DOPA) and dopamine (DA) in rat brain microdialysate of Parkinson's disease (PD) is an essential tool for the clinical study and attenuated synergistic drug screening for L-DOPA from traditional Chinese medicines. Using d0/d3-10-methyl-acridone-2-sulfonyl chloride (d0/d3-MASC) as stable isotope derivatization reagent, a novel ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for L-DOPA and DA by stable isotope- coded derivatization coupled with ultrasonic-assisted dispersive liquid-liquid microextraction (UA-DLLME). d3-MASC (light) and d3-MASC (heavy) were used as derivatization reagents for microdialysate samples and standards, respectively. Mixtures of the two solutions were prepared by UA-DLLME for UHPLC-MS/MS analysis with multiple reaction monitoring (MRM) mode. With d3-MASC heavy derivatives as internal standards for corresponding light derivatives from samples, the stable isotope internal standard quantification for L-DOPA and DA was carried out. The stable derivatives were obtained in aqueous acetonitrile (pH 10.8 sodium carbonate-sodium bicarbonate buffer) at 37 °C for 3.0 min, and then were separated within 2.0 min using gradient elution. Linear range was 0.20-1500.0 nmol/L (R > 0.994). LODs were 0.005 and 0.009 nmol/L for DA and L-DOPA (S/N = 3), respectively. This method was validated, and it showed obvious advantages in comparing with the reported methods in terms of sensitivity, analysis speed and anti-matrix interference. This method has been successfully applied to the study of effect of Shouwu Fang on L-DOPA and DA concentration fluctuations in PD rat brain microdialysate.

  3. Synthesis of N-formyl-3,4-di-t-butoxycarbonyloxy-6(trimethylstannyl)-L-phenylalanine ethyl ester and its regioselective radiofluorodestannylation to 6-[{sup 18}F]fluoro-L-dopa

    Satyamurthy, N.; Barrio, J.R.; Bishop, A.J.; Namavari, M.


    A protected 6-trimethylstannyl dopa derivative has been synthesized for the as a precursor for the preparation of 6-[{sup 18}F]fluoro-L-dopa. The tin derivative readily reacts with electrophilic radiofluorinating agents such as [{sup 18}F]F{sub 2}, [{sup 18}F]OF{sub 2} and [{sup 18}F]AcOF. The [{sup 18}F]fluoro intermediate was easily hydrolyzed with HBr and the product 6-[{sup 18}F]fluoro-L-dopa was isolated after HPLC purification in a maximum radiochemical yield of 23%, ready for human use. 1 fig.

  4. Synthesis of N-formyl-3,4-di-t-butoxycarbonyloxy-6-(trimethylstannyl)-L-phenylalanine ethyl ester and its regioselective radiofluorodestannylation to 6-[.sup.18 F]fluoro-L-dopa

    Satyamurthy, Nagichettiar; Barrio, Jorge R.; Bishop, Allyson J.; Namavari, Mohammad


    A protected 6-trimethylstannyl dopa derivative has been synthesized for the as a precursor for the preparation of 6-[.sup.18 F]fluoro-L-dopa. The tin derivative readily reacts with electrophilic radiofluorinating agents such as [.sup.18 F]F.sub.2, [.sup.18 F]OF.sub.2 and [.sup.18 F]AcOF. The [.sup.18 F]fluoro intermediate was easily hydrolyzed with HBr and the product 6-[.sup.18 F]fluoro-L-dopa was isolated after HPLC purification in a maximum radiochemical yield of 23%, ready for human use.

  5. Automated production at the curie level of no-carrier-added 6-[(18)F]fluoro-L-dopa and 2-[(18)F]fluoro-L-tyrosine on a FASTlab synthesizer.

    Lemaire, C; Libert, L; Franci, X; Genon, J-L; Kuci, S; Giacomelli, F; Luxen, A


    An efficient, fully automated, enantioselective multi-step synthesis of no-carrier-added (nca) 6-[(18)F]fluoro-L-dopa ([(18)F]FDOPA) and 2-[(18)F]fluoro-L-tyrosine ([(18)F]FTYR) on a GE FASTlab synthesizer in conjunction with an additional high- performance liquid chromatography (HPLC) purification has been developed. A PTC (phase-transfer catalyst) strategy was used to synthesize these two important radiopharmaceuticals. According to recent chemistry improvements, automation of the whole process was implemented in a commercially available GE FASTlab module, with slight hardware modification using single use cassettes and stand-alone HPLC. [(18)F]FDOPA and [(18)F]FTYR were produced in 36.3 ± 3.0% (n = 8) and 50.5 ± 2.7% (n = 10) FASTlab radiochemical yield (decay corrected). The automated radiosynthesis on the FASTlab module requires about 52 min. Total synthesis time including HPLC purification and formulation was about 62 min. Enantiomeric excesses for these two aromatic amino acids were always >95%, and the specific activity of was >740 GBq/µmol. This automated synthesis provides high amount of [(18)F]FDOPA and [(18)F]FTYR (>37 GBq end of synthesis (EOS)). The process, fully adaptable for reliable production across multiple PET sites, could be readily implemented into a clinical good manufacturing process (GMP) environment.

  6. Study of the coumarate decarboxylase and vinylphenol reductase activities of Dekkera bruxellensis (anamorph Brettanomyces bruxellensis) isolates.

    Godoy, L; Garrido, D; Martínez, C; Saavedra, J; Combina, M; Ganga, M A


    To evaluate the coumarate descarboxylase (CD) and vinylphenol reductase (VR) activities in Dekkera bruxellensis isolates and study their relationship to the growth rate, protein profile and random amplified polymorphic DNA (RAPD) molecular pattern. CD and VR activities were quantified, as well, the growth rate, intracellular protein profile and molecular analysis (RAPD) were determined in 12 isolates of D. bruxellensis. All the isolates studied showed CD activity, but only some showed VR activity. Those isolates with the greatest growth rate did not present a different protein profile from the others. The FASC showed a relationship between RAPD molecular patterns and VR activity. CD activity is common to all of the D. bruxellensis isolates. This was not the case with VR activity, which was detected at a low percentage in the analysed micro-organisms. A correlation was observed between VR activity and the RAPD patterns. This is the first study that quantifies the CD and VR enzyme activities in D. bruxellensis, demonstrating that these activities are not present in all isolates of this yeast.

  7. Antagonism of haloperidol-induced swim impairment in L-dopa and caffeine treated mice: a pre-clinical model to study Parkinson's disease.

    Luthra, Pratibha Mehta; Barodia, Sandeep Kumar; Raghubir, Ram


    Parkinson's disease (PD) exhibits symptoms of motor dysfunction such as tremor, akinesia and rigidity. Agents that selectively disrupt or destroy catecholaminergic systems, such as reserpine, methamphetamine, 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, have been used to develop PD models and to study the animal behavior like catalepsy, akinesia, swim-test, etc. The major apprehension while working with these chemicals is their irreversible neuro-toxic effect. Haloperidol is a classical antipsychotic drug, which produces extra-pyrimidal Parkinson's symptoms (EPS). Measuring catalepsy and akinesia in the treated mice monitored the haloperidol-induced EPS. Alternatively, swimming disability was tested as a new parameter to monitor haloperidol-induced EPS. The results showed that the restoration of swimming disability in haloperidol-induced L-dopa and caffeine pre-treated mice could be used as pre-clinical model to study PD.

  8. Preformulation Study on L-Dopa Methyl Ester Hydrochloride%盐酸左旋多巴甲酯处方前研究

    羊鸣; 蒋伟哲; 黄兴振; 许崇摇


    目的:对盐酸左旋多巴甲酯进行处方前研究,为设计优良的处方提供参考.方法:考察3批盐酸左旋多巴甲酯外观、溶解度、熔点、晶型、粒度分布、休止角、堆密度、紧密度、压缩度、引湿性、吸湿平衡时间与临界相对湿度等相关理化性质,为处方研究提供参考.结果:3批盐酸左旋多巴甲酯样品均为白色结晶性粉末,无臭,无味,易溶于水、甲醇、乙醇,熔点分别为170.3~171.1C、170.5~171.4℃、170.2~171.2℃;样品晶态下不存在分子内氢键,晶态下分子以盐键、氢键、和范德华力维系在空间的稳定排列;粒径<420 μm者占66.15%,<74 μm者占0.97%;休止角>38.00°;流动性较差,可压性随着粒度的减小而降低;引湿性增质量百分率为7.7%;第7天吸湿基本达到平衡;25℃时的临界相对湿度约为74%.结论:处方前研究表明盐酸左旋多巴甲酯水溶性好,流动性较差,且具有较强的引湿性,考虑宜制成片剂.%OBJECTIVE: To provide reference for optimal formulation design by conducting preformulation study on L-dopa methyl ester hydrochloride. METHODS: Related physicochemical properties of 3 batches of L-dopa methyl ester hydrochloride were investigated to provide reference for formulation research, such as appearance, melting point, solubility, crystal type, particle size distribution, angle of repose, bulk density, compacted density, degree of compression, hygroscopicity, moisture absorption equilibrium time and critical relative humidity. RESULTS: 3 batches of L-dopa methyl ester hydrochloride were white crystalline powder which was odorless, tasteless and easily soluble in water, methanol and ethanol. The melting points of them were 170.3-171.1℃, 170.5-171.4℃ and 170.2-171.2 ℃, respectively; there was no intramolecular hydrogen bond under crystalline state, and the molecules were held together by salt bond, hydrogen bond and van der Waals' forces; particle

  9. Synthesis of polymer-bound 6-mercuric carboxylate DOPA precursors and solid phase labelling method of 6-radioiodinated L-DOPA.

    Kawai, K; Ohta, H; Channing, M A; Kubodera, A; Eckelman, W C


    In order to avoid separating unreacted mercury precursor and other mercury-containing compounds after the halodemercuration of a 6-mercury DOPA precursor, we developed a polymer-bound mercury precursor for the preparation of 6-halogenated DOPA. In this study, polymer-bound 6-mercuric carboxylate DOPA derivatives were synthesized from ion-exchange resin and Merrifield-type resin. Iododemercuration of polymer-bound 6-mercuric carboxylate DOPA derivatives gave higher yields (49-54%) compared with monomeric 6-mercuric trifluoroacetate protected DOPA. The radioiodination of the resin with no-carrier added iodine-125 afforded protected 6-[125I]I-L-DOPA with labelling efficiency of 92-97% with both polymer-bound 6-mercuric carboxylate DOPA derivatives.

  10. Mucuna pruriens and its major constituent L-DOPA recover spermatogenic loss by combating ROS, loss of mitochondrial membrane potential and apoptosis.

    Akhand Pratap Singh

    Full Text Available BACKGROUND: The Ayurvedic medicinal system claims Mucuna pruriens (MP to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD, and finding the possible mechanism of action thereof in a rat model. METHODOLOGY/FINDINGS: Ethinyl estradiol (EE was administered at a rate of 3 mg/kg body weight (BW/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15(th day for a period of 56 days, and the results were compared with an auto-recovery (AR group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS, mitochondrial membrane potential (MMP, apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP, recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility. CONCLUSION/SIGNIFICANCE: M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro

  11. Protective effect of two essential oils isolated from Rosa damascena Mill. and Lavandula angustifolia Mill, and two classic antioxidants against L-dopa oxidative toxicity induced in healthy mice.

    Nikolova, Galina; Karamalakova, Yanka; Kovacheva, Natasha; Stanev, Stanko; Zheleva, Antoaneta; Gadjeva, Veselina


    Levodopa (L-dopa) is a "gold standard" and most effective symptomatic agent in the Parkinson's disease (PD) treatment. The several treatments have been developed in an attempt to improve PD treatment, but most patients were still levodopa dependent. The issue of toxicity was raised in vitro studies, and suggests that L-dopa can be toxic to dopaminergic neurons, but it is not yet entirely proven. L-dopa prolonged treatment is associated with motor complications and some limitations. Combining the L-dopa therapy with antioxidants can reduce related sideeffects and provide symptomatic relief. The natural antioxidants can be isolated from any plant parts such as seeds, leaves, roots, bark, etc., and their extracts riched in phenols can retard the oxidative degradation of the lipids, proteins and DNA. Thus, study suggests that combination of essential oils (Rose oil and Lavender oil), Vitamin C and Trolox with Ldopa can reduce oxidative toxicity, and may play a key role in ROS/RNS disarm.

  12. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil


    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

  13. A dopa decarboxylase modulating the immune response of scallop Chlamys farreri.

    Zhi Zhou

    Full Text Available BACKGROUND: Dopa decarboxylase (DDC is a pyridoxal 5-phosphate (PLP-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate. METHODOLOGY: The full-length cDNA encoding DDC (designated CfDDC was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P<0.05 at 3 h and reached the peak at 12 h (9.8-fold, P<0.05, and then recovered to the baseline level. The recombinant protein of CfDDC (rCfDDC was expressed in Escherichia coli BL21 (DE3-Transetta, and 1 mg rCfDDC could catalyze the production of 1.651±0.22 ng dopamine within 1 h in vitro. When the haemocytes were incubated with rCfDDC-coated agarose beads, the haemocyte encapsulation to the beads was increased significantly from 70% at 6 h to 93% at 24 h in vitro in comparison with that in the control (23% at 6 h to 25% at 24 h, and the increased haemocyte encapsulation was repressed by the addition of rCfDDC antibody (which is acquired via immunization 6-week old rats with rCfDDC. After the injection of DDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (P<0.05 of blank group at 12 h and 0.47-fold (P<0.05 at 24 h, respectively. CONCLUSIONS: These results collectively suggested that CfDDC, as a homologue of DDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc.

  14. Extraction of Velvet Bean L-Dopa by Membrane Technology%膜工艺提取猫豆左旋多巴的研究

    罗栋源; 魏璇; 龚琴; 吴莎; 万端极


    针对传统工艺存在的收率低、有毒试剂用量大、污染严重等问题,研究了膜技术在猫豆左旋多巴提取中的应用,考察了各级膜工作过程及分离效果,探讨了膜技术工艺可行性.结果表明,猫豆提取液经过膜技术除杂、分离、浓缩三级膜过程,左旋多巴收率由传统工艺的50%提高至77.98%、纯度达到99.72%,超过国家药用要求,达到欧洲标准水平,且水回用率接近90%,整个工艺过程能耗低、污染少、不使用有毒试剂,实现了清洁生产目标.表明采用膜技术提取猫豆左旋多巴是切实可行的.%For traditional technology exists some problems such as low yield, using toxic agents and serious pollution, this paper mainly studied on the application of membrane technology to extract velvet bean L-dopa.The separation effects of various membrane processes were investigated to explore the feasibility of the membrane technology. The results showed that, treating the extract of velvet bean by three membrane processes of removing impurities, separation and concentration, the yield of L-dopa increased from 50% of the traditional technology to 77.98% and its purity reached 99.72%, which was over the national standard and was up to the European standard, furthurmore, the reused degree of water reached 90%. The whole process realized the cleaner production with low energy consumption, less pollution and didn't use toxic agents. So the membrane technology is feasible.

  15. Confined Flocculation of Ionic Pollutants by Poly(L-dopa)-Based Polyelectrolyte Complexes in Hydrogel Beads for Three-Dimensional, Quantitative, Efficient Water Decontamination.

    Yu, Li; Liu, Xiaokong; Yuan, Weichang; Brown, Lauren Joan; Wang, Dayang


    The development of simple and recyclable adsorbents with high adsorption capacity is a technical imperative for water treatment. In this work, we have successfully developed new adsorbents for the removal of ionic pollutants from water via encapsulation of polyelectrolyte complexes (PECs) made from positively charged poly(allylamine hydrochloride) (PAH) and negatively charged poly(l-3,4-dihydroxyphenylalanine) (PDopa), obtained via the self-polymerization of l-3,4-dihydroxyphenylalanine (l-Dopa). Given the outstanding mass transport through the hydrogel host matrixes, the PDopa-PAH PEC guests loaded inside can effectively and efficiently remove various ionic pollutants, including heavy metal ions and ionic organic dyes, from water. The adsorption efficiency of the PDopa-PAH PECs can be quantitatively correlated to and tailored by the PDopa-to-PAH molar ratio. Because PDopa embodies one catechol group, one carboxyl group, and one amino group in each repeating unit, the resulting PDopa-PAH PECs exhibit the largest capacity of adsorption of heavy metal ions compared to available adsorbents. Because both PDopa and PAH are pH-sensitive, the PDopa-PAH PEC-loaded agarose hydrogel beads can be easily and completely recovered after the adsorption of ionic pollutants by adjusting the pH of the surrounding media. The present strategy is similar to the conventional process of using PECs to flocculate ionic pollutants from water, while in our system flocculation is confined to the agarose hydrogel beads, thus allowing easy separation of the resulting adsorbents from water.

  16. An unusual decarboxylative Maillard reaction between L-DOPA and D-glucose under biomimetic conditions: factors governing competition with Pictet-Spengler condensation.

    Manini, P; d'Ischia, M; Prota, G


    In 0.1 M phosphate buffer at pH 7.4 and 37 degrees C, the tyrosine metabolite L-3,4-dihydroxyphenylalanine (L-DOPA) reacts smoothly with D-glucose to afford, besides diastereoisomeric tetrahydroisoquinolines 1 and 2 by Pictet-Spengler condensation, a main product shown to be the unexpected decarboxylated Amadori compound N-(1-deoxy-D-fructos-1-yl)-dopamine (3). Under similar conditions, dopamine gave only tetrahydroisoquinoline products 4 and 5, whereas L-tyrosine gave exclusively the typical Amadori compound 6. Fe(3+) and Cu(2+) ions, which accumulate in relatively high levels in parkinsonian substantia nigra, both inhibited the formation of 3. Cu(2+) ions also inhibited the formation of 1 and 2 to a similar degree, whereas Fe(3+) ions increased the yields of 1 and 2. Apparently, the formation of 3 would not be compatible with a simple decarboxylation of the initial Schiff base adduct, but would rather involve the decarboxylative decomposition of a putative oxazolidine-5-one intermediate assisted by the catechol ring. These results report the first decarboxylative Maillard reaction between an amino acid and a carbohydrate under biomimetic conditions and highlight the critical role of transition metal ions in the competition with Pictet-Spengler condensation.

  17. Effect of low-frequency repetitive transcranial magnetic stimulation combined with physical therapy on L-dopa-induced painful off-period dystonia in Parkinson's disease.

    Kodama, Mitsuhiko; Kasahara, Takashi; Hyodo, Masaki; Aono, Koji; Sugaya, Mutsumi; Koyama, Yuji; Hanayama, Kozo; Masakado, Yoshihisa


    Previous research has shown that low-frequency repetitive transcranial magnetic stimulation over the primary motor area and supplementary motor area can reduce L-dopa-induced dyskinesias in Parkinson's disease; however, it involved only patients with peak-dose or diphasic dyskinesia. We report a case of a patient with severely painful off-period dystonia in the unilateral lower limb who underwent 0.9-Hz subthreshold repetitive transcranial magnetic stimulation over contralateral primary motor area and supplementary motor area. Repetitive transcranial magnetic stimulation over the primary motor area significantly reduced the painful dystonia and walking disturbances but repetitive transcranial magnetic stimulation over the supplementary motor area did not. The cortical silent period also prolonged after repetitive transcranial magnetic stimulation over the primary motor area. At 5 mos of approximately once a week repetitive transcranial magnetic stimulation over the primary motor area, the Unified Parkinson's Disease Rating Scale motor score also improved. This report shows that repetitive transcranial magnetic stimulation over the inhibitory primary motor area can be useful for rehabilitating patients with Parkinson's disease with off-period dystonia and suggests that this treatment should be further verified in such patients.

  18. Development of validated RP-HPLC method for the estimation of L-Dopa from Mucuna pruriens, its extracts and in Aphrodisiac formulation.

    Bhumika G. Rathod


    Full Text Available In the present study, reversed phase HPLC method was developed for the estimation of L-Dopa from Mucuna Prurines, its extract and in Aphrodisiac formulation. HPLC analysis was performed on C18 column using a mixture of Water: Acetonitrile: Methanol containing 0.2 % Triethylamine PH adjusted to 3.3 as isocratic mobile phase at a flow rate of 1.0 ml per minute at detection wavelength of 280 nm. The method was validated for accuracy, precision, linearity, specificity and sensitivity in accordance with International conference on Harmonization guidelines. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r2 > 0.999 was obtained for calibration plots in the range of 10 – 80 μg/ml. Intraday and Interday RSD of retention times and peak areas were less than 2.0 %. Average Percent Recovery was 98.83 %. The method was successfully used for quantitative analysis of this marker compound in Polyherbal formulation.

  19. Positron emission tomography in drug evaluation: Influence of three different catechol-O-methyltransferase inhibitors on metabolism of [NCA] 6-[{sup 18}F]fluoro-l-dopa in Rhesus monkey

    Guenther, I.; Psylla, M.; Reddy, G.N.; Antonini, A.; Vontobel, P.; Reist, H.W.; Zollinger, A.; Nickles, R.J.; Beer, H.-F.; Schubiger, P.A.; Leenders, K.L


    We compared the influence of three different catechol-O-methyltransferase (COMT) inhibitors (CGP 28014, OR-611 and Ro 40-7592) on the metabolism of no-carrier-added (NCA) 6-[{sup 18}F]fluoro-l-dopa (6-FDOPA) in one Rhesus monkey. All three COMT inhibitors improved 6-FDOPA availability in plasma, increased the specific uptake in the brain and thus improved 6-FDOPA uptake measurements using positron emission tomography (PET). Best results were obtained with Ro 40-7592.

  20. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A


    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  1. Factors affecting the hydroxycinnamate decarboxylase/vinylphenol reductase activity of dekkera/brettanomyces: application for dekkera/brettanomyces control in red wine making.

    Benito, S; Palomero, F; Morata, A; Calderón, F; Suárez-Lepe, J A


    The growth of Dekkera/Brettanomyces yeasts during the ageing of red wines-which can seriously reduce the quality of the final product-is difficult to control. The present study examines the hydroxycinnamate decarboxylase/vinylphenol reductase activity of different strains of Dekkera bruxellensis and Dekkera anomala under a range of growth-limiting conditions with the aim of finding solutions to this problem. The yeasts were cultured in in-house growth media containing different quantities of growth inhibitors such as ethanol, SO(2), ascorbic acid, benzoic acid and nicostatin, different sugar contents, and at different pHs and temperatures. The reduction of p-coumaric acid and the formation of 4-ethylphenol were periodically monitored by HPLC-PDA. The results of this study allow the optimization of differential media for detecting/culturing these yeasts, and suggest possible ways of controlling these organisms in wineries.

  2. 13C magnetic resonance spectroscopy measurements with hyperpolarized [1‐13C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo

    Dzien, Piotr; Tee, Sui‐Seng; Kettunen, Mikko I.; Lyons, Scott K.; Larkin, Timothy J.; Timm, Kerstin N.; Hu, De‐En; Wright, Alan; Rodrigues, Tiago B.; Serrao, Eva M.; Marco‐Rius, Irene; Mannion, Elizabeth; D'Santos, Paula; Kennedy, Brett W. C.


    Purpose Dissolution dynamic nuclear polarization can increase the sensitivity of the 13C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize 13C‐labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC, which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. Methods Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using 13C MRS measurements of the conversion of hyperpolarized [1‐13C] pyruvate to H13 CO3–. Results Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two‐fold increase in the H13 CO3–/[1‐13C] pyruvate signal ratio following intravenous injection of hyperpolarized [1‐13C] pyruvate. Conclusion We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized 13C MRS. Magn Reson Med 76:391–401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMID:26388418

  3. Striatal activation by optogenetics induces dyskinesias in the 6-hydroxydopamine rat model of Parkinson disease.

    F Hernández, Ledia; Castela, Ivan; Ruiz-DeDiego, Irene; Obeso, Jose A; Moratalla, Rosario


    Long-term levodopa (l-dopa) treatment is associated with the development of l-dopa-induced dyskinesias in the majority of patients with Parkinson disease (PD). The etiopathogonesis and mechanisms underlying l-dopa-induced dyskinesias are not well understood. We used striatal optogenetic stimulation to induce dyskinesias in a hemiparkinsonian model of PD in rats. Striatal dopamine depletion was induced unilaterally by 6-hydroxydopamine injection into the medial forebrain bundle. For the optogenetic manipulation, we injected adeno-associated virus particles expressing channelrhodopsin to stimulate striatal medium spiny neurons with a laser source. Simultaneous optical activation of medium spiny neurons of the direct and indirect striatal pathways in the 6-hydroxydopamine lesion but l-dopa naïve rats induced involuntary movements similar to l-dopa-induced dyskinesias, labeled here as optodyskinesias. Noticeably, optodyskinesias were facilitated by l-dopa in animals that did not respond initially to the laser stimulation. In general, optodyskinesias lasted while the laser stimulus was applied, but in some instances remained ongoing for a few seconds after the laser was off. Postmortem tissue analysis revealed increased FosB expression, a molecular marker of l-dopa-induced dyskinesias, primarily in medium spiny neurons of the direct pathway in the dopamine-depleted hemisphere. Selective optogenetic activation of the dorsolateral striatum elicits dyskinesias in the 6-hydroxydopamine rat model of PD. This effect was associated with a preferential activation of the direct striato-nigral pathway. These results potentially open new avenues in the understanding of mechanisms involved in l-dopa-induced dyskinesias. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

  4. The effects of carbidopa on uptake of 6-18F-Fluoro-L-DOPA in PET of pheochromocytoma and extraadrenal abdominal paraganglioma.

    Timmers, H.J.L.M.; Hadi, M.; Carrasquillo, J.A.; Chen, C.C.; Martiniova, L.; Whatley, M.; Ling, A.; Eisenhofer, G.; Adams, K.T.; Pacak, K.


    6-(18)F-fluoro-l-3,4-dihydroxyphenylalanine ((18)F-DOPA) PET is a useful tool for the detection of certain neuroendocrine tumors, especially with the preadministration of carbidopa, an inhibitor of DOPA decarboxylase. Whether carbidopa also improves (18)F-DOPA PET of adrenal pheochromocytomas and ex

  5. L-DOPA-Coated Manganese Oxide Nanoparticles as Dual MRI Contrast Agents and Drug-Delivery Vehicles.

    McDonagh, Birgitte Hjelmeland; Singh, Gurvinder; Hak, Sjoerd; Bandyopadhyay, Sulalit; Augestad, Ingrid Lovise; Peddis, Davide; Sandvig, Ioanna; Sandvig, Axel; Glomm, Wilhelm Robert


    Manganese oxide nanoparticles (MONPs) are capable of time-dependent magnetic resonance imaging contrast switching as well as releasing a surface-bound drug. MONPs give T2/T2* contrast, but dissolve and release T1-active Mn(2+) and L-3,4-dihydroxyphenylalanine. Complementary images are acquired with a single contrast agent, and applications toward Parkinson's disease are suggested.

  6. L-dopa, biperideno e excreção sebácea na doença de Parkinson

    João C. B. Villares


    Full Text Available A excreção sebácea frontal de 47 parkinsonianos «de novo» antes e após tratamento com anticolinérgico (biperideno, levodopa + IDAA e bromocriptina foi avaliada pelo método do ácido ósmico. Outros 100 parkinsonianos sob terapêutica crônica com biperideno, levodopa + IDAA ou associação de ambos foram avaliados. Parkinsonianos «de novo» do sexo masculino apresentam valores de excreção sebácea significativamente mais elevados em relação às mulheres. Verificou-se que biperideno não foi eficaz em reduzir o grau de excreção sebácea. Já, em relação a L-dopa + IDAA constatou-se que a droga foi efetiva em reduzir o grau de excreção sebácea (NC e TRE tanto no sexo masculino quanto no feminino. Em relação à bromocriptina (l0mg/dia também constatou-se que houve redução da excreção sebácea no sexo masculino. Correlação significante positiva foi verificada entre o NC, tremor, bradicinesia, hipertonia, alterações da marcha e postura e incapacidade funcional, entre parkinsonianos do sexo masculino e faixa etária 50-59 anos, no período pré-tratamento. Após o período de tratamento não mais havia correlação entre excreção sebácea e as manifestações neurológicas da doença de Parkinson. Entre parkinsonianos sob terapêutica crônica verificou-se correlação positiva e significante entre excreção sebácea e bradicinesia. O grau de excreção sebácea de parkinsonianos «de novo» sem tratamento não difere do grau daqueles sob tratamento crônico, exceção feita a parkinsonianos com idade > 60 anos, em que verificou-se maior grau de excreção sebácea (NC e TRE em relação ao mesmo sexo e faixa etária, sem tratamento. L-dopa + IDAA foi eficiente em reduzir o grau de excreção sebácea de parkinsonianos «de novo», tornando-a significativamente menor em relação àqueles sob tratamento crônico. Não há diferença entre o grau de excreção sebácea de parkinsonianos «de novo» sem tratamento e

  7. Selection and Test of L-histidine Decarboxylase Enzyme Activity of Six Isolates of Histamine Forming Bacteria

    Romauli Aya Sophia


    Full Text Available Six isolates of histamine forming bacteria were screened to see the degree of ability in producing histamine on modified Niven's medium. The result showed that the six bacteria were able to produce histamine by giving a pinkish color on the medium, which could be used as a preliminary identification of histamine-forming bacteria (HFB. The isolates were grown in liquid modified Niven medium to measure the production of histamine. The histamine produced were determined by Hardy and Smith method. The result showed that all of the isolates produced high level of histamine (92.35 - 305.49 mg/100 ml of the medium. From all of them, Enterobacter spp. produced the highest level of histamine (305.49 mg/100 ml. A synthetic medium was used to measure the growth pattern and optimum time required by Enterobacter spp and Morganella morganii (as control bacteria to produce the L-histidine decarboxylase enzyme (HDC which is responsible for histamine production. The result showed that for both bacteria, the optimum enzim production was 8 hours after incubation.

  8. Relation between coumarate decarboxylase and vinylphenol reductase activity with regard to the production of volatile phenols by native Dekkera bruxellensis strains under 'wine-like' conditions.

    Sturm, M E; Assof, M; Fanzone, M; Martinez, C; Ganga, M A; Jofré, V; Ramirez, M L; Combina, M


    Dekkera/Brettanomyces bruxellensis is considered a major cause of wine spoilage, and 4-ethylphenol and 4-ethylguaiacol are the most abundant off-aromas produced by this species. They are produced by decarboxylation of the corresponding hydroxycinnamic acids (HCAs), followed by a reduction of the intermediate 4-vinylphenols. The aim of the present study was to examine coumarate decarboxylase (CD) and vinylphenol reductase (VR) enzyme activities in 5 native D. bruxellensis strains and determine their relation with the production of ethylphenols under 'wine-like' conditions. In addition, biomass, cell culturability, carbon source utilization and organic acids were monitored during 60 days. All strains assayed turned out to have both enzyme activities. No significant differences were found in CD activity, whilst VR activity was variable among the strains. Growth of D. bruxellensis under 'wine-like' conditions showed two growth phases. Sugars were completely consumed during the first growth phase. Transformation of HCAs into ethylphenols also occurred during active growth of the yeast. No statistical differences were observed in volatile phenol levels produced by the strains growing under 'wine-like' conditions, independently of the enzyme activity previously recorded. Furthermore, our results demonstrate a relationship between the physiological state of D. bruxellensis and its ability to produce ethylphenols. Inhibition of growth of D. bruxellensis in wine seems to be the most efficient way to avoid ethylphenol production and the consequent loss of wine quality. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Keto-isovalerate decarboxylase enzymes and methods of use thereof

    McElvain, Jessica; O'Keefe, Daniel P.; Paul, Brian James; Payne, Mark S.; Rothman, Steven Cary; He, Hongxian


    Provided herein are polypeptides and polynucleotides encoding such polypeptides which have ketoisovalerate decarboxylase activity. Also provided are recombinant host cells comprising such polypeptides and polynucleotides and methods of use thereof.

  10. Gene cloning of phenolic acid decarboxylase from Bacillus subtilis ...



    Aug 16, 2010 ... 1College of Food Engineering and Biotechnology, Tianjin University of ... Bacillus subtilis and ligated with a shuttle vector YEp352 to generate a novel plasmid YPADC. ... phenolic acid decarboxylase activity and its functions.

  11. Characterization of glutamate decarboxylase from Lactobacillus plantarum and its C-terminal function for the pH dependence of activity.

    Shin, Sun-Mi; Kim, Hana; Joo, Yunhye; Lee, Sang-Jae; Lee, Yong-Jik; Lee, Sang Jun; Lee, Dong-Woo


    The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454-Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0-8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values.

  12. Colonic ornithine decarboxylase in inflammatory bowel disease: ileorectal activity gradient, guanosine triphosphate stimulation, and association with epithelial regeneration but not the degree of inflammation and clinical features.

    Allgayer, Hubert; Roisch, Ulla; Zehnter, Elmar; Ziegenhagen, Dieter J; Dienes, Hans P; Kruis, Wolfgang


    The role of colonic mucosal ornithine decarboxylase (ODC) in inflammatory bowel disease (IBD) remains controversial. This study assessed mucosal ODC activity in IBD patients segment by segment with regard to patient characteristics, disease activity/duration, medication, degree of mucosal inflammation, and presence/absence of epithelial regeneration and guanosine triphosphate (GTP) stimulation. Mucosal ODC activity was determined in biopsy specimens from the terminal ileum, cecum/ascending, transverse, and descending colon, and the sigmoid/rectum of 35 patients with IBD (18 with Crohn's disease, 17 with ulcerative colitis) and 29 controls, using the amount of 14CO2 liberated from (carboxyl-14C)ornithine hydrochloride. GTP-stimulatable activity was expressed as the ratio of ODC activity in the presence and absence of GTP (70 micromol/L). Mucosal inflammation was assessed endoscopically/microscopically with previously described criteria. Presence/absence of mucosal regeneration also was determined by predefined criteria. Mucosal ODC-activity did not significantly differ in IBD patients and controls. There was a 4.4-fold activity gradient from the ileum to the rectum. Mucosal ODC activity was significantly higher in areas with epithelial regeneration compared to those without regeneration, and was stimulated by GTP by a factor of 1.42 in Crohn's disease and 1.19 in ulcerative colitis patients compared to controls (p < 0.004). On the other hand, there was no significant association/relationship of mucosal ODC activity with disease activity/duration and the endoscopic/histologic degree of mucosal inflammation. The observation of unchanged mucosal ODC activity in patients with IBD and the absence of a significant relationship with clinical and endoscopic/histologic disease characteristics speaks against a major role of ODC in IBD as a major disease marker. The role of the ileorectal gradient, the enhanced activity in areas with epithelial regeneration, and the GTP

  13. Inhibition of ileal and colonic ornithine decarboxylase activity by alpha-difluoromethylornithine in rats: transient atrophic changes and loss of postresectional adaptive growth.

    Kingsnorth, A N; Abu-Khalaf, M; LaMuraglia, G M; McCann, P P; Diekema, K A; Ross, J S; Malt, R A


    To determine the role of putrescine synthesis in adaptive hyperplasia of the ileum and colon, DL-alpha-difluoromethylornithine (DFMO), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase (ODC), the enzyme controlling putrescine biosynthesis, was fed to rats after excision of the proximal half of the small bowel. A rise in ODC activity (280% in the proximal ileum, 62% in the proximal colon) and a rise in putrescine content (220% in the proximal ileum, 250% in the proximal colon) normally accompanied characteristic cytochemical adaptive increases in the ileum and colon at day 6. Inclusion of 1% DFMO (2.1 gm/kg/day) in drinking water for 12 hours before operation and for 14 days thereafter decreased ODC activity by 85% to 96%, reduced levels of putrescine and spermidine and measurements of the adaptive response by 50% in the ileum, and abolished the adaptive response in the colon. During the first 10 days of DFMO feeding, villous atrophy and other hypoplastic changes occurred in control rats, but by 14 days of DFMO feeding atrophy and hypoplasia were no longer present. Although DFMO inhibits adaptive hyperplasia occurring in the ileum and colon of rats after resection of the proximal half of the small bowel, spontaneous recovery of villous atrophy occurs during further DFMO feeding and may protect the host during chemotherapy.

  14. Brain histaminergic system in mast cell-deficient (Ws/Ws) rats: histamine content, histidine decarboxylase activity, and effects of (S) alpha-fluoromethylhistidine.

    Sugimoto, K; Maeyama, K; Alam, K; Sakurai, E; Onoue, H; Kasugai, T; Kitamura, Y; Watanabe, T


    The mast cell-deficient [Ws/Ws (White spotting in the skin)] rat was investigated with regard to the origin of histamine in the brain. No mast cells were detected in the pia mater and the perivascular region of the thalamus of Ws/Ws rats by Alcian Blue staining. The histamine contents and histidine decarboxylase (HDC) activities of various brain regions of Ws/Ws rats were similar to those of +/+ rats except the histamine contents of the cerebral cortex and cerebellum. As the cerebral cortex and cerebellum have meninges that are difficult to remove completely, the histamine contents of these two regions may be different between Ws/Ws and +/+ rats. We assume that the histamine content of whole brain with meninges in Ws/Ws rats is < 60% of that in +/+ rats. So we conclude that approximately half of the histamine content of rat brain is derived from mast cells. Next, the effects of (S) alpha-fluoromethylhistidine (FMH), a specific inhibitor of HDC, on the histamine contents and HDC activities of various regions of the brain were examined in Ws/Ws rats. In the whole brain of Ws/Ws rats, 51 and 37% of the histamine content of the control group remained 2 and 6 h, respectively, after FMH administration (100 mg/kg of body weight). Therefore, we suggest that there might be other histamine pools including histaminergic neurons in rat brain.

  15. Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

    Park, Jae Hee; Lee, Chang Ki [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Oral Cancer Research Institute, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Hwang, Young Sun [Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Park, Kwang-Kyun [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Chung, Won-Yoon [Department of Oral Biology, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of); Department of Applied Life Science and Brain Korea 21 Project, Yonsei University College of Dentistry, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul 120-752 (Korea, Republic of)], E-mail:


    Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H{sub 2}O{sub 2} formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-{kappa}B) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-{kappa}B activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-{kappa}B signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.

  16. Regional haemodynamic effects of dopamine and its prodrugs L-dopa and gludopa in the rat and in the glycerol-treated rat as a model for acute renal failure.

    Drieman, J. C.; van Kan, F. J.; Thijssen, H H; Van Essen, H.; Smits, J. F.; Struijker Boudier, H. A.


    1. In this study the renal selectivity of dopamine and its prodrugs L-dopa and gludopa, with respect to their effects on regional blood flow, vascular resistance and central haemodynamics was investigated in normal rats and in rats with glycerol-induced acute renal failure (ARF). 2. In normal, anaesthetized rats, dopamine as well as its prodrugs caused a dose-dependent reduction of vascular resistance in the kidney (RR), mesentery (MR) and hindquarters (HQR) (dose range: dopamine: 0.1-5 mumol...

  17. Conformational Changes in Orotidine 5’-Monophosphate Decarboxylase: A Structure-Based Explanation for How the 5’-Phosphate Group Activates the Enzyme†

    Desai, Bijoy J.; Wood, McKay; Fedorov, Alexander A.; Fedorov, Elena V.; Goryanova, Bogdana; Amyes, Tina L.; Richard, John P.; Almo, Steven C.; Gerlt, John A.


    The binding of a ligand to orotidine 5’-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (Eo) to a closed, active conformation (Ec). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions are enforced that destabilize the carboxylate group of the substrate as well as stabilize the intermediate (in the Ec•S‡ complex). Focusing on the OMPDC from Methanothermobacter thermautotrophicus, the “remote” 5’-phosphate group of the substrate activates the enzyme 2.4 × 108-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that 1) directly contact the 5’-phosphate group; 2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from solvent; and 3) form hydrogen-bonding interactions across the interface between the “mobile” and “fixed” half-barrel domains of the (β/α8-barrel structure. Our data support a model in which the IBE provided by the 5’-phosphate group is used to enable interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec•S and Ec•S‡ complexes relative to the Eo•S complex. The conclusion that the IBE of the 5’-phosphate group provides stabilization of both the Ec•S and Ec•S‡ complexes, not just the Ec•S‡ complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions. PMID:23030629

  18. Tyrosine decarboxylase activity of enterococci grown in media with different nutritional potential: tyramine and 2-phenylethylamine accumulation and tyrDC gene expression.

    Bargossi, Eleonora; Tabanelli, Giulia; Montanari, Chiara; Lanciotti, Rosalba; Gatto, Veronica; Gardini, Fausto; Torriani, Sandra


    The ability to accumulate tyramine and 2-phenylethylamine by two strains of Enterococcus faecalis and two strains Enterococcus faecium was evaluated in two cultural media added or not with tyrosine. All the enterococcal strains possessed a tyrosine decarboxylase (tyrDC) which determined tyramine accumulation in all the conditions tested, independently on the addition of high concentration of free tyrosine. Enterococci differed in rate and level of biogenic amines accumulation. E. faecalis EF37 and E. faecium FC12 produced tyramine in high amount since the exponential growth phase, while 2-phenylethylamine was accumulated when tyrosine was depleted. E. faecium FC12 and E. faecalis ATCC 29212 showed a slower tyraminogenic activity which took place mainly in the stationary phase up to 72 h of incubation. Moreover, E. faecalis ATCC 29212 produced 2-phenylethylamine only in the media without tyrosine added. In BHI added or not with tyrosine the tyrDC gene expression level differed considerably depending on the strains and the growth phase. In particular, the tyrDC gene expression was high during the exponential phase in rich medium for all the strains and subsequently decreased except for E. faecium FC12. Even if tyrDC presence is common among enterococci, this study underlines the extremely variable decarboxylating potential of strains belonging to the same species, suggesting strain-dependent implications in food safety.

  19. Inhibitory effects of brown algae extracts on histamine production in mackerel muscle via inhibition of growth and histidine decarboxylase activity of Morganella morganii.

    Kim, Dong Hyun; Kim, Koth Bong Woo Ri; Cho, Ji Young; Ahn, Dong Hyun


    This study was performed to investigate the inhibitory effects of brown algae extracts on histamine production in mackerel muscle. First, antimicrobial activities of brown algae extracts against Morganella morganii were investigated using a disk diffusion method. An ethanol extract of Ecklonia cava (ECEE) exhibited strong antimicrobial activity. The minimum inhibitory concentration (MIC) of ECEE was 2 mg/ml. Furthermore, the brown algae extracts were examined for their ability to inhibit crude histidine decarboxylase (HDC) of M. morganii. The ethanol extract of Eisenia bicyclis (EBEE) and ECEE exhibited significant inhibitory activities (19.82% and 33.79%, respectively) at a concentration of 1 mg/ml. To obtain the phlorotannin dieckol, ECEE and EBEE were subjected to liquid-liquid extraction, silica gel column chromatography, and HPLC. Dieckol exhibited substantial inhibitory activity with an IC50 value of 0.61 mg/ml, and exhibited competitive inhibition. These extracts were also tested on mackerel muscle. The viable cell counts and histamine production in mackerel muscle inoculated with M. morganii treated with ≥2.5 MIC of ECEE (weight basis) were highly inhibited compared with the untreated sample. Furthermore, treatment of crude HDC-inoculated mackerel muscle with 0.5% ECEE and 0.5% EBEE (weight basis), which exhibited excellent inhibitory activities against crude HDC, reduced the overall histamine production by 46.29% and 56.89%, respectively, compared with the untreated sample. Thus, these inhibitory effects of ECEE and EBEE should be helpful in enhancing the safety of mackerel by suppressing histamine production in this fish species.

  20. Biosynthetic arginine decarboxylase in phytopathogenic fungi.

    Khan, A J; Minocha, S C


    It has been reported that while bacteria and higher plants possess two different pathways for the biosynthesis of putrescine, via ornithine decarboxylase (ODC) and arginine decarboxylase (ADC); the fungi, like animals, only use the former pathway. We found that contrary to the earlier reports, two of the phytopathogenic fungi (Ceratocystis minor and Verticillium dahliae) contain significant levels of ADC activity with very little ODC. The ADC in these fungi has high pH optimum (8.4) and low Km (0.237 mM for C. minor, 0.103 mM for V. dahliae), and is strongly inhibited by alpha-difluoromethylarginine (DFMA), putrescine and spermidine, further showing that this enzyme is probably involved in the biosynthesis of polyamines and not in the catabolism of arginine as in Escherichia coli. The growth of these fungi is strongly inhibited by DFMA while alpha-difluoromethylornithine (DFMO) has little effect.

  1. Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.

    Zeng, X; Farrenkopf, B; Hohmann, S; Dyda, F; Furey, W; Jordan, F


    Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

  2. [Inhibitory effect of essential oils, food additives, peracetic acid and detergents on bacterial histidine decarboxylase].

    Kamii, Eri; Terada, Gaku; Akiyama, Jyunki; Isshiki, Kenji


    The aim of this study is to examine whether various essential oils, food additives, peracetic acid and detergents inhibit bacterial histidine decarboxylase. Crude extract of Morganella morganii NBRC3848 was prepared and incubated with various agents. Histidine decarboxylase activity was significantly inhibited (pperacetic acid caused slight decomposition. Histidine and histamine were stable in the presence of the other 24 agents. These results indicated that 25 of the agents examined were inhibitors of histidine decarboxylase.

  3. Structures of Bacterial Biosynthetic Arginine Decarboxylases

    F Forouhar; S Lew; J Seetharaman; R Xiao; T Acton; G Montelione; L Tong


    Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. The TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.

  4. 5-HT1A Receptor Activation Improves Anti-Cataleptic Effects of Levodopa in 6-Hydroxydopamine-Lesioned Rats

    S. Reyhani-Rad


    Full Text Available Background and the purpose of the study: In Parkinsons disease (PD prolong use of L-DOPA causes some motor disorders such as wearing-off and L-DOPA induced dyskinesia (LID. In this investigation the effect of 8-OHDAPT, as a 5-HT1A agonist on anti-cataleptic effect of L-DOPA in 6-hydroxydopamine (6-OHDA lesioned male Wistar rats was investigated. Methods: Catalepsy was induced by unilateral injection of 6-OHDA (8 μg/2μl/rat into the central region of the SNc. After 3 weeks as a recovery period, animals received intraperitoneally (i.p. L-DOPA (15 mg/kg twice daily for 20 days, and anti-cataleptic effect of L-DOPA was assessed by bar-test at days of 5, 10, 15 and 20. Results and major conclusion: The results showed that L-DOPA had anti-cataleptic effect only until the day of 15, and its effect was decreased on the day of 20. On the day of 21, rats were co-injected with three different doses of 8-OHDAPT (0.1, 0.5 and 2.5 mg/kg, i.p. and L-DOPA (15 mg/kg, ip. 8-Hydroxy-2-(di-n-propylamino tetralin (8-OHDAPT improved anti-cataleptic effect of L-DOPA at the dose of 0.5 mg/kg. Moreover the effect of 8-OHDAPT on anti-cataleptic effect of L-DOPA (15 mg/kg, ip was abolished by 1-(2-methyoxyphenyl-4-[4-(2-phthalamido butyl] piperazine hydrobromide (NAN-190; 0.5 mg/kg, i.p. as a 5-HT1A receptor antagonist. According to the obtained results, it may be concluded that activation of 5-HT1A receptors by 8-OHDAPT may improve anti-cataleptic effect of L-DOPA in a 6-OHDA- induced rat model of PD. Further studies are required to clarify the exact mechanism of interaction between 5-HT1A and dopaminergic neurons.

  5. The effects of protaglandin E sub 2 and cyclooxygenase inhibition on ornithine decarboxylase activation and DNA synthesis during carbon tetrachloride-induced liver regeneration

    Shilstone, J.J.


    Increases in prostaglandin E{sub 2} (PGE{sub 2}) and ornithine decarboxylase (ODC) activity are necessary for liver regeneration following surgical partial hepatectomy (SPH). The purpose of this study was to examine liver regeneration induced by carbon tetrachloride (CCl{sub 4}) to determine whether DNA synthesis initiation mechanisms involving PGE{sub 2} and ODC operated in a similar manner to that seen in SPH. The rat chemical partial hepatectomy (CPH) model was established in our laboratory as a method to examine regenerative processes. A characteristic time course of {sup 3}H thymidine incorporation into DNA was demonstrated which peaked 48 hours following CPH. Increases in liver specific serum sorbitol dehydrogenase (sSDH) and glutamate-pyruvate transaminase (sGPT) indicated that significant necrotic damage had occurred in the liver as a result of CCl{sub 4} toxicity. Increased DNA synthesis and necrotic damage in the liver satisfied criteria for use of this procedure as a model of regeneration. Hepatic PGE{sub 2} synthesis was measured using radioimmunoassay (RIA) during the 12 hr period following CPH. Increases in PGE{sub 2} concentration were seen at 2, 4, 6, and 8 hrs. Indomethacin (50 mg/kg) administered intraperitoneally 90 minutes prior to CPH inhibited increases in PGE{sub 2}. Therefore, increased PGE{sub 2} seen during this time is due to cyclooxygenase. Indomethacin administration did not inhibit DNA synthesis measured by {sup 3}H thymidine incorporation into DNA at 24, 48, 72, and 96 hrs. Thus the increased PGE{sub 2} concentrations seen in the period immediately following CPH are not required for DNA synthesis. Therefore, different mechanisms of DNA synthesis initiation are operative in CPH and SPH.

  6. Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

    Yan Dong

    Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

  7. High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities

    Volke, A; Wegener, Gregers; Vasar, E


    regulate NOS activity. We aimed to develop a HPLC-based method to measure simultaneously the products of these three enzymes. Traditionally, the separation of amino acids and related compounds with HPLC has been carried out with precolumn derivatization and reverse phase chromatography. We describe here...... a simple and fast HPLC method with radiochemical detection to separate radiolabeled L-arginine, L-citrulline, L-ornithine, and agmatine. 3H-labeled L-arginine, L-citrulline, agmatine, and 14C-labeled L-citrulline were used as standards. These compounds were separated in the normal phase column (Allure...

  8. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

    The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

  9. 左旋多巴激发试验对儿童生长激素缺乏症的临床价值%Clinical Value of L-dopa Provocation Test in Children with Growth Hormone Dificiency

    王鹏; 程俊; 王永; 孔桂莲


    目的 探讨左旋多巴激发试验在诊断儿童生长激素缺乏症(GHD)的临床价值.方法 对330例身材矮小儿童应用左旋多巴激发试验,采用化学发光法进行生长激素(GH)检测.以测得的GH最高值为峰值,峰值≥10ng/ml为GH不缺乏,激发试验阳性;10ng/ml>峰值≥5ng/ml为GH部分缺乏,峰值<5ng/ml为GH缺乏,激发试验阴性.结果 激发后峰值强度为(12.23±8.10)ng/ml;峰值出现在(30~90)min者占96%,出现在120min者占4%(阳性3例),两者峰值有显著性差异(P<0.01);GH完全缺乏者占21%,部分缺乏者占22%,完全不缺乏者占57%.结论 左旋多巴激发试验可应用于临床GHD的诊断,但其诊断敏感度低,需要联合其它激发方式和其它指标对GHD患者进行综合评价.%Objective To investigate the clinical value of L-dopa provocation test on the diagnosis of growth hormone deficiency ( GHD) children. Methods The L-dopa provocation tests were performed in 330 subjects with short stature. Serum GH level was determined by chemilumineacence immunoassay. The highest GH value after provocation was termed as the peak value, the peak value was 10ng/ml or above as provocation teat positive (no GH deficiency ) ; 5ng/ml or above as part deficiency and lower than 5ng/ml as GH deficiency, provocation test negative. Results The peak value of serum GH after L-dopa provocation test was (12.23 ± 8.10) ng/ml;mostly focused on period of 30 to 90 min (96% ) ,only 4% were distributed at 120min after administration, the peak values were significant different between them(P<0.01). GH deficiency accouted for 21% , part deficiency accouted 22% individually, no GH deficiency accouted 57%. Conclusion The L-dopa provocation test could be used in clinical diagnosis of GHD, but as the low diagnostic sensitivity, it shoud be combined with other provocation testa or other indicators to comprehensive evaluated GHD.

  10. [Effect of a controlled low-protein diet on the pharmacological response to levodopa and on the plasma levels of L-dopa and amino acids in patients with Parkinson's disease].

    Sanchís, G; Mena, M A; Martín del Río, R; Morales, B; Casarejo, M J; de Yébenes, M J; Tabernero, C; Jiménez, A; de Yébenes, J G


    Levodopa is the treatment of choice in Parkinson's disease, but a high percentage of patients develop complications in the response, including fluctuations, after some years of treatment. Although the origin of fluctuations is unknown, these could be, at least partly, attributed to pharmacokinetic factors. Aromatic aminoacids interfere in the absorption and brain penetration of levodopa, and lowering protein intake improves the quality of the response. The continuation of a low-protein diet is difficult for some patients. In this way, to know if these diet effects are noticeable in an acute period would be interesting, in order to select groups of patients who were susceptible to improve with this treatment. In this report we have studied the acute effect of a low protein diet on the pharmacological response to levodopa, and the plasmatic levels of L-Dopa, 3-OM-Dopa and large neutral aminoacids. Protein restriction improves clinical response to levodopa, although the mechanisms of this improvement remain unknown.

  11. DL-alpha-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of DL-alpha-difluoromethylarginine to DL-alpha-difluoromethylornithine.

    Slocum, R D; Bitonti, A J; McCann, P P; Feirer, R P


    DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [( 3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.

  12. Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation.

    Burtin, D; Martin-Tanguy, J; Paynot, M; Rossin, N


    We studied the effects of dl-alpha-difluoromethylarginine (DFMA) and dl-alpha-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.

  13. Análise acústica da prosódia em mulheres com doença de Parkinson: efeito da Levodopa Acoustic analysis of prosody in females with Parkinson's disease: effect of L-dopa

    Luciana L. Azevedo


    Full Text Available MOTIVO DO ESTUDO: Pacientes com doença de Parkinson (DP apresentam anormalidades da fala caracterizadas por pequena variação da frequência fundamental (Fo e maior amplitude. O papel dos sistemas dopaminérgicos na patogênese dessas alterações e o efeito terapêutico da L-dopa não se encontram claramente determinados. O objetivo desta investigação é avaliar o efeito da L-dopa nas caraterísticas da prosódia de mulheres com DP. MÉTODO: Nós estudamos 8 mulheres com DP (68,4 ± 6,4 anos e 8 mulheres controles (63,5 ± 6,8 anos. As pacientes (estágio H-Y 2, uma; estágio 2,5, quatro; estágio 3, três foram examinadas quando off e on. A frequência fundamental intensidade e duração da fala de sentenças afirmativas foram analisadas com o programa WinPitch 1.8 (Philippe Martin®. Os parâmetros Fo analisados foram: Fo usual (Fo us, Fo máxima (Fo max, Fo mínima (Fo min, Fo max da tônica prénuclear (Fo max PNT, Fo min da tônica prénuclear (Fo min PNT, Fo max da tônica nuclear (Fo max NT, Fo min da tônica nuclear (Fo min TN, velocidade da variação melódica da PNT (VPNT, velocidade da variação melódica da NT (VNT, amplitude da variação melódica da PNT (APNT, e amplitude da variação melódica da NT (ANT. Duração da sentença (D, duração da PNT (DPNT, duração da NT (DNT e número de sílabas por segundo (NSS foram os parâmetros de duração investigados. Foram estudadas as seguintes variáveis da intensidade: intensidade média (MI, intensidade máxima (I max, intensidade mínima (I min, intensidade da PNT (IPNT e intensidade da NT (INT. Diferenças foram estatisticamente significativas se pBACKGROUND: Untreated Parkinson's disease (PD patients display speech abnormalities characterized by narrow range of fundamental frequency (Fo variation and higher amplitude. The role of dopaminergic systems in the pathogenesis of these changes as well as the therapeutic effect of L-dopa have not been clearly determined. The aim

  14. Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS Are Active In Vivo in Anemic Baboons and Transgenic Mice.

    Michael S Boosalis

    Full Text Available High-level fetal (γ globin expression ameliorates clinical severity of the beta (β hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

  15. Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

    Taupin, P; Ben-Ari, Y; Roisin, M P


    Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

  16. Crystal structure and Hirshfeld surface analysis of 1-carboxy-2-(3,4-dihydroxyphenylethan-1-aminium chloride 2-ammonio-3-(3,4-dihydroxyphenylpropanoate: a new polymorph of l-dopa HCl and isotypic with its bromide counterpart

    Perumal Kathiravan


    Full Text Available The title molecular salt, C9H12NO4+·Cl−·C9H11NO4, is isotypic with that of the bromide counterpart [Kathiravan et al. (2016. Acta Cryst. E72, 1544–1548]. The title salt is a second monoclinic polymorph of the l-dopa HCl structure reported earlier in the monoclinic space group P21 [Jandacek & Earle (1971. Acta Cryst. B27, 841–845; Mostad & Rømming (1974. Acta Chemica Scand. B28, 1161–1168]. In the title compound, monoclinic space group I2, one of the dopa molecules has a positive charge with a protonated α-amino group and the α-carboxylic acid group uncharged, while the second dopa molecule has a neutral charge, the α-amino group is protonated and the α-carboxylic acid is deprotonated. In the previously reported form, a single dopa molecule is observed in which the α-amino group is protonated and the α-carboxylic acid group is uncharged. The invariant and variations of various types of intermolecular interactions present in these two forms of dopa HCl structures are discussed with the aid of two-dimensional fingerprint plots.

  17. Characterization of arginine decarboxylase from Dianthus caryophyllus.

    Ha, Byung Hak; Cho, Ki Joon; Choi, Yu Jin; Park, Ky Young; Kim, Kyung Hyun


    Arginine decarboxylase (ADC, EC is a key enzyme in the biosynthesis of polyamines in higher plants, whereas ornithine decarboxylase represents the sole pathway of polyamine biosynthesis in animals. Previously, we characterized a genomic clone from Dianthus caryophyllus, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 78 kDa. In the present study, the ADC gene was subcloned into the pGEX4T1 expression vector in combination with glutathione S-transferase (GST). The fusion protein GST-ADC was water-soluble and thus was purified by sequential GSTrap-arginine affinity chromatography. A thrombin-mediated on-column cleavage reaction was employed to release free ADC from GST. Hiload superdex gel filtration FPLC was then used to obtain a highly purified ADC. The identity of the ADC was confirmed by immunoblot analysis, and its specific activity with respect to (14)C-arginine decarboxylation reaction was determined to be 0.9 CO(2) pkat mg(-1) protein. K(m) and V(max) of the reaction between ADC and the substrate were 0.077 +/- 0.001 mM and 6.0 +/- 0.6 pkat mg(-1) protein, respectively. ADC activity was reduced by 70% in the presence of 0.1 mM Cu(2+) or CO(2+), but was only marginally affected by Mg(2+), or Ca(2+) at the same concentration. Moreover, spermine at 1 mM significantly reduced its activity by 30%.

  18. Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis



    In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continueddevelopment of the injected embryos. These results indicate that cells overexpressed with SAMDC undergoapoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a "fail-safe" mechanism to eliminatephysiologically-severely damaged cells to save the rest of the embryo.

  19. Simultaneous determination of L- DOPA, DA and its metabolites,hydroxyl free radical in brain microdialysis of rats%大鼠脑微透析样品中左旋多巴、多巴胺及其代谢产物、羟自由基的测定

    吴兆恩; 牛晓红; 张美玉; 王志国; 李鹏; 王丹巧


    Objective: To establish a rapid, accurate HPLC - ECD method to simultaneously detect the L - di-hydroxyphenylalanine(L - DOPA) ,dopamine( DA) and its metabolites,hydroxyl free radical. Method: The ana-lytes were separated on a Antec Leyden BV C18 column(3 μm,2. 1 mm × 100 mm) under the same mobile phase. The electrochemical detector was equipped with a glass carbon working electrode and a Ag/AgCl reference electrode. The working voltage was 0. 52 V. Result: The extracellular L - dihydroxyphenylalanine( L - DOPA ) , DA and its metabolites, hydroxyl free radical in striatum of free - moving rats can be separated in 15 min. The calibration curves were linear within the limited concentration ranges. Conclusion:This method can be used to determine simultaneously the concentrations of L - DOPA, DA and its metabolites from the brain microdialysis of rats, providing the possible method of simultaneously measuring the concentration of L - DOPA in brain, target efficacy and studying the side effects of drugs.%目的:建立快速、准确、同时测定大鼠脑微透析样品中左旋多巴(L-DOPA)、多巴胺(DA)及其代谢产物、羟自由基指标的高效液相色谱-电化学检测方法.方法:应用玻璃碳工作电极、银/氯化银( Ag/AgCl)参比电极、Antec Leyden BV C18分析柱,工作电压为0.52 V,单一流动相,检测大鼠脑微透析样品.结果:清醒自由活动大鼠纹状体微透析液中L-DOPA、DA及其代谢产物、羟自由基指标等10种物质在15 min内分离.标准曲线在一定浓度内线性良好.结论:所建方法可用于大鼠脑微透析样品中L- DOPA、DA及其代谢产物等的同时测定,为脑内L-DOPA药物浓度-药物效应-药物副作用同步研究提供可用的方法.

  20. Arginine decarboxylase as the source of putrescine for tobacco alkaloids

    Tiburcio, A. F.; Galston, A. W.


    The putrescine which forms a part of nicotine and other pyrrolidine alkaloids is generally assumed to arise through the action of ornithine decarboxylase (ODC). However, we have previously noted that changes in the activity of arginine decarboxylase (ADC), an alternate source of putrescine, parallel changes in tissue alkaloids, while changes in ODC activity do not. This led us to undertake experiments to permit discrimination between ADC and ODC as enzymatic sources of putrescine destined for alkaloids. Two kinds of evidence presented here support a major role for ADC in the generation of putrescine going into alkaloids: (a) A specific 'suicide inhibitor' of ADC effectively inhibits the biosynthesis of nicotine and nornicotine in tobacco callus, while the analogous inhibitor of ODC is less effective, and (b) the flow of 14C from uniformly labelled arginine into nicotine is much more efficient than that from ornithine.

  1. Combined Sepiapterin Reductase and Methylmalonyl-CoA Epimerase Deficiency in a Second Patient: Cerebrospinal Fluid Polyunsaturated Fatty Acid Level and Follow-Up Under L-DOPA, 5-HTP and BH4 Trials.

    Mazzuca, Michel; Maubert, Marie-Anne; Damaj, Léna; Clot, Fabienne; Cadoudal, Marylène; Dubourg, Christele; Odent, Sylvie; Benoit, Jean François; Bahi-Buisson, Nadia; Christa, Laurence; de Lonlay, Pascale


    Objective/context: We describe the second patient presenting the combination of two homoallelic homozygous nonsense mutations in two genes distant from 1.8 Mb in the chromosome 2p13-3, the methylmalonyl-CoA epimerase gene (MCEE) and the sepiapterin reductase gene (SPR). The patient was born from consanguineous parents. He has presented a moderate but constant methylmalonic acid (MMA) excretion in urine associated with a mental retardation. The first homozygous mutation was identified in the MCEE gene (c.139C>T; p.Arg47*). Progressive dystonia and cataplexy narcolepsy led to diagnose the second homozygous mutation in the SPR gene: c.751A>T; p.Lys251*. Sepiapterin reductase deficiency (SRD) was characterized by a defect in tetrahydrobiopterin (BH4), the cofactor of several hydroxylases needed for the synthesis of neurotransmitters. A treatment with L-DOPA/carbidopa and 5-HTP dramatically improved the dystonic posture, the mood and the hypersomnia, proving that the pathogenesis was due to SRD. A supplementation with BH4 did not induce additional clinical benefit, although HVA and HIAA increased in CSF. The polyunsaturated fatty acids were measured in CSF as the markers of the neuronal stress. We have shown that DHA and its precursor EPA were high before and during the time course of the different treatments. The patient has inherited two copies of the two mutations from his consanguineous parents in the MCEE and SPR genes in the chromosome 2p13-3. DHA and EPA increased in CSF as a response to the neuronal stress induced by the defect in neurotransmitters or the altered metabolism of the odd-chain fatty acids and cholesterol.

  2. 6-[18F]Fluoro-L-DOPA: A Well-Established Neurotracer with Expanding Application Spectrum and Strongly Improved Radiosyntheses

    M. Pretze


    Full Text Available For many years, the main application of [18F]F-DOPA has been the PET imaging of neuropsychiatric diseases, movement disorders, and brain malignancies. Recent findings however point to very favorable results of this tracer for the imaging of other malignant diseases such as neuroendocrine tumors, pheochromocytoma, and pancreatic adenocarcinoma expanding its application spectrum. With the application of this tracer in neuroendocrine tumor imaging, improved radiosyntheses have been developed. Among these, the no-carrier-added nucleophilic introduction of fluorine-18, especially, has gained increasing attention as it gives [18F]F-DOPA in higher specific activities and shorter reaction times by less intricate synthesis protocols. The nucleophilic syntheses which were developed recently are able to provide [18F]F-DOPA by automated syntheses in very high specific activities, radiochemical yields, and enantiomeric purities. This review summarizes the developments in the field of [18F]F-DOPA syntheses using electrophilic synthesis pathways as well as recent developments of nucleophilic syntheses of [18F]F-DOPA and compares the different synthesis strategies regarding the accessibility and applicability of the products for human in vivo PET tumor imaging.

  3. Occurrence of pre-MBT synthesis of caspase-8 mRNA and activation of caspase-8 prior to execution of SAMDC (S-adenosylmethionine decarboxylase)-induced, but not p53-induced, apoptosis in Xenopus late blastulae.

    Shiokawa, Koichiro; Takayama, Eiji; Higo, Takayasu; Kuroyanagi, Shinsaku; Kaito, Chikara; Hara, Hiroshi; Kajitani, Masayuki; Sekimizu, Kazuhisa; Tadakuma, Takushi; Miura, Kin-Ichiro; Igarashi, Kazuei; Yaoita, Yoshio


    Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.

  4. Expression of arginine decarboxylase and ornithine decarboxylase genes in apple cells and stressed shoots.

    Hao, Yu-Jin; Kitashiba, Hiroyasu; Honda, Chikako; Nada, Kazuyoshi; Moriguchi, Takaya


    Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) are two important enzymes responsible for putrescine biosynthesis. In this study, a full-length ADC cDNA (MdADC) was isolated from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. Meanwhile, a partial ODC (pMdODC) could be amplified only by a second RCR from the RT-PCR products, whereas a full-length ODC could not be obtained by either cDNA library screening or 5'- and 3'-RACEs, suggesting quite low expression. Moreover, D-arginine, an ADC inhibitor, caused a decrease in ADC activity and severely inhibited the growth of apple callus, which could be partially resumed by exogenous addition of putrescine, whereas alpha-difluoromethylornithine (DFMO), an inhibitor for ODC, caused the incomplete repression of callus growth without changing ODC activity. RNA gel blot showed that the expression level of MdADC was high in young tissues/organs with rapid cell division and was positively induced by chilling, salt, and dehydration, implying its involvement in both cell growth and these stress responses. By contrast, the transcript of ODC could not be detected by RNA gel blot analysis. Based on the present study, it is possible to conclude that (i) the ODC pathway is active in apple, although the expression level of the pMdODC gene homologous with its counterparts found in other plant species is quite low; and (ii) MdADC expression correlates with cell growth and stress responses to chilling, salt, and dehydration, suggesting that ADC is a primary biosynthetic pathway for putrescine biosynthesis in apple.

  5. 精氨酸、可乐定、精氨酸联合左旋多巴不同激发试验对GH分泌的影响%Influence of different motivation test with arginine,clonidine,arginine combined with L-dopa,clonidine in GH secretion

    吴红玲; 陈雪梅; 陈爱劳; 钟林; 钟梅珍; 刘军平; 罗锦成


    Objective To compare the effect of different motivation test with arginine,clonidine, arginine combined with L-dopa in GH secretion of dwarfism children. Methods 60 GH children patients from June 2013 to December 2014 of our hospital were selected and retrospectively analyzed.Patients were divided into GHD group (n=30) and non GHD group (n=30) by diagnosis results.Patients were treated with drug motivation test by using arginine,clonidine,arginine combined with L-dopa.The impact of different motivation test on GH secretion was compared. Results The peak value of arginine,clonidine and arginine combined with L-dopa in GHD group were lower than non GHD group,and the dif-ferences were statistically significant (P<0.05).The peak value of arginine combined with L-dopa in two groups were higher than arginine and clonidine,and the differences were statistically significant (P<0.05).The sensitivity,specificity and accuracy of arginine combined with L-dopa were higher than arginine and clonidine,and the differences were sta-tistically significant (P<0.05). Conclusion The sensitivity,specificity and accuracy of arginine combined with L-dopa is higher.The clinical value is higher in checking GH secretion of dwarfism children.%目的:比较精氨酸、可乐定、精氨酸联合左旋多巴不同激发试验对矮小症儿童脑垂体生长激素(GH)分泌的影响。方法选取我院自2013年6月~2014年12月收治的GH患儿60例进行回顾性分析,根据诊断结果进行分组,共分为两组:生长激素缺乏症(GHD)组(30例)和非GHD组(30例),对其进行精氨酸药物激发试验、可乐定药物激发试验精、氨酸联合左旋多巴药物激发试验,比较不同激发试验对GH分泌的影响。结果 GHD组精氨酸联合左旋多巴、精氨酸、可乐定的峰值均低于非GHD组,差异有统计学意义(P<0.05)。两组精氨酸联合左旋多巴的峰值均高于精氨酸、可乐定,差异有统计学意义(P<0.05)

  6. Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate.

    Kuo, D J; Jordan, F


    (E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.

  7. Crystal structure and Hirshfeld surface analysis of 1-carboxy-2-(3,4-dihydroxyphenyl)ethan-1-aminium chloride 2-ammonio-3-(3,4-dihydroxyphenyl)propanoate: a new polymorph of l-dopa HCl and isotypic with its bromide counterpart

    Perumal Kathiravan; Thangavelu Balakrishnan; Perumal Venkatesan; Kandasamy Ramamurthi; María Judith Percino; Subbiah Thamotharan


    The title molecular salt, C9H12NO4+·Cl−·C9H11NO4, is isotypic with that of the bromide counterpart [Kathiravan et al. (2016). Acta Cryst. E72, 1544–1548]. The title salt is a second monoclinic polymorph of the l-dopa HCl structure reported earlier in the monoclinic space group P21 [Jandacek & Earle (1971). Acta Cryst. B27, 841–845; Mostad & Rømming (1974). Acta Chemica Scand. B28, 1161–1168]. In the title compound, monoclinic space group I2, one of the dopa molecules has a positive charge wit...

  8. Crystal structure and Hirshfeld surface analysis of 1-carb­oxy-2-(3,4-di­hydroxy­phen­yl)ethan-1-aminium chloride 2-ammonio-3-(3,4-di­hydroxy­phen­yl)propano­ate: a new polymorph of l-dopa HCl and isotypic with its bromide counterpart

    Kathiravan, Perumal; Balakrishnan, Thangavelu; Venkatesan, Perumal; Ramamurthi, Kandasamy; Percino, María Judith; Thamotharan, Subbiah


    The title mol­ecular salt, C9H12NO4 +·Cl−·C9H11NO4, is isotypic with that of the bromide counterpart [Kathiravan et al. (2016 ▸). Acta Cryst. E72, 1544–1548]. The title salt is a second monoclinic polymorph of the l-dopa HCl structure reported earlier in the monoclinic space group P21 [Jandacek & Earle (1971 ▸). Acta Cryst. B27, 841–845; Mostad & Rømming (1974 ▸). Acta Chemica Scand. B28, 1161–1168]. In the title compound, monoclinic space group I2, one of the dopa mol­ecules has a positive c...

  9. Sustained increase of PKA activity in the postcommissural putamen of dyskinetic monkeys.

    Azkona, Garikoitz; Marcilla, Irene; López de Maturana, Rakel; Sousa, Amaya; Pérez-Navarro, Esther; Luquin, Maria-Rosario; Sanchez-Pernaute, Rosario


    Levodopa-induced dyskinesias (LID) are a frequent complication of Parkinson's disease pharmacotherapy that causes significant disability and narrows the therapeutic window. Pharmacological management of LID is challenging partly because the precise molecular mechanisms are not completely understood. Here, our aim was to determine molecular changes that could unveil targetable mechanisms underlying this drug complication. We examined the expression and downstream activity of dopamine receptors (DR) in the striatum of 1-methyl-4-phenyl-1,2,3,6 tetrahydropiridine (MPTP)-lesioned monkeys with and without L-DOPA treatment. Four monkeys were made dyskinetic and other four received a shorter course of L-DOPA and did not develop LID. Our results show that L-DOPA treatment induces an increase in DRD2 and DRD3 expression in the postcommissural putamen, but only DRD3 is correlated with the severity of LID. Dyskinetic monkeys show a hyperactivation of the canonical DRD1-signaling pathway, measured by an increased phosphorylation of protein kinase A (PKA) and its substrates, particularly DARPP32. In contrast, activation of the DRD2-signaling pathway, visible in the levels of Akt phosphorylated on Thr308 and GSK3β on Ser9, is associated with L-DOPA treatment, independently of the presence of dyskinesias. Our data clearly demonstrate that dyskinetic monkeys present a dysregulation of the DRD3 receptor and the DRD1 pathway with a sustained increase of PKA activity in the postcommissural putamen. Importantly, we found that all signaling changes related to long-term L-DOPA administration are exquisitely restricted to the postcommissural putamen, which may be related to the recurrent failure of pharmacological approaches.

  10. Mutation of His465 Alters the pH-dependent Spectroscopic Properties of Escherichia coli Glutamate Decarboxylase and Broadens the Range of Its Activity toward More Alkaline pH

    Pennacchietti, E.; Lammens, T.M.; Capitani, G.; Franssen, M.C.R.; John, R.A.; Bossa, F.; Biase, De D.


    Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5'-phosphate-dependent enzyme catalyzing CO2 release from the a-carboxyl group of l-glutamate to yield ¿-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperat

  11. Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

    Maupin-Furlow, Julie A [Gainesville, FL; Talarico, Lee Ann [Gainesville, FL; Raj, Krishnan Chandra [Tamil Nadu, IN; Ingram, Lonnie O [Gainesville, FL


    The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

  12. Comparison of ultraviolet light-induced skin carcinogenesis and ornithine decarboxylase activity in sencar and hairless SKH-1 mice fed a constant level of dietary lipid varying in corn and coconut oil.

    Berton, T R; Fischer, S M; Conti, C J; Locniskar, M F


    To investigate the effect of various levels of corn oil and coconut oil on ultraviolet (UV) light-induced skin tumorigenesis and ornithine decarboxylase (ODC) activity, Sencar and SKH-1 mice were fed one of three 15% (weight) fat semipurified diets containing three ratios of corn oil to coconut oil: 1.0%:14.0%, 7.9%:7.1%, and 15.0%:0.0% in Diets A, B, and C, respectively. Groups of 30 Sencar and SKH-1 mice were fed one of the diets for three weeks before UV irradiation; then both strains were UV irradiated with an initial dose of 90 mJ/cm2. The dose was given three times a week and increased 25% each week. For Sencar mice (irradiated 33 wks for a total dose of 48 J/cm2), tumor incidence reached a maximum of 60%, 60%, and 53% for Diets A, B, and C, respectively, with an overall average of one to two tumors per tumor-bearing animal. For the SKH-1 mice (irradiated 29 wks for a total dose of 18 J/cm2), all diet groups reached 100% incidence by 29 weeks, with approximately 12 tumors per tumor-bearing mouse. No significant effect of dietary corn oil/coconut oil was found for tumor latency, incidence, or yield in either strain. The effect of increasing corn oil on epidermal ODC activity in chronically UV-irradiated Sencar and SKH-1 mice was assessed. Three groups of mice from each strain were fed one of the experimental diets and UV irradiated for six weeks. Sencar mice showed no increase in ODC activity until six weeks of treatment, when the levels of ODC activity in the UV-irradiated mice fed Diet A were significantly higher than those in mice fed Diet B or Diet C: 1.27, 0.55, and 0.52 nmol/mg protein/hr, respectively. In the SKH-1 mice, ODC activity was increased by the first week of UV treatment, and by three weeks of treatment a dietary effect was observed; ODC activity was significantly higher in mice fed Diet C (0.70 nmol/mg protein/hr) than in mice fed Diet A (0.18 nmol/mg protein/hr). Although there was no significant effect of dietary corn oil/coconut oil on UV

  13. Resolution of brewers' yeast pyruvate decarboxylase into two isozymes.

    Kuo, D J; Dikdan, G; Jordan, F


    A novel purification method was developed for brewers' yeast pyruvate decarboxylase (EC that for the first time resolved the enzyme into two isozymes on DEAE-Sephadex chromatography. The isozymes were found to be distinct according to sodium dodecyl sulfate polyacrylamide gel electrophoresis: the first one to be eluted gave rise to one band, the second to two bands. The isozymes were virtually the same so far as specific activity, KM, inhibition kinetics and irreversible binding properties by the mechanism-based inhibitor (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid are concerned. This finding resolves a longstanding controversy concerning the quaternary structure of this enzyme.

  14. Conformational Stabilization of Rat S-Adenosylmethionine Decarboxylase by Putrescine

    和田, 牧子; 白幡, 晶


    The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using m...

  15. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P; Geller, Alfred I


    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine

  16. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P.; Geller, Alfred I.


    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an ∼9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported ∼90 % glutamatergic neuron-specific expression. The GAD67 promoter supported ∼90 % GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic

  17. Glucocorticoids modulate the response of ornithine decarboxylase to unilateral removal of the dorsal hippocampus

    De Kloet, E R; Cousin, M A; Veldhuis, H D; Voorhuis, T D; Lando, D


    The effect of unilateral removal of the dorsal hippocampus and of glucocorticoid administration was measured on the activity of ornithine decarboxylase (ODC) in the remaining contralateral hippocampus lobe. Unilateral hippocampectomy (Hx) resulted in a rapid rise of ODC activity in the contralateral

  18. Chromosomal Integration and Expression of Two Bacterial alpha-Acetolactate Decarboxylase Genes in Brewer's Yeast.

    Blomqvist, K; Suihko, M L; Knowles, J; Penttilä, M


    A bacterial gene encoding alpha-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the alpha-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the alpha-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.

  19. Retina maturation following administration of thyroxine in developing rats: effects on polyamine metabolism and glutamate decarboxylase.

    Macaione, S; Di Giorgio, R M; Nicotina, P A; Ientile, R


    The effects of subcutaneous daily treatment with thyroxine on cell proliferation, differentiation, polyamines, and gamma-aminobutyric acid metabolism in the rat retina were studied during the first 20 postnatal days. The retinal layers of the treated rats displayed an enhanced cell differentiation which reached its maximum 9-12 days from birth; but this effect stopped very quickly and was finished by the 20th postnatal day. Primarily there was an increase in ornithine decarboxylase activity which was accompanied by an increase in putrescine, spermidine, and spermine levels. S-Adenosylmethionine decarboxylase was induced later than ODC; corresponding with the enhanced synaptogenesis, glutamate decarboxylase increased 15-fold between the fourth and 15th days. Our data are consistent with the hypothesis that thyroxine may exert some of its effects by inducing the enzymes which regulate polyamine metabolism and synaptogenesis.

  20. Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a.

    Vanderslice, P; Copeland, W C; Robertus, J D


    Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram-positive organisms.

  1. Effects of hyperbaric oxygen combined with acupuncture and low-dose L-dopa on the improvement of life quality of the patients with Parkinson's disease%高压氧、针灸联合小剂量美多巴改善帕金森综合征患者生活质量的临床研究

    闫各; 崔倩倩; 孔敏


    Objective To investigate the effects of hyperbaric oxygen ( HBO ) combined with acupuncture and low-dose L-dopa on the improvement of motor and non-motor symptoms and life quality of the patients with Parkinson's disease.Methods One hundred and forty PD patients were chosen for our study . With the knowledge and consent of the patients and their dependents , the patients were divided into 4 groups, by different treatment methods:group A (the acupuncture plus small-dose L-dopa group, n=20 cases), group B ( the HBO plus small-dose L-dopa group , n=20 cases ) , group C ( the HBO plus acupuncture and plus small-dose L-dopa group , n =60 cases ) , and group D ( the small-dose L-dopa group , n =40 cases ) . Improvement of motor and non-motor symptoms and their life quality of the patients were assessed by using the EQ-5D, Uniform Parkinson's Disease Rating Scale (UPDRS), Hoehn-Yahr (H-Y) Rating Scale, Parkinson's Disease Sleep Scale ( PDSS) , Hamilton Anxiety Rating Scale ( HAMA) and Hamilton Depression Rating Scale (HAMD).Results (1)After one month of treatment, the scores of UPDRS I, UPDRS II and UPDRSⅢfor group A were respectively 7.71, 6.14 and 6.50, the scores for group B were respectively 6.87, 6.09 and 6.66, the scores for group C were respectively 6.69, 6.66 and 9.68, and the scores for group D were respectively 7.79, 6.07 and 6.41.The scores of HAMA, HAMD, PDSS and EQ-5D were significantly improved as compared with those before treatment (P<0.05), indicating that the 4 treatment profiles within a relatively short time could all improve the motor and non-motor symptoms and life quality of the PD patients The scores of UPDRSⅢ, HAMA, HAMD and PDSS for group C were more significantly improved (P<0.05).(2) After 6 months of prolonged treatment , statistical significance could be noted in the scores of UPDRS Ⅲ, HAMA, HAMD and PDSS in the patients of group C (P <0.05).(3) Following 6 months of prolonged treatment , the life quality of the patients in group C

  2. Tyrosine decarboxylase activity of enterococci grown in media with different nutritional potential: tyramine and 2-phenylethylamine accumulation and tyrDC gene expression

    Eleonora eBargossi


    Full Text Available The ability to accumulate tyramine and 2-phenylethylamine by two strains of Enterococcus faecalis and two strains Enterococcus faecium was evaluated in two cultural media added or not with tyrosine. All the enterococcal strains possessed a tyrDC which determined tyramine accumulation in all the conditions tested, independently on the addition of high concentration of free tyrosine. Enterococci differed in rate and level of biogenic amines accumulation. E. faecalis EF37 and E. faecium FC12 produced tyramine in high amount since the exponential growth phase, while 2-phenylethylamine was accumulated when tyrosine was depleted. Enterococcus faecium FC12 and E. faecalis ATCC 29212 showed a slower tyraminogenic activity which took place mainly in the stationary phase up to 72 h of incubation. Moreover, E. faecalis ATCC 29212 produced 2-phenylethylamine only in the media without tyrosine added. In BHI added or not with tyrosine the tyrDC gene expression level differed considerably depending on the strains and the growth phase. In particular, the tyrDC gene expression was high during the exponential phase in rich medium for all the strains and subsequently decreased except for E. faecium FC12. Even if tyrDC presence is common among enterococci, this study underlines the extremely variable decarboxylating potential of strains belonging to the same species, suggesting strain-dependent implications in food safety.

  3. The effect of lactose, NaCl and an aero/anaerobic environment on the tyrosine decarboxylase activity of Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis.

    Buňková, Leona; Buňka, František; Pollaková, Eva; Podešvová, Tereza; Dráb, Vladimír


    The aim of this work was to study, under model conditions, combined effects of the concentration of lactose (0-1% w/v), NaCl (0-2% w/v) and aero/anaerobiosis on the growth and tyramine production in 3 strains of Lactococcus lactis subsp. lactis and 2 strains of L. lactis subsp. cremoris. The levels of the factors tested were chosen with respect to the conditions which can occur during the real process of natural cheese production, including the culture temperature (10 ± 1°C). In all strains tested, tyrosine decarboxylation was most influenced by NaCl concentration; the highest production of tyramine was obtained within the culture with the highest (2% w/v) salt concentration applied. Two of the strains L. lactis subsp. lactis produced tyramine only in broth with the highest NaCl concentration tested. In the remaining 3 strains of L. lactis, tyramine was detected under all conditions applied. The tested concentration of lactose and aero/anaerobiosis had a less significant effect on tyramine decarboxylation. However, it was also found that at the same concentrations of NaCl and lactose, a higher amount of tyramine was detected under anaerobic conditions. In all strains tested, tyramine decarboxylation started during the active growth phase of the cells.

  4. The ornithine decarboxylase gene of Caenorhabditis elegans: Cloning, mapping and mutagenesis

    Macrae, M.; Coffino, P. [Univ. of California, San Francisco, CA (United States); Plasterk, R.H.A. [Netherlands Cancer Institute, Amsterdam (Netherlands)


    The gene (odc-1) encoding ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned and characterized. Two introns interrupt the coding sequence of the gene. The deduced protein contains 442 amino acids and is homologous to ornithine decarboxylases of other eukaryotic species. In vitro translation of a transcript of the cDNA yielded an enzymatically active product. The mRNA is 1.5 kb in size and is formed by trans-splicing to SL1, a common 5{prime} RNA segment. odc-1 maps to the middle of LG V, between dpy-11 and unc-42 and near a breakpoint of the nDf32 deficiency strain. Enzymatic activity is low in starved 1 (L1) larva and, after feeding, rises progressively as the worms develop. Targeted gene disruption was used to create a null allele. Homozygous mutants are normally viable and show no apparent defects, with the exception of a somewhat reduced brood size. In vitro assays for ornithine decarboxylase activity, however, show no detectable enzymatic activity, suggesting that ornithine decarboxylase is dispensible for nematode growth in the laboratory. 37 refs., 6 figs., 1 tab.

  5. Evidence for the existence of mammalian acetoacetate decarboxylase: with special reference to human blood serum

    Stekelenburg, Gerard J. van; Koorevaar, Gerrit

    In this article evidence is presented for the existence of mammalian acetoacetate decarboxylase (acetoacetate carboxy-lyase: E.G. From experiments with human blood serum the presence of a non-ultrafiltrable activator, accelerating the decomposition of acetoacetate into acetone and carbon

  6. Evidence for the existence of mammalian acetoacetate decarboxylase: with special reference to human blood serum

    Stekelenburg, Gerard J. van; Koorevaar, Gerrit


    In this article evidence is presented for the existence of mammalian acetoacetate decarboxylase (acetoacetate carboxy-lyase: E.G. From experiments with human blood serum the presence of a non-ultrafiltrable activator, accelerating the decomposition of acetoacetate into acetone and carbon d

  7. Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases.

    Moral, Mario E G; Tu, Chingkuang; Richards, Nigel G J; Silverman, David N


    Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the nonoxidative reaction but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO(2) and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO(2) from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO(2) using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O(2) using MIMS also estimates the oxidase activity. The use of isotope-labeled substrate ((13)C(2)-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product (13)CO(2) (m/z 45), avoiding inaccuracies due to endogenous (12)CO(2). Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Complex evolution of orthologous and paralogous decarboxylase genes.

    Sáenz-de-Miera, L E; Ayala, F J


    The decarboxylases are involved in neurotransmitter synthesis in animals, and in pathways of secondary metabolism in plants. Different decarboxylase proteins are characterized for their different substrate specificities, but are encoded by homologous genes. We study, within a maximum-likelihood framework, the evolutionary relationships among dopa decarboxylase (Ddc), histidine decarboxylase (Hdc) and alpha-methyldopa hypersensitive (amd) in animals, and tryptophan decarboxylase (Wdc) and tyrosine decarboxylase (Ydc) in plants. The evolutionary rates are heterogeneous. There are differences between paralogous genes in the same lineages: 4.13 x 10(-10) nucleotide substitutions per site per year in mammalian Ddc vs. 1.95 in Hdc; between orthologous genes in different lineages, 7.62 in dipteran Ddc vs. 4.13 in mammalian Ddc; and very large temporal variations in some lineages, from 3.7 up to 54.9 in the Drosophila Ddc lineage. Our results are inconsistent with the molecular clock hypothesis.

  9. Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

    French, Jarrod B.; Ealick, Steven E. (Cornell)


    The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K{sub i} of 30 {+-} 2 {micro}m. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.

  10. Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella.

    Julie P M Viala

    Full Text Available During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i to survive an extreme acid shock, (ii to grow at mild acidic pH and (iii to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.

  11. Cell density-correlated induction of pyruvate decarboxylase under aerobic conditions in the yeast Pichia stipitis.

    Mergler, M; Klinner, U


    During the aerobic batch cultivation of P. stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner. Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation. This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.

  12. Optimization of Culture Conditions for Recombinant E.coliContaining High Activity of L-DOPA Synthesis%高左旋多巴合成活性的重组大肠杆菌培养条件优化

    李华钟; 孙伟; 陈坚


    对重组大肠杆菌进行培养条件的研究,得到细胞合成L-DOPA最佳培养条件为:L-tyrosine 0.2%、KH2PO4 0.2%、MgSO4*7H2O 0.1%、FeSO4*7H2O 0.001%、ZnSO4 0.001%、磷酸吡哆醛 0.005%、牛肉膏 0.5%、蛋白胨 2%、DL-Alanine 0.1%、温度37 ℃、pH值7.0、培养时间16 h、摇瓶转速150 r/min.%Culture conditions for recombinant E.coli containing high tyrosine phenol lyase were studied. Optimum condition of enzyme formation was cells cultured at 37 ℃for 16hours in the medium containing 0.2% L-tyrosine, 0.2%KH2PO4, 0.1% MgSO4*7H2O, 0.001% FeSO4*7H2O, 0.001% ZnSO4*7H2O, 0.005% pyridoxal phosphate, 0.5% meat extract, 2% polypeptone, and 0.1% DL-Alanine. Initial pH and rotation rate were 7.0 and 150 r/min respectively.

  13. 司来吉兰与左旋多巴联用治疗帕金森病运动障碍的临床观察%Clinical observation of Selegiline in combination with L-dopa on patients with parkinson movement disorders

    叶虹; 罗丽霞; 李飞; 刘清阁


    Objective To explore the clinical effect and safety of Selegiline in combination with L-dopa on Parkinson movement disorders therapy. Methods 108 patients in the Affiliated Nanhai People’s Hospital of Southern Medical U-niversity were chosen as the research object, all the patients were in accordance with the national seminar for ex-trapyramidal disease diagnostic criteria of Parkinson's, and ruled out other diseases such as familial and senile tremor, they were divided into control group and experiment group with 54 cases in each group, two groups of patients in the general data, such as the illness, age and gender were comparable. Two groups of patients were given the same intra-venous drip based treatment of 10% glucose solution. While the control group on the basis of the above were given L-dopa 3 pills/d and Pramipexole Hydrochloride 1 piece/d; the experiment group on the basis of the application of L-dopa were given Pramipexole Hydrochloride 1 time/d, 1 h after the breakfast, with the dose of 0.5 mg/time in the first two weeks, and 1 mg/time since the third week. The whole course of the treatment were 8 weeks for the two groups. Clinical symptoms improving and adverse reactions occurence were observed and recorded before and after treatment in the two groups, the clinical curative effect was evaluated. UPDRS score of two groups before and after treatment were observed, SPSS 17.0 software was used for statistical analysis. Results There was no significant difference of UPDRS between two groups before the treatment (P0.05),治疗后,两组患者的UPDRS评分均较治疗前显著下降,差异有统计学意义(P<0.05);治疗2、4、8周后,实验组UPDRS评分下降程度均对照组明显(P<0.05);治疗8周后,实验组和对照组治疗总有效率分别为75.9豫和40.7豫,实验组显著优于对照组,差异有统计学意义(P<0.05);两组均无严重不良反应出现。结论采用司来吉兰联合左旋多巴治疗帕金森病

  14. Perturbation of the Monomer-Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer

    Andrews, Forest H.; Rogers, Megan P.; Paul, Lake N.; McLeish, Michael J. [IUPUI; (Purdue)


    The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer–monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer–tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

  15. Tissue and regional distribution of cysteic acid decarboxylase. A new assay method.

    Wu, J Y; Moss, L G; Chen, M S


    A sensitive and rapid assay method method for cysteic acid decarboxylase was develped which combined the selectivity of ion exchange resin (a complete retention of the substrate, cysteic acid, and exclusion of the product, taurine) with the speed of a vacuum filtration. The synthesis and purification of 35S-labeled cysteic acid were described. The validity of the assay was established by the identification of the reaction product as taurine. With this new method, the decarboxylase activity was measured in discrete regions of bovine brain. Putamen had the highest activity, 172 pmol taurine formed/min/mg protein (100%), followed by caudate nucleus, 90%; cerebral cortex, 82%; hypothalamus, 81%; cerebellar cortex, 79%; cerebellar peduncle, 59%; thalamus, 42%; brain stem, 25%; pons, 10%; and corpus callosum, 3%. The decarboxylase activity in various mouse tissues was also determined as follows: liver, 403; brain, 145; kidney, 143; spinal cord, 59; lung, 21; and spleen, 10 pmol taurine formed/min/mg. No activity could be detected in skeleton muscle and heart, suggesting a different biosynthetic pathway for taurine synthesis in these tissues. The advantages and disadvantages of the new assay method are also discussed.

  16. Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

    Michael, A J; Furze, J M; Rhodes, M J; Burtin, D


    A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

  17. Inhibitory effect of 2,4,2',4'-tetrahydroxy-3-(3-methyl-2-butenyl)-chalcone on tyrosinase activity and melanin biosynthesis.

    Zhang, Xiaodong; Hu, Xiao; Hou, Aijun; Wang, Heyao


    2,4,2',4'-Tetrahydroxy-3-(3-methyl-2-butenyl)-chalcone (TMBC), a naturally occurring compound from Morus nigra, modulated melanogenesis by inhibiting tyrosinase. TMBC inhibited the L-dopa oxidase activity of mushroom tyrosinase with an IC(50) value of 0.95+/-0.04 microM, which was more potent than kojic acid (IC(50)=24.88+/-1.13 microM), a well-known tyrosinase inhibitor. The kinetic studies of tyrosinase inhibition revealed that TMBC acts as a competitive inhibitor of mushroom tyrosinase with L-dopa as the substrate. Furthermore, TMBC effectively inhibited both cellular tyrosinase activity and melanin biosynthesis in B16 melanoma cells without significant cytotoxicity. The inhibitory effect of TMBC on melanogenesis was attributed to the direct inhibition of tyrosinase activity, rather than the suppression of tyrosinase gene expression. These results indicated that TMBC may be a new promising pigmentation-altering agent for cosmetic or therapeutic applications.

  18. Dopamine signaling leads to loss of Polycomb repression and aberrant gene activation in experimental parkinsonism.

    Erik Södersten


    Full Text Available Polycomb group (PcG proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq in combination with expression analysis by RNA-sequencing (RNA-seq showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease.

  19. The Effect of Dopaminergic Activity on Aldosterone Secretion in Edematous State

    Han, Bong Heon; Ro, Heung Kyu [Chungnam National University College of Medicine, Daejeon (Korea, Republic of)


    To evaluate the effect of dopaminergic activity on aldosterone secretion, the plasma renin activity, serum cortisol and aldosterone were measured by radioimmunoassay in 6 normal controls and 12 patients who had hyponatremia and generalized edema or ascites with possible condition with secondary aldosteronism before and after(15, 30, and 60 min) 15 mg of metaclopramide by iv bolus injection and same method with 500 mg of L-dopa by per oral in 6 normal controls and 12 patients with edema ascites. The result were as follows; l) The basal level of PRA was higher in patients rather than normal controls but PRA was not influenced by MC or L-dopa administration on both normal controls and patients group. 2) The serum cortisol level was significantly elevated at 30 min after MC injection compared with basal level in normal controls but no significant change was not patients group. After L-dopa administration the serum cortisol level was noted in changed in both normal controls and patients group, 3) The serum aldosterone level was significantly elevated in 15, 30 and 60 min after MC injection in normal controls, and there also same tendency of aldosterone secretion was noticed in patients group. On the other hands, there was no changes in aldosterone level in both normal controls and patients group with L-dopa administration. Above result means that MC stimulate aldosterone secretion by dopaminergic antagonist and aldosterone secretion in normal subject is controlled by maximal tonic dopaminergic inhibition. In edematous patients, however, both of the dopaminergic inhibitory and stimulating effect of PRA, ACTH etc on the aldosterone secretion seems to be variable.

  20. Crystal structure and Hirshfeld surface analysis of 1-carb­oxy-2-(3,4-di­hydroxy­phen­yl)ethan-1-aminium chloride 2-ammonio-3-(3,4-di­hydroxy­phen­yl)propano­ate: a new polymorph of l-dopa HCl and isotypic with its bromide counterpart

    Kathiravan, Perumal; Balakrishnan, Thangavelu; Venkatesan, Perumal; Ramamurthi, Kandasamy; Percino, María Judith; Thamotharan, Subbiah


    The title mol­ecular salt, C9H12NO4 +·Cl−·C9H11NO4, is isotypic with that of the bromide counterpart [Kathiravan et al. (2016 ▸). Acta Cryst. E72, 1544–1548]. The title salt is a second monoclinic polymorph of the l-dopa HCl structure reported earlier in the monoclinic space group P21 [Jandacek & Earle (1971 ▸). Acta Cryst. B27, 841–845; Mostad & Rømming (1974 ▸). Acta Chemica Scand. B28, 1161–1168]. In the title compound, monoclinic space group I2, one of the dopa mol­ecules has a positive charge with a protonated α-amino group and the α-carb­oxy­lic acid group uncharged, while the second dopa mol­ecule has a neutral charge, the α-amino group is protonated and the α-carb­oxy­lic acid is deprotonated. In the previously reported form, a single dopa mol­ecule is observed in which the α-amino group is protonated and the α-carb­oxy­lic acid group is uncharged. The invariant and variations of various types of inter­molecular inter­actions present in these two forms of dopa HCl structures are discussed with the aid of two-dimensional fingerprint plots. PMID:27840723

  1. Crystal structure and Hirshfeld surface analysis of 1-carb-oxy-2-(3,4-di-hydroxy-phen-yl)ethan-1-aminium chloride 2-ammonio-3-(3,4-di-hydroxy-phen-yl)propano-ate: a new polymorph of l-dopa HCl and isotypic with its bromide counterpart.

    Kathiravan, Perumal; Balakrishnan, Thangavelu; Venkatesan, Perumal; Ramamurthi, Kandasamy; Percino, María Judith; Thamotharan, Subbiah


    The title mol-ecular salt, C9H12NO4(+)·Cl(-)·C9H11NO4, is isotypic with that of the bromide counterpart [Kathiravan et al. (2016 ▸). Acta Cryst. E72, 1544-1548]. The title salt is a second monoclinic polymorph of the l-dopa HCl structure reported earlier in the monoclinic space group P21 [Jandacek & Earle (1971 ▸). Acta Cryst. B27, 841-845; Mostad & Rømming (1974 ▸). Acta Chemica Scand. B28, 1161-1168]. In the title compound, monoclinic space group I2, one of the dopa mol-ecules has a positive charge with a protonated α-amino group and the α-carb-oxy-lic acid group uncharged, while the second dopa mol-ecule has a neutral charge, the α-amino group is protonated and the α-carb-oxy-lic acid is deprotonated. In the previously reported form, a single dopa mol-ecule is observed in which the α-amino group is protonated and the α-carb-oxy-lic acid group is uncharged. The invariant and variations of various types of inter-molecular inter-actions present in these two forms of dopa HCl structures are discussed with the aid of two-dimensional fingerprint plots.

  2. Experimental Evidence and In Silico Identification of Tryptophan Decarboxylase in Citrus Genus

    Luigi De Masi


    Full Text Available Plant tryptophan decarboxylase (TDC converts tryptophan into tryptamine, precursor of indolealkylamine alkaloids. The recent finding of tryptamine metabolites in Citrus plants leads to hypothesize the existence of TDC activity in this genus. Here, we report for the first time that, in Citrus x limon seedlings, deuterium labeled tryptophan is decarboxylated into tryptamine, from which successively deuterated N,N,N-trimethyltryptamine is formed. These results give an evidence of the occurrence of the TDC activity and the successive methylation pathway of the tryptamine produced from the tryptophan decarboxylation. In addition, with the aim to identify the genetic basis for the presence of TDC, we carried out a sequence similarity search for TDC in the Citrus genomes using as a probe the TDC sequence reported for the plant Catharanthus roseus. We analyzed the genomes of both Citrus clementina and Citrus sinensis, available in public database, and identified putative protein sequences of aromatic l-amino acid decarboxylase. Similarly, 42 aromatic l-amino acid decarboxylase sequences from 23 plant species were extracted from public databases. Potential sequence signatures for functional TDC were then identified. With this research, we propose for the first time a putative protein sequence for TDC in the genus Citrus.

  3. Insulin and phorbol myristic acetate induce ornithine decarboxylase in Reuber H35 rat hepatoma cells by different mechanisms.

    Goodman, S A; Esau, B; Koontz, J W


    Reuber H35 rat hepatoma cells respond to insulin or to tumor promoting phorbol esters with an increase in ornithine decarboxylase enzyme activity. This occurs in a time- and dose-dependent manner with both types of agonist. We report here that the increase in ornithine decarboxylase activity with optimal concentrations of both agonists is additive. Furthermore, the initial increase is dependent on continued RNA and protein synthesis. We also find that both of these agonists cause an increase in mRNA coding for ornithine decarboxylase in a time- and dose-dependent manner which suggests that the increase in enzyme activity can be accounted for by the increase in transcript levels. The difference in the time course of induction by the agonists, the additivity of induction by the two agonists, the differential sensitivity of induction to cycloheximide and RNA synthesis inhibitors, and the observation that phorbol myristic acetate causes a further increase in ornithine decarboxylase activity and transcript levels in cells already maximally induced by insulin suggest that these two agonists act through separate mechanisms.

  4. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community.

    Zargar, K; Saville, R; Phelan, R M; Tringe, S G; Petzold, C J; Keasling, J D; Beller, H R


    Anaerobic bacterial biosynthesis of toluene from phenylacetate was reported more than two decades ago, but the biochemistry underlying this novel metabolism has never been elucidated. Here we report results of in vitro characterization studies of a novel phenylacetate decarboxylase from an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (CsdBC). However, the toluene synthase under study appears to be able to catalyze both phenylacetate and p-hydroxyphenylacetate decarboxylation. Observations suggesting that phenylacetate and p-hydroxyphenylacetate decarboxylation in complex cell-free extracts were catalyzed by the same enzyme include the following: (i) the specific activity for both substrates was comparable in cell-free extracts, (ii) the two activities displayed identical behavior during chromatographic separation of cell-free extracts, (iii) both activities were irreversibly inactivated upon exposure to O2, and (iv) both activities were similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding and ultimately harnessing it for making bio-based toluene.

  5. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    Kanerva, Kristiina; Maekitie, Laura T. [Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); Baeck, Nils [Department of Anatomy, Institute of Biomedicine, University of Helsinki, Helsinki (Finland); Andersson, Leif C., E-mail: [Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); HUSLAB, Helsinki (Finland); Department of Oncology and Pathology, Karolinska Institutet, Stockholm (Sweden)


    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  6. A porphodimethene chemical inhibitor of uroporphyrinogen decarboxylase.

    Kenneth W Yip

    Full Text Available Uroporphyrinogen decarboxylase (UROD catalyzes the conversion of uroporphyrinogen to coproporphyrinogen during heme biosynthesis. This enzyme was recently identified as a potential anticancer target; its inhibition leads to an increase in reactive oxygen species, likely mediated by the Fenton reaction, thereby decreasing cancer cell viability and working in cooperation with radiation and/or cisplatin. Because there is no known chemical UROD inhibitor suitable for use in translational studies, we aimed to design, synthesize, and characterize such a compound. Initial in silico-based design and docking analyses identified a potential porphyrin analogue that was subsequently synthesized. This species, a porphodimethene (named PI-16, was found to inhibit UROD in an enzymatic assay (IC50 = 9.9 µM, but did not affect porphobilinogen deaminase (at 62.5 µM, thereby exhibiting specificity. In cellular assays, PI-16 reduced the viability of FaDu and ME-180 cancer cells with half maximal effective concentrations of 22.7 µM and 26.9 µM, respectively, and only minimally affected normal oral epithelial (NOE cells. PI-16 also combined effectively with radiation and cisplatin, with potent synergy being observed in the case of cisplatin in FaDu cells (Chou-Talalay combination index <1. This work presents the first known synthetic UROD inhibitor, and sets the foundation for the design, synthesis, and characterization of higher affinity and more effective UROD inhibitors.

  7. Overexpression, purification, crystallization and preliminary structural studies of p-coumaric acid decarboxylase from Lactobacillus plantarum

    Rodríguez, Héctor; Rivas, Blanca de las; Muñoz, Rosario [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Mancheño, José M., E-mail: [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain)


    The enzyme p-coumaric acid decarboxylase (PDC) from L. plantarum has been recombinantly expressed, purified and crystallized. The structure has been solved at 2.04 Å resolution by the molecular-replacement method. The substrate-inducible p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum has been overexpressed in Escherichia coli, purified and confirmed to possess decarboxylase activity. The recombinant His{sub 6}-tagged enzyme was crystallized using the hanging-drop vapour-diffusion method from a solution containing 20%(w/v) PEG 4000, 12%(w/v) 2-propanol, 0.2 M sodium acetate, 0.1 M Tris–HCl pH 8.0 with 0.1 M barium chloride as an additive. Diffraction data were collected in-house to 2.04 Å resolution. Crystals belonged to the tetragonal space group P4{sub 3}, with unit-cell parameters a = b = 43.15, c = 231.86 Å. The estimated Matthews coefficient was 2.36 Å{sup 3} Da{sup −1}, corresponding to 48% solvent content, which is consistent with the presence of two protein molecules in the asymmetric unit. The structure of PDC has been determined by the molecular-replacement method. Currently, the structure of PDC complexed with substrate analogues is in progress, with the aim of elucidating the structural basis of the catalytic mechanism.

  8. Carboxyspermidine decarboxylase of the prominent intestinal microbiota species Bacteroides thetaiotaomicron is required for spermidine biosynthesis and contributes to normal growth.

    Sakanaka, Mikiyasu; Sugiyama, Yuta; Kitakata, Aya; Katayama, Takane; Kurihara, Shin


    Recent studies have indicated that polyamines produced by gut microbes significantly influence host health; however, little is known about the microbial polyamine biosynthetic pathway except for that in Escherichia coli, a minor component of the gastrointestinal microbiota. Here, we investigated the polyamine biosynthetic ability of Bacteroides thetaiotaomicron, a predominant gastrointestinal bacterial species in humans. High-performance liquid chromatography analysis revealed that B. thetaiotaomicron cultured in polyamine-free minimal medium accumulated spermidine intracellularly at least during the mid-log and stationary phases. Deletion of the gene encoding a putative carboxyspermidine decarboxylase (casdc), which converts carboxyspermidine to spermidine, resulted in the depletion of spermidine and loss of decarboxylase activity in B. thetaiotaomicron. The Δcasdc strain also showed growth defects in polyamine-free growth medium. The complemented Δcasdc strain restored the spermidine biosynthetic ability, decarboxylase activity, and growth. These results indicate that carboxyspermidine decarboxylase is essential for synthesizing spermidine in B. thetaiotaomicron and contributes to the growth of this species.

  9. Characterization of the Entamoeba histolytica ornithine decarboxylase-like enzyme.

    Anupam Jhingran

    Full Text Available BACKGROUND: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC, the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. METHODOLOGY AND RESULTS: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF of approximately 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size approximately 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO, an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. CONCLUSION: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from

  10. Dynamic evaluation of growth hormone (GH) and prolactin (hPRL) secretion in active acromegaly with high and low GH output.

    Tolis, G; Kovacs, L; Friesen, H; Martin, J B


    Ten patients with active acromegaly were studied. In 9 plasma GH levels failed to suppress after glucose (OGTT), in 8 an increase in serum GH occurred after thyrotropin releasing hormone (TRH). After L-Dopa, 4 patients showed no change in serum GH, 3 exhibited a decrease and in 3 an increase in serum hGH occurred. With a combined insulin (ITT) and arginine (ATT) test, 2 patients exhibited an increase in hGH, and in 6 no change occurred. Fasting serum GH concentration was less than 11 ng/ml in 5 patients. Basal prolactin (hPRL) levels were normal in all patients including two with galactorrhea. L-Dopa suppressed and TRH stimulated hPRL secretion in all, but the responses which were seen were subnormal. Hydrocortisone infusion in two acromegalics did not affect the prolactin induced increase after TRH but blunted the GH increase after TRH.

  11. A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

    Vogel Constantin


    Full Text Available Abstract Background Standard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD is still missing. Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult. Results We developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8 was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED and was used to systematically analyze functionally and structurally relevant

  12. Characterization of a second ornithine decarboxylase isolated from Morganella morganii.

    De Las Rivas, Blanca; González, Ramón; Landete, José María; Muñoz, Rosario


    The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC) encoded a 722-amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF). The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

  13. Role of ornithine decarboxylase in breast cancer

    Wensheng Deng; Xian Jiang; Yu Mei; Jingzhong Sun; Rong Ma; Xianxi Liu; Hui Sun; Hui Tian; Xueying Sun


    Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis that decarboxylates ornithine to putrescine, has become a promising target for cancer research. The aim of this study is to investigate the role of ODC in breast cancer. We detected expression of ODC in breast cancer tissues and four breast cancer cell lines, and transfected breast cancer cells with an adenoviral vector carrying antisense ODC (rAd-ODC/Ex3as) and examined their growth and migration.ODC was overexpressed in breast cancer tissues and cell lines compared with non-tumor tissues and normal breast epithelial celis,and there was a positive correlation between the level of ODC mRNA and the staging of tumors.The expression of ODC correlated with cyclin D1,a cell cycle protein,in synchronized breast cancer MDA-MB-231 cells.Gene transfection of rAd-ODC/Ex3as markedly down-regulated expression Of ODC and cyclin D1,resulting in suppression of proliferation and cell cycle arrest at G0-G1 phase,and the inhibifion of colony formation,an anchorage-independent growth pattern,and the migratory ability of MDA-MB-231 cells.rAd-ODC/Ex3as also markedly reduced the concentration of putrescine,but not spermidine or spermine,in MDA-MB-231 cells.The results suggested that the ODC gene might act as aprognostic factor for breast cancer and it could be a promising therapeutic target.

  14. Fusion of pyruvate decarboxylase and alcohol dehydrogenase increases ethanol production in Escherichia coli.

    Lewicka, Aleksandra J; Lyczakowski, Jan J; Blackhurst, Gavin; Pashkuleva, Christiana; Rothschild-Mancinelli, Kyle; Tautvaišas, Dainius; Thornton, Harry; Villanueva, Hugo; Xiao, Weike; Slikas, Justinas; Horsfall, Louise; Elfick, Alistair; French, Christopher


    Ethanol is an important biofuel. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (AdhB) increases ethanol production in Escherichia coli. A fusion of PDC and ADH was generated and expressed in E. coli. The fusion enzyme was demonstrated to possess both activities. AdhB activity was significantly lower when fused to PDC than when the two enzymes were expressed separately. However, cells expressing the fusion protein generated ethanol more rapidly and to higher levels than cells coexpressing Pdc and AdhB, suggesting a specific rate enhancement due to the fusion of the two enzymes.

  15. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus 1

    Legaz, María Estrella; Vicente, Carlos


    Arginase (EC, l-arginine decarboxylase (EC, and agmatine amidinohydrolase (EC activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation. PMID:16662821

  16. Endogenous Inactivators of Arginase, l-Arginine Decarboxylase, and Agmatine Amidinohydrolase in Evernia prunastri Thallus.

    Legaz, M E; Vicente, C


    Arginase (EC, l-arginine decarboxylase (EC, and agmatine amidinohydrolase (EC activities spontaneously decay in Evernia prunastri thalli incubated on 40 millimolar l-arginine used as inducer of the three enzymes if dithiothreitol is not added to the media. Lichen thalli accumulate both chloroatranorin and evernic acid in parallel to the loss of activity. These substances behave as inactivators of the enzymes at a range of concentrations between 2 and 20 micromolar, whereas several concentrations of dithiothreitol reverse, to some extent, the in vitro inactivation.

  17. Conformational stabilization of rat s-adenosylmethionine decarboxylase by putrescine.

    Wada, Makiko; Shirahata, Akira


    The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The activity of AdoMetDC treated with IAA in the absence of putrescine was reduced, but about 80% of its activity remained after treatment with IAA in the presence of putrescine. In the presence of putrescine, IAA incorporation was 1.9 mol IAA/mol of AdoMetDC α-subunit, while there was no incorporation of IAA in the β-subunit of AdoMetDC. In the absence of putrescine, 5.0 mol of IAA/mol of α-subunit and 0.9 mol of IAA/mol of β-subunit were incorporated. Only Cys292 and Cys310 were carboxymethylated by IAA in the presence of putrescine. In contrast, in the absence of putrescine all cysteines were carboxymethylated by IAA. In addition, putrescine slowed the rate of AdoMetDC degradation by trypsin. These results demonstrate that the conformation of AdoMetDC purified from rat prostate is stabilized by putrescine.

  18. Differential distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs in the entopeduncular nucleus of the rat.

    Yuan, P Q; Grånäs, C; Källström, L; Yu, J; Huhman, K; Larhammar, D; Albers, H E; Johnson, A E


    The entopeduncular nucleus is one of the major output nuclei of the basal ganglia, with topographically organized projections to both motor and limbic structures. Neurons of the entopeduncular nucleus use GABA as the principal transmitter, and glutamic acid decarboxylase (the GABA synthetic enzyme) is widely distributed throughout the region. Previous studies have shown that glutamate decarboxylase exists in two forms (glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67), and that the messenger RNAs for these different enzymes are widely distributed in rat brain. The purpose of the present experiment was to describe the distribution of glutamic acid decarboxylase-65 and glutamic decarboxylase-67 messenger RNAs throughout the entopeduncular nucleus using recently developed oligodeoxynucleotide probes and in situ hybridization histochemical methods. In agreement with previous studies, northern analysis of rat brain poly(A)+ messenger RNA preparations showed that the glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 probes used in the present study hybridized to messenger RNAs of approximately 5.7 and 3.7 kb, respectively. Film autoradiographic analysis revealed large region-dependent, isoform-specific differences in the levels of expression of the two messenger RNAs, with glutamic acid decarboxylase-65 messenger RNA predominating in rostral and medial regions of the entopeduncular nucleus and glutamic acid decarboxylase-67 messenger RNA most abundant in the caudal region. Cellular analysis showed that these region-dependent differences in labelling were due to differences in the relative amounts of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs expressed per cell rather than the number of cells expressing each form of glutamic acid decarboxylase messenger RNA. The differences in the distribution of glutamic acid decarboxylase-65 and glutamic acid decarboxylase-67 messenger RNAs are closely related to the

  19. Cellular target recognition of perfluoroalkyl acids: In vitro evaluation of inhibitory effects on lysine decarboxylase

    Wang, Sufang; Lv, Qiyan; Yang, Yu, E-mail:; Guo, Liang-Hong, E-mail:; Wan, Bin; Zhao, Lixia


    Perfluoroalkyl acids (PFAAs) have been shown to bind with hepatic peroxisome proliferator receptor α, estrogen receptors and human serum albumin and subsequently cause some toxic effects. Lysine decarboxylase (LDC) plays an important role in cell growth and developmental processes. In this study, the inhibitory effect of 16 PFAAs, including 13 perfluorinated carboxylic acids (PFCAs) and 3 perfluorinated sulfonic acids (PFSAs), on lysine decarboxylase (LDC) activity was investigated. The inhibition constants obtained in fluorescence enzyme assays fall in the range of 2.960 μM to 290.8 μM for targeted PFCAs, and 41.22 μM to 67.44 μM for targeted PFSAs. The inhibitory effect of PFCAs increased significantly with carbon chain (7–18 carbons), whereas the short chain PFCAs (less than 7 carbons) did not show any effect. Circular dichroism results showed that PFAA binding induced significant protein secondary structural changes. Molecular docking revealed that the inhibitory effect could be rationalized well by the cleft binding mode as well as the size, substituent group and hydrophobic characteristics of the PFAAs. At non-cytotoxic concentrations, three selected PFAAs inhibited LDC activity in HepG2 cells, and subsequently resulted in the decreased cadaverine level in the exposed cells, suggesting that LDC may be a possible target of PFAAs for their in vivo toxic effects. - Highlights: • Inhibitory effects of PFAAs on lysine decarboxylase activity were evaluated. • Four different methods were employed to investigate the mechanisms. • The long chain PFAAs showed inhibitory effect compare with 4–6 carbon chain. • The long chain PFAAs bound with LDC differently from the short ones. • The results in cells correlate with those obtained from fluorescence assay.

  20. Acquisition of a heat stable enzyme; S-adenosylmethionine decarboxylase from selenomonas ruminantium

    Ko, Kyong Cheol; Park, Sang Hyun [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Kamio, Yoshiyuku [Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University (Japan)


    In Selenomoans ruminantium, a strictly anaerobic and gram negative bacterium, cadaverine and putrescine are the essential constituents of its peptidoglycan. S. ruminantium does not contain both free and bound types of lipoprotein, but it contains cadaverine as a component of its peptidoglycan. S-adenosylmethionine decarboxylase (SAMDC) is a key enzyme for a synthesis of spermidine and spermine in S. ruminantium. The crude extract of S. ruminantium was preincubated at 100 degrees Celcius and its SAMDC activity was measured by using a {sup 14}C labeled substrate. We report here on a heat stable SAMDC which is able to withstand a temperature up to 100 degrees Celcius.

  1. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    Nilsson, Tatjana; Bogdanovic, Nenad; Volkman, Inga


    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD...

  2. Genetics Home Reference: malonyl-CoA decarboxylase deficiency

    ... use fatty acids as a major source of energy. Mutations in the MLYCD gene reduce or eliminate the function of malonyl-CoA decarboxylase. A shortage of this enzyme disrupts the normal balance of fatty acid formation and breakdown in the ...

  3. Aromatic L-Aminoacid Decarboxylase Deficiency A Defect of the Neurotransmitter Metabolism with Severe Neurological Impairment

    Sequeira, S.; Calado, E.; Maia, AL; Pacheco, L.


    A descarboxilase dos L-aminoácidos aromáticos, um enzima piridoxina dependente, é responsável pela conversão da L-dopa em dopamina e do 5 hidroxitriptofano em serotonina. A deficiência desse enzima, um erro inato do metabolismo dos neurotransmissores, resulta numa doença autossómica recessiva com manifestações neurológicas graves. Os dois casos apresentados de deficiência da descarboxilase dos L-aminoácidos aromáticos, entidade pela primeira vez descrita no nosso país, apresentam caracterí...

  4. Cerebellar Ataxia and Glutamic Acid Decarboxylase Antibodies

    Ariño, Helena; Gresa-Arribas, Nuria; Blanco, Yolanda; Martínez-Hernández, Eugenia; Sabater, Lidia; Petit-Pedrol, Mar; Rouco, Idoia; Bataller, Luis; Dalmau, Josep O.; Saiz, Albert; Graus, Francesc


    IMPORTANCE Current clinical and immunologic knowledge on cerebellar ataxia (CA) with glutamic acid decarboxylase 65 antibodies (GAD65-Abs) is based on case reports and small series with short-term follow-up data. OBJECTIVE To report the symptoms, additional antibodies, prognostic factors, and long-term outcomes in a cohort of patients with CA and GAD65-Abs. DESIGN, SETTING, AND PARTICIPANTS Retrospective cohort study and laboratory investigations at a center for autoimmune neurologic disorders among 34 patients with CA and GAD65-Abs, including 25 with long-term follow-up data (median, 5.4 years; interquartile range, 3.1-10.3 years). MAIN OUTCOMES AND MEASURES Analysis of clinicoimmunologic features and predictors of response to immunotherapy. Immunochemistry on rat brain, cultured neurons, and human embryonic kidney cells expressing GAD65, GAD67, α1-subunit of the glycine receptor, and a repertoire of known cell surface autoantigens were used to identify additional antibodies. Twenty-eight patients with stiff person syndrome and GAD65-Abs served as controls. RESULTS The median age of patients was 58 years (range, 33-80 years); 28 of 34 patients (82%) were women. Nine patients (26%) reported episodes of brainstem and cerebellar dysfunction or persistent vertigo several months before developing CA. The clinical presentation was subacute during a period of weeks in 13 patients (38%). Nine patients (26%) had coexisting stiff person syndrome symptoms. Systemic organ-specific autoimmunities (type 1 diabetes mellitus and others) were present in 29 patients (85%). Twenty of 25 patients with long-term follow-up data received immunotherapy (intravenous immunoglobulin in 10 and corticosteroids and intravenous immunoglobulin or other immunosuppressors in 10), and 7 of them (35%) improved. Predictors of clinical response included subacute onset of CA (odds ratio [OR], 0.50; 95% CI, 0.25-0.99; P = .047) and prompt immunotherapy (OR, 0.98; 95% CI, 0.96-0.99; P = .01). Similar

  5. Catalysis of acetoin formation by brewers' yeast pyruvate decarboxylase isozymes.

    Stivers, J T; Washabaugh, M W


    Catalysis of C(alpha)-proton transfer from 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC from Saccharomyces carlsbergensis was investigated by determining the steady-state kinetics of the reaction of [1-L]acetaldehyde (L = H, D, or T) to form acetoin and the primary kinetic isotope effects on the reaction. The PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) have different steady-state kinetic parameters and isotope effects for acetoin formation in the presence and absence of the nonsubstrate allosteric effector pyruvamide: pyruvamide activation occurs by stabilization of the acetaldehyde/PDC ternary complex. The magnitudes of primary L(V/K)-type (L = D or T) isotope effects on C(alpha)-proton transfer from alpha 4-PDC-bound HETDP provide no evidence for significant breakdown of the Swain-Schaad relationship that would indicate partitioning of the putative C(alpha)-carbanion/enamine intermediate between HETDP and products. The substrate concentration dependence of the deuterium primary kinetic isotope effects provides evidence for an intrinsic isotope effect of 4.1 for C(alpha)-proton transfer from alpha 4-PDC-bound HETDP. A 1.10 +/- 0.02-fold 14C isotope discrimination against [1,2-14C]acetaldehyde in acetoin formation is inconsistent with a stepwise mechanism, in which the addition step occurs after rate-limiting formation of the C(alpha)-carbanion/enamine as a discrete enzyme-bound intermediate, and provides evidence for a concerted reaction mechanism with an important component of carbon-carbon bond formation in the transition state.

  6. Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase.

    Kellett, Whitney F; Brunk, Elizabeth; Desai, Bijoy J; Fedorov, Alexander A; Almo, Steven C; Gerlt, John A; Rothlisberger, Ursula; Richards, Nigel G J


    The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report quantum mechanical/molecular mechenical calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 °C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect.

  7. Decarboxylase gene expression and cadaverine and putrescine production by Serratia proteamaculans in vitro and in beef.

    De Filippis, Francesca; Pennacchia, Carmela; Di Pasqua, Rosangela; Fiore, Alberto; Fogliano, Vincenzo; Villani, Francesco; Ercolini, Danilo


    Studies of the molecular basis of microbial metabolic activities that are important for the changes in food quality are valuable in order to help in understanding the behavior of spoiling bacteria in food. The growth of a psychrotrophic Serratia proteamaculans strain was monitored in vitro and in artificially inoculated raw beef. Two growth temperatures (25°C and 4°C) were tested in vitro, while growth at 15°C and 4°C was monitored in beef. During growth, the expression of inducible lysine and ornithine-decarboxylase genes was evaluated by quantitative reverse transcription-PCR (qRT-PCR), while the presence of cadaverine and putrescine was quantified by LC-ESI-MS/MS. The expression of the decarboxylase genes, and the consequent production of cadaverine and putrescine were shown to be influenced by the temperature, as well as by the complexity of the growth medium. Generally, the maximum gene expression and amine production took place during the exponential and early stationary phase, respectively. In addition, lower temperatures caused slower growth and gene downregulation. Higher amounts of cadaverine compared to putrescine were found during growth in beef with the highest concentrations corresponding to microbial loads of ca. 9CFU/g. The differences found in gene expression evaluated in vitro and in beef suggested that such activities are more reliably investigated in situ in specific food matrices.

  8. The yeast Dekkera bruxellensis genome contains two orthologs of the ARO10 gene encoding for phenylpyruvate decarboxylase.

    de Souza Liberal, Anna Theresa; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante; Simões, Diogo Ardaillon; de Morais, Marcos Antonio


    The yeast Dekkera bruxellensis possesses important physiological traits that enable it to grow in industrial environments as either spoiling yeast of wine production or a fermenting strain used for lambic beer, or fermenting yeast in the bioethanol production process. In this work, in silico analysis of the Dekkera genome database allowed the identification of two paralogous genes encoding for phenylpyruvate decarboxylase (DbARO10) that represents a unique trait among the hemiascomycetes. The molecular analysis of the theoretical protein confirmed its protein identity. Upon cultivation of the cell in medium containing phenylpyruvate, both increases in gene expression and in phenylpyruvate decarboxylase activity were observed. Both genes were differentially expressed depending on the culture condition and the type of metabolism, which indicated the difference in the biological function of their corresponding proteins. The importance of the duplicated DbARO10 genes in the D. bruxellensis genome was discussed and represents the first effort to understand the production of flavor by this yeast.

  9. Spinal cord hemisection facilitates aromatic L-amino acid decarboxylase cells to produce serotonin in the subchronic but not the chronic phase

    Azam, Bushra; Wienecke, Jacob; Jensen, Dennis Bo


    Neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT) and noradrenalin, play an essential role in regulating the motor and sensory functions in the spinal cord. We have previously shown that in the rat spinal cord the activity of aromatic L-amino acid decarboxylase (AADC) cells to produce...

  10. Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

    Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi


    We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

  11. Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni.

    Bonnin-Jusserand, Maryse; Grandvalet, Cosette; David, Vanessa; Alexandre, Hervé


    Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.

  12. Does the aromatic L-amino acid decarboxylase contribute to thyronamine biosynthesis?

    Hoefig, Carolin S; Renko, Kostja; Piehl, Susanne; Scanlan, Thomas S; Bertoldi, Mariarita; Opladen, Thomas; Hoffmann, Georg Friedrich; Klein, Jeannette; Blankenstein, Oliver; Schweizer, Ulrich; Köhrle, Josef


    Thyronamines (TAM), recently described endogenous signaling molecules, exert metabolic and pharmacological actions partly opposing those of the thyromimetic hormone T(3). TAM biosynthesis from thyroid hormone (TH) precursors requires decarboxylation of the L-alanine side chain and several deiodination steps to convert e.g. L-thyroxine (T(4)) into the most potent 3-T(1)AM. Aromatic L-amino acid decarboxylase (AADC) was proposed to mediate TAM biosynthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC, which actively catalyzes dopamine production from DOPA, with several TH. Under all reaction conditions tested, AADC failed to catalyze TH decarboxylation, thus challenging the initial hypothesis. These in vitro observations are supported by detection of 3-T(1)AM in plasma of patients with AADC-deficiency at levels (46 ± 18 nM, n=4) similar to those of healthy controls. Therefore, we propose that the enzymatic decarboxylation needed to form TAM from TH is catalyzed by another unique, perhaps TH-specific, decarboxylase. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  13. Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

    Han, Q.; Ding, H; Robinson, H; Christensen, B; Li, J


    3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  14. Molecular docking studies and anti-tyrosinase activity of Thai mango seed kernel extract.

    Nithitanakool, Saruth; Pithayanukul, Pimolpan; Bavovada, Rapepol; Saparpakorn, Patchreenart


    The alcoholic extract from seed kernels of Thai mango (Mangifera indica L. cv. 'Fahlun') (Anacardiaceae) and its major phenolic principle (pentagalloylglucopyranose) exhibited potent, dose-dependent inhibitory effects on tyrosinase with respect to L-DOPA. Molecular docking studies revealed that the binding orientations of the phenolic principles were in the tyrosinase binding pocket and their orientations were located in the hydrophobic binding pocket surrounding the binuclear copper active site. The results indicated a possible mechanism for their anti-tyrosinase activity which may involve an ability to chelate the copper atoms which are required for the catalytic activity of tyrosinase.

  15. Heterologous expression of a plant arginine decarboxylase gene in Trypanosoma cruzi.

    Carrillo, Carolina; Serra, María P; Pereira, Claudio A; Huber, Alejandra; González, Nélida S; Algranati, Israel D


    Wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity. However, the transformation of these parasites with a recombinant plasmid containing the oat ADC cDNA coding region gave rise to the transient heterologous expression of the enzyme, suggesting the absence of endogenous mechanisms that could inhibit the expression of a hypothetical own ADC gene or the assay used to measure its enzymatic activity. The foreign ADC enzyme expressed in the transgenic T. cruzi was characterized by identification of the products, the stoichiometry of the catalysed reaction, the specific inhibition by alpha-difluoromethylarginine (DFMA) and the study of its metabolic turnover. The half-life of the heterologous ADC activity in T. cruzi was about 150 min. Bioinformatics studies and polymerase chain reaction (PCR) analyses seem to indicate the absence of ADC-like DNA sequences in the wild-type T. cruzi genome.

  16. Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

    Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan; Poduch, Ewa; Bello, Angelica M.; Mishra, Ram K.; Pai, Emil F.; Kotra, Lakshmi P. (TGRI); (Toronto)


    In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

  17. Cell biology, physiology and enzymology of phosphatidylserine decarboxylase.

    Di Bartolomeo, Francesca; Wagner, Ariane; Daum, Günther


    Phosphatidylethanolamine is one of the most abundant phospholipids whose major amounts are formed by phosphatidylserine decarboxylases (PSD). Here we provide a comprehensive description of different types of PSDs in the different kingdoms of life. In eukaryotes, type I PSDs are mitochondrial enzymes, whereas other PSDs are localized to other cellular compartments. We describe the role of mitochondrial Psd1 proteins, their function, enzymology, biogenesis, assembly into mitochondria and their contribution to phospholipid homeostasis in much detail. We also discuss briefly the cellular physiology and the enzymology of Psd2. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum.

  18. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    Verimli, Ural; Sehirli, Umit S


    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  19. Identification and molecular cloning of glutamate decarboxylase gene from Lactobacillus casei

    Yasaman Tavakoli


    Full Text Available Gamma-amino butyric acid (GABA possesses several physiological functions such as neurotransmission, induction of hypotension, diuretic and tranquilizer effects. Production of GABA-enriched products by lactic acid bacteria has been a focus of different researches in recent years because of their safety and health-promoting specifities. In this study, glutamate decarboxylase (gad gene of a local strains Lactobacillus casei was identified and cloned. In order to clone the gad gene from this strain, the PCR was carried out using primers designed based on conserved regions. The PCR product was purified and ligated into PGEM-T vector. Comparison of obtained sequences shows that this fragment codes the pyridoxal 5′-phosphate binding region. This strain could possibly be used for the industrial GABA production and also for development of functional fermented foods. Gad gene manipulation can also either decrease or increase the activity of enzyme in bacteria.

  20. Glutamate alteration of glutamic acid decarboxylase (GAD) in GABAergic neurons: the role of cysteine proteases.

    Monnerie, Hubert; Le Roux, Peter D


    Brain cell vulnerability to neurologic insults varies greatly, depending on their neuronal subpopulation. Among cells that survive a pathological insult such as ischemia or brain trauma, some may undergo morphological and/or biochemical changes that could compromise brain function. We previously reported that surviving cortical GABAergic neurons exposed to glutamate in vitro displayed an NMDA receptor (NMDAR)-mediated alteration in the levels of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) [Monnerie, H., Le Roux, P., 2007. Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro. Exp. Neurol. 205, 367-382]. In this study, we examined the mechanisms by which glutamate excitotoxicity caused a change in cortical GABAergic neurons' GAD protein levels. Removing extracellular calcium prevented the NMDAR-mediated decrease in GAD protein levels, measured using Western blot techniques, whereas inhibiting calcium entry through voltage-gated calcium channels had no effect. Glutamate's effect on GAD protein isoforms was significantly attenuated by preincubation with the cysteine protease inhibitor N-Acetyl-L-Leucyl-L-Leucyl-L-norleucinal (ALLN). Using class-specific protease inhibitors, we observed that ALLN's effect resulted from the blockade of calpain and cathepsin protease activities. Cell-free proteolysis assay confirmed that both proteases were involved in glutamate-induced alteration in GAD protein levels. Together these results suggest that glutamate-induced excitotoxic stimulation of NMDAR in cultured cortical neurons leads to altered GAD protein levels from GABAergic neurons through intracellular calcium increase and protease activation including calpain and cathepsin. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation

  1. Downstream reporter gene imaging for signal transduction pathway of dopamine type 2 receptor

    Le, Uyenchi N.; Min, Jung Joon; Moon, Sung Min; Bom, Hee Seung [School of Midicine, Chonnam National University, Gwangju (Korea, Republic of)


    The Dopamine 2 receptor (D2R) signal pathway regulates gene expression by phosphorylation of proteins including cAMP reponse element-binding protein (CREB), a transcription factor. In this study, we developed a reporter strategy using the GAL4 fusion CREB to assess the phosphorylation of CREB, one of the targets of the D2R signal transduction pathway. We used three plasmids: GAL4 fusion transactivator (pCMV-CREB), firefly luciferase reporter with GAL4 binding sites (pG5-FLUC), and D2R plasmid (pCMV-D2R). Group 1 293T cells were transiently transfected with pCMV-CREB and pG5-FLUC, and group 2 cells were transfected with all three plasmids. Transfected cells were stimulated with different concentrations of dopamine (0-200 M). For animal studies, group 1 and 2 cells (1x10{sup 6}) were subcutaneously injected on the left and right thigh of six nude mice, respectively. Dopamine stimiulation was performed with intraperitoneal injection of L-DOPA incombination with carbidopa, a peripheral DOPA decarboxylase inhibitor. Bioluminescence optical imaging studies were performed before and after L-DOPA injection. In cell culture studies, group 1 cells showed strong luciferase activity which implies direct activation of the signaling pathway due to growth factors contained in culture medium. Group 2 cells showed strong luciferase activity and a further increase after administration of dopamine. In animal studies, group 1 and 2 cells showed bioluminescence signal before L-DOPA injection, but signal from group 2 cells significantly increased 12 h after L-DOPA injection. The signal from group 1 cells disappeared thereafter, but group 2 cells continued to show signal until 36 h of L-DOPA injection. This study demonstrates imaging of the D2R signal transduction pathway and should be useful for noninvasive imaging of downstream effects of G-coupled protein pathways.

  2. DMEM enhances tyrosinase activity in B16 mouse melanoma cells and human melanocytes

    Panpen Diawpanich


    Full Text Available Media components may affect the activities of cultured cells. In this study, tyrosinase activity was evaluated by using B16-F10 mouse melanoma cell lines (B16-F10 and primary human melanocytes cultured in different media. An optical density measurement and a L-dopa reaction assay were used as the determination of the tyrosinase activity. The study of B16-F10 found the optical density to be 2010, 2246 and 2961 in cells cultured in RPMI Medium 1640 (RPMI1640,Minimum Essential Medium (MEM and Dulbecco’s Modified Eagle Medium (DMEM, respectively. Moreover, compared to RPMI 1640 and MEM, DMEM showed the darkest color of melanin formation in culture media and in cells after the L-dopa reaction assay. Addition of kojic acid showed a significant inhibitory effect on tyrosinase activity in all media.Whereas MCDB153 showed no significant effect on human melanocytes, DMEM caused a dramatic increase in tyrosinase activity after 4 days of cultivation. Addition of kojic acid showed a significant tyrosinase inhibitory effect in DMEM only. Furthermore, an active ingredient in green tea, epigallocathechin gallate (EGCG could inhibit tyrosinase activity in both B16-F10 and human melanocytes cultured in DMEM. In summary, these results suggest that DMEM is a suitable medium that provides high detection sensitivity in a tyrosinase inhibition assay.

  3. Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine.

    Vuosku, Jaana; Jokela, Anne; Läärä, Esa; Sääskilahti, Mira; Muilu, Riina; Sutela, Suvi; Altabella, Teresa; Sarjala, Tytti; Häggman, Hely


    In this study, we show that both arginine decarboxylase (ADC) protein and mRNA transcript are present at different phases of mitosis in Scots pine (Pinus sylvestris) zygotic embryogenesis. We also examined the consistency of polyamine (PA) profiles with the effective temperature sum, the latter indicating the developmental stage of the embryos. PA metabolism was analyzed by fitting statistical regression models to the data of free and soluble conjugated PAs, to the enzyme activities of ADC and ornithine decarboxylase (ODC), as well as to the gene expression of ADC. According to the fitted models, PAs typically had the tendency to increase at the early stages but decrease at the late stages of embryogenesis. Only the free putrescine fraction remained stable during embryo development. The PA biosynthesis strongly preferred the ADC pathway. Both ADC gene expression and ADC enzyme activity were substantially higher than putative ODC gene expression or ODC enzyme activity, respectively. ADC gene expression and enzyme activity increased during embryogenesis, which suggests the involvement of transcriptional regulation in the expression of ADC. Both ADC mRNA and ADC protein localized in dividing cells of embryo meristems and more specifically within the mitotic spindle apparatus and close to the chromosomes, respectively. The results suggest the essential role of ADC in the mitosis of plant cells.

  4. Partial purification and characterization of a novel histidine decarboxylase from Enterobacter aerogenes DL-1.

    Zou, Yu; Hu, Wenzhong; Jiang, Aili; Tian, Mixia


    Histidine decarboxylase (HDC) from Enterobacter aerogenes DL-1 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 52.4 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH for HDC activity was 6.5, and the enzyme was stable between pH 4 and 8. Enterobacter aerogenes HDC had optimal activity at 40°C and retained most of its activity between 4 and 50°C. HDC activity was reduced in the presence of numerous tested compounds. Particularly with SDS, it significantly (p < 0.01) inhibited enzyme activity. Conversely, Ca(2+) and Mn(2+) showed prominent activation effects (p < 0.01) with activity increasing to 117.20% and 123.42%, respectively. The Lineweaver-Burk plot showed that K m and V max values of the enzyme for L-histidine were 0.21 mM and 71.39 µmol/min, respectively. In comparison with most HDCs from other microorganisms and animals, HDC from E. aerogenes DL-1 displayed higher affinity and greater reaction velocity toward L-histidine.

  5. Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders.

    Pearce, David A; Atkinson, Mark; Tagle, Danilo A


    Degenerative diseases of the CNS, such as stiff-person syndrome (SPS), progressive cerebellar ataxia, and Rasmussen encephalitis, have been characterized by the presence of autoantibodies. Recent findings in individuals with Batten disease and in animal models for the disorder indicate that this condition may be associated with autoantibodies against glutamic acid decarboxylase (GAD), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Anti-GAD autoantibodies could result in excess excitatory neurotransmitters, leading to the seizures and other symptoms observed in patients with Batten disease. The pathogenic potential of GAD autoantibodies is examined in light of what is known for other autoimmune disorders, such as multiple sclerosis, SPS, Rasmussen encephalitis, and type 1 diabetes, and may have radical implications for diagnosis and management of Batten disease.

  6. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.


    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  7. Potential role of tyrosine hydroxylase in the loss of psychostimulant effect of amphetamine under conditions of impaired dopamine transporter activity.

    Janenaite, Egle; Vengeliene, Valentina; Bespalov, Anton; Behl, Berthold


    Amphetamine and methylphenidate are known to have stimulatory effect in healthy subjects but not in humans with attention deficit hyperactivity disorder and in rodents with impaired dopamine transporter (DAT) function. This phenomenon is called the paradoxical calming effect of psychostimulants. It has been previously demonstrated that psychostimulants may regulate the enzymatic activity of tyrosine hydroxylase (TH). Hence, the objective of the present study was to determine whether the lack of activity-stimulating effects of amphetamine in hyperactive rats is associated with changes in TH activity. To model hyperactivity in rats, acute administration of DAT inhibitor GBR12909 was used. Changes in TH activity, assessed as L-DOPA accumulation and TH phosphorylation levels, were measured in amphetamine treated rats with or without pretreatment with GBR12909. Our results showed that amphetamine treatment alone increased locomotor activity in rats, whereas pretreatment of rats with GBR12909 counteracted this effect, a finding consistent with the paradoxical calming effect. GBR12909, while having no effect on its own, blocked amphetamine-induced elevation of TH activity in dorsal striatum and nucleus accumbens, measured as increased tissue L-DOPA concentration. However, the phosphorylation levels of TH were not affected by treatment with amphetamine, GBR12909 or the combination of both. Our findings indicate that other mechanisms than phosphorylation-regulated TH activity changes are responsible for the paradoxical calming effect of amphetamine under conditions of impaired DAT activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Urinary dopamine in aromatic L-amino acid decarboxylase deficiency: the unsolved paradox.

    Wassenberg, T.; Willemsen, M.H.; Geurtz, P.B.; Lammens, M.M.Y.; Verrijp, K.; Wilmer, M.J.G.; Lee, W.T.; Wevers, R.A.; Verbeek, M.M.


    INTRODUCTION: In aromatic L-amino acid decarboxylase (AADC) deficiency, a neurotransmitter biosynthesis defect, paradoxical normal or increased levels of urinary dopamine have been reported. Genotype/phenotype correlations or alternative metabolic pathways may explain this remarkable finding, but

  9. Structural Basis of the Substrate Specificity and Enzyme Catalysis of a Papaver somniferum Tyrosine Decarboxylase

    Guan, Huai; Song, Shuaibao; Robinson, Howard; Liang, Jing; Ding, Haizhen; Li, Jianyong; Han, Qian


    Tyrosine decarboxylase (TyDC), a type II pyridoxal 5′-phosphate decarboxylase, catalyzes the decarboxylation of tyrosine. Due to a generally high sequence identity to other aromatic amino acid decarboxylases (AAADs), primary sequence information is not enough to understand substrate specificities with structural information. In this study, we selected a typical TyDC from Papaver somniferum as a model to study the structural basis of AAAD substrate specificities. Analysis of the native P. somniferum TyDC crystal structure and subsequent molecular docking and dynamics simulation provide some structural bases that explain substrate specificity for tyrosine. The result confirmed the previous proposed mechanism for the enzyme selectivity of indolic and phenolic substrates. Additionally, this study yields the first crystal structure for a plant type II pyridoxal-5'-phosphate decarboxylase. PMID:28232911

  10. Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.

    Kanjanaphachoat, Parawee; Wei, Bi-Yin; Lo, Shuen-Fang; Wang, I-Wen; Wang, Chang-Sheng; Yu, Su-May; Yen, Ming-Liang; Chiu, Sheng-Hsien; Lai, Chien-Chen; Chen, Liang-Jwu


    A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

  11. Purification and Properties of L-Arginine Decarboxylase of Evernia prunastri

    Carlos, Vicente; Estrella, Legaz; Catedra de Fisiologia Vegetal, Facultad de Biologia, Universidad Complutense


    An L-arginine decarboxylase was isolated from Evernia prunastri thallus. The enzyme was purified about 117-fold and showed a pH optimum of 7.1 and a temperature optimum at 26℃. Its molecular weight was estimated as 300,000. The Evernia arginine decarboxylase was significantly inhibited by L-ornithine, urea and putrescine. The K_m for L-arginine was about 12.5 mM.

  12. Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier.

    Peper, Stephanie; Kara, Selin; Long, Wei Sing; Liese, Andreas; Niemeyer, Bernd


    If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460I-F464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme.

  13. Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes.

    Bassez, T; Paris, J; Omilli, F; Dorel, C; Osborne, H B


    The level at which ornithine decarboxylase expression is regulated in growing oocytes has been investigated. Immunoprecipitation of the in vivo labelled proteins showed that ornithine decarboxylase accumulated less rapidly in stage IV oocytes than in previtellogenic stage I + II oocytes. Quantitative Northern analysis showed that ornithine decarboxylase mRNA is abundant in oocytes (about 8 x 10(8) transcripts/cell) and this number does not significantly change during oogenesis. Polysome analysis showed that this mRNA is present in polysomes in stage I + II oocytes but has passed into puromycin-insensitive mRNP particles by stage IV of oogenesis. Therefore, during the growth phase of oogenesis, ornithine decarboxylase expression is regulated at a translational level. These results are discussed relative to the temporal expression of ornithine decarboxylase and of other proteins whose expression also decreases during oogenesis. In order to perform these experiments, the cDNA (XLODC1) corresponding to Xenopus laevis ornithine decarboxylase mRNA was cloned and sequenced.

  14. Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in Vivo

    Guilan Chen; Elisabeth M. Perchellet; Xiao Mei Gao; Steven W. Newell; richard W. Hemingway; Vittorio Bottari; Jean-Pierre Perchellet


    m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce ODC activity at least as much as 3 ug of 12-O-tetradecanoylphorbol-13-acetate (TPA). ODC induction peaks...

  15. [Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase].

    Maskevich, S A; Maskevich, A A; Kivach, L N; Chernikevich, I P; Zabrodskaia, S V; Oparin, D A


    Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.

  16. The role of arginine decarboxylase in modulating the sensitivity of barley to ozone.

    Rowland-Bamford, A J; Borland, A M; Lea, P J; Mansfield, T A


    Polyamines (PA) are known to be involved in the areas of plant physiology and biochemistry which are related to the response of a plant to air pollution. This study examines the role of arginine decarboxylase (ADC), an important rate-limiting enzyme in polyamine synthesis, in barley plants exposed to ozone (O(3)). The activity of ADC increased significantly in O(3)-treated leaves when visible injury was hardly apparent. The increase in ADC activity may be a mechanism to increase the PA levels in O(3)-treated leaves and so minimize the damaging effects of O(3). Supporting this, foliar applications of DL-alpha-difluoromethylarginine (DFMA), a specific inhibitor of ADC, prevented the rise in ADC activity and visible injury was considerable on exposure to O(3). This damage was not due to the foliar sprays, as little visible injury was seen in leaves in the O(3)-free controls. The results are discussed in terms of the roles of PA in conferring O(3) resistance in plants.

  17. Complexes of Thermotoga maritima S-adenosylmethionine decarboxylase provide insights into substrate specificity

    Bale, Shridhar; Baba, Kavita; McCloskey, Diane E.; Pegg, Anthony E.; Ealick, Steven E.


    The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-adenosylmethionine methyl ester and 5{prime}-deoxy-5{prime}-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.

  18. Production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde using partially purified pyruvate decarboxylase (PDC).

    Shin, H S; Rogers, P L


    Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) as a key intermediate for L-ephedrine synthesis has been evaluated using pyruvate decarboxylase (PDC) partially purified from Candida utilis. PDC activity was enhanced by controlled fermentative metabolism and pulse feeding of glucose prior to the enzyme purification. With partially purified PDC, several enzymatic reactions occurred simultaneously and gave rise to by-products (acetaldehyde and acetoin) as well as L-PAC production. Optimal reaction conditions were determined for temperature, pH, addition of ethanol, PDC activity, benzaldehyde, and pyruvate:benzaldehyde ratio to maximize L-PAC, and minimize by-products. The highest L-PAC concentration of 28.6 g/L (190.6 mM) was achieved at 7 U/mL PDC activity and 200 mM benzaldehyde with 2.0 molar ratio of pyruvate to benzaldehyde in 40 mM potassium phosphate buffer (pH 7.0) containing 2.0 M ethanol at 4 degrees C.

  19. Effects of Teucrium polium aerial parts extracts on malonyl-CoA decarboxylase level.

    Durdi Qujeq


    Full Text Available Malonyl-CoA decarboxylase (MCD is an enzyme involved in the decarboxylation of malonyl-CoA to acetyl-CoA. In order to explore the hypothesis that the changing plant materials’ MCD activity level can serve as therapy to diabetics, the effect of Teucrium polium compounds was studied in a diabetic rat model. In this experimental study, two groups of rats, a control and a diabetic group, each including six rats, were used. At the end of the experiment, all rats were exterminated by ether anesthesia, their pancreases removed and dissected. Isolated rat pancreas was cultured in buffers with or without 100-500µg/l T. polium aerial parts extracts containing arginine and leucine. MCD and insulin levels were measured after culture at 37°C and 5% CO2, for 1, 3 and 5 days. Results showed that T. polium aqueous and the alcoholic extract decreased MCD activity. Present data also indicate that incubation of pancreatic tissue at a concentration of 2.8 and 16.7 mmol/L glucose stimulated insulin release. For the first time it seems that aqueous and alcoholic extracts of this plant decreased MCD activity.

  20. [Ornithine decarboxylase in mammalian organs and tissues at hibernation and artificial hypobiosis].

    Logvinovich, O S; Aksenova, G E


    Ornithine decarboxylase (ODC, EC is a short-lived and dynamically regulated enzyme of polyamines biosynthesis. Regulation of functional, metabolic and proliferative state of organs and tissues involves the modifications of the ODC enzymatic activity. The organ-specific changes in ODC activity were revealed in organs and tissues (liver, spleen, bone marrow, kidney, and intestinal mucosa) of hibernating mammals - squirrels Spermophilus undulates - during the hibernating season. At that, a positive correlation was detected between the decline and recovery of the specialized functions of organs and tissues and the respective modifications of ODC activity during hibernation bouts. Investigation of changes in ODC activity in organs and tissues of non-hibernating mammals under artificial hypobiosis showed that in Wistar rats immediately after exposure to hypothermia-hypoxia-hypercapnia (hypobiosis) the level of ODC activity was low in thymus, spleen, small intestine mucosa, neocortex, and liver. The most marked reduction in enzyme activity was observed in actively proliferating tissues: thymus, spleen, small intestine mucosa. In bone marrow of squirrels, while in a state of torpor, as well as in thymus of rats after exposure to hypothermia-hypoxia-hypercapnia, changes in the ODC activity correlated with changes in the rate of cell proliferation (by the criterion of cells distribution over cell cycle). The results obtained, along with the critical analysis of published data, indicate that the ODC enzyme is involved in biochemical adaptation of mammals to natural and artificial hypobiosis. A decline in the ODC enzymatic activity indicates a decline in proliferative, functional, and metabolic activity of organs and tissues of mammals (bone marrow, mucosa of small intestine, thymus, spleen, neocortex, liver, kidneys) when entering the state of hypobiosis.

  1. Fatal malonyl CoA decarboxylase deficiency due to maternal uniparental isodisomy of the telomeric end of chromosome 16.

    Malvagia, S; Papi, L; Morrone, A; Donati, M A; Ciani, F; Pasquini, E; la Marca, G; Scholte, H R; Genuardi, M; Zammarchi, E


    Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.

  2. Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b.

    Streit, Bennett R; Celis, Arianna I; Shisler, Krista; Rodgers, Kenton R; Lukat-Rodgers, Gudrun S; DuBois, Jennifer L


    A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O2 and H2O2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O2 to the ferric state. The subsequent second-order reaction between the ferric complex and H2O2 is slow, pH-dependent, and further decelerated by D2O2 (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H2O2. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H2O2 cleavage is therefore unclear. From a cellular perspective, the use of H2O2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.

  3. Expression of ornithine decarboxylase in precancerous and cancerous gastric lesions

    Xin-Pu Miao; Jian-Sheng Li; Hui-Yan Li; Shi-Ping Zeng; Ye Zhao; Jiang-Zheng Zeng


    AIM:To investigate the expression of ornithine decarboxylase (ODC) in precancerous and cancerous gastric lesions.METHODS: We studied the expression of ODC in gastric mucosa from patients with chronic superficial gastritis (CSG, n = 32), chronic atrophic gastritis [CAG, n = 43;15 with and 28 without intestinal metaplasia (IM)],gastric dysplasia (DYS, n = 11) and gastric cancer (GC,n = 48) tissues using immunohistochemical staining. All 134 biopsy specimens of gastric mucosa were collected by gastroscopy.METHODS: The positive rate of ODC expression was 34.4%, 42.9%, 73.3%, 81.8% and 91.7% in cases with CSG, CAG without IM, CAG with IM, DYS and GC, respectively (P < 0.01), The positive rate of ODC expression increased in the order of CSG < CAG (without IM) < CAG (with IM) < DYS and finally, GC. In addition,ODC positive immunostaining rate was lower in welldifferentiated GC than in poorly-differentiated GC (P <0.05).CONCLUSION: The expression of ODC is positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. This finding indicates that ODC may be used as a good biomarker in the screening and diagnosis of precancerous lesions.

  4. Ornithine decarboxylase gene is overexpressed in colorectal carcinoma

    Hai-Yan Hu; Bing Zhang; Xian-Xi Liu; Chun-Ying Jiang; Yi Lu; Shi-Lian Liu; Ji-Feng Bian; Xiao-Ming Wang; Zhao Geng; Yan Zhang


    AIM: To investigate the ornithine decarboxylase (ODC)gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology.METHODS: Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR.ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay.RESULTS: A cell line, which could steadily secrete antiODC mAb, was selected through subcloning four times.Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6±1.03%vs 5.26±5%, P<0.01).CONCLUSION: ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.

  5. Polyamines and plant stress - Activation of putrescine biosynthesis by osmotic shock

    Flores, H. E.; Galston, A. W.


    The putrescine content of oat leaf cells and protoplasts increases up to 60-fold within 6 hours of exposure to osmotic stress (0.4 to 0.6 molar sorbitol). Barley, corn, wheat, and wild oat leaves show a similar response. Increased arginine decarboxylase activity parallels the rise in putrescine, whereas ornithine decarboxylase remains unchanged. DL-alpha-Difluoromethylarginine, a specific irreversible inhibitor of arginine decarboxylase, prevents the stress-induced rise in increase in arginine decarboxylase activity and putrescine synthesis, indicating the preferential activation of this pathway.

  6. Role of glutamate decarboxylase-like protein 1 (GADL1) in taurine biosynthesis.

    Liu, Pingyang; Ge, Xiaomei; Ding, Haizhen; Jiang, Honglin; Christensen, Bruce M; Li, Jianyong


    This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

  7. Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.

    Werther, Tobias; Spinka, Michael; Tittmann, Kai; Schütz, Anja; Golbik, Ralph; Mrestani-Klaus, Carmen; Hübner, Gerhard; König, Stephan


    The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

  8. Structure of lpg0406, a carboxymuconolactone decarboxylase family protein possibly involved in antioxidative response from Legionella pneumophila.

    Chen, Xiaofang; Hu, Yanjin; Yang, Bo; Gong, Xiaojian; Zhang, Nannan; Niu, Liwen; Wu, Yun; Ge, Honghua


    Lpg0406, a hypothetical protein from Legionella pneumophila, belongs to carboxymuconolactone decarboxylase (CMD) family. We determined the crystal structure of lpg0406 both in its apo and reduced form. The structures reveal that lpg0406 forms a hexamer and have disulfide exchange properties. The protein has an all-helical fold with a conserved thioredoxin-like active site CXXC motif and a proton relay system similar to that of alkylhydroperoxidase from Mycobacterium tuberculosis (MtAhpD), suggesting that lpg0406 might function as an enzyme with peroxidase activity and involved in antioxidant defense. A comparison of the size and the surface topology of the putative substrate-binding region between lpg0406 and MtAhpD indicates that the two enzymes accommodate the different substrate preferences. The structural findings will enhance understanding of the CMD family protein structure and its various functions.

  9. Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma.

    Lozier, Ann M; Rich, Maria E; Grawe, Anissa Pedersen; Peck, Anderson S; Zhao, Ping; Chang, Anthony Ting-Tung; Bond, Jeffrey P; Sholler, Giselle Saulnier


    LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.

  10. Partial purification and characterization of arginine decarboxylase from avocado fruit, a thermostable enzyme.

    Winer, L; Vinkler, C; Apelbaum, A


    A partially purified preparation of arginine decarboxylase (EC, a key enzyme in polyamine metabolism in plants, was isolated from avocado (Persea americana Mill. cv Fuerte) fruit. The preparation obtained from the crude extract after ammonium sulfate precipitation, dialysis, and heat treatment, had maximal activity between pH 8.0 and 9.0 at 60 degrees C, in the presence of 1.2 millimolar MnCl(2), 2 millimolar dithiothreitol, and 0.06 millimolar pyridoxal phosphate. The K(m), of arginine for the decarboxylation reaction was determined for enzymes prepared from the seed coat of both 4-week-old avocado fruitlet and fully developed fruit, and was found to have a value of 1.85 and 2.84 millimolar, respectively. The value of V(app) (max) of these enzymes was 1613 and 68 nanomoles of CO(2) produced per milligram of protein per hour for the fruitlet and the fully developed fruit, respectively. Spermine, an end product of polyamine metabolism, caused less than 5% inhibition of the enzyme from fully developed fruit and 65% inhibition of the enzyme from the seed coat of 4-week-old fruitlets at 1 millimolar under similar conditions. The effect of different inhibitors on the enzyme and the change in the nature of the enzyme during fruit development are discussed.

  11. Cloning and Characterization of Two Pyruvate Decarboxylase Genes from Pichia stipitis CBS 6054

    Lu, Ping; Davis, Brian P.; Jeffries, Thomas W.


    In Pichia stipitis, fermentative and pyruvate decarboxylase (PDC) activities increase with diminished oxygen rather than in response to fermentable sugars. To better characterize PDC expression and regulation, two genes for PDC (PsPDC1 and PsPDC2) were cloned and sequenced from P. stipitis CBS 6054. Aside from Saccharomyces cerevisiae, from which three PDC genes have been characterized, P. stipitis is the only organism from which multiple genes for PDC have been identified and characterized. PsPDC1 and PsPDC2 have diverged almost as far from one another as they have from the next most closely related known yeast gene. PsPDC1 contains an open reading frame of 1,791 nucleotides encoding 597 amino acids. PsPDC2 contains a reading frame of 1,710 nucleotides encoding 570 amino acids. An 81-nucleotide segment in the middle of the β domain of PsPDC1 codes for a unique segment of 27 amino acids, which may play a role in allosteric regulation. The 5′ regions of both P. stipitis genes include two putative TATA elements that make them similar to the PDC genes from S. cerevisiae, Kluyveromyces marxianus, and Hanseniaspora uvarum. PMID:9435065

  12. Characterization of an avian histidine decarboxylase and localization of histaminergic neurons in the chicken brain.

    Bessho, Yuki; Iwakoshi-Ukena, Eiko; Tachibana, Tetsuya; Maejima, Sho; Taniuchi, Shusuke; Masuda, Keiko; Shikano, Kenshiro; Kondo, Kunihiro; Furumitsu, Megumi; Ukena, Kazuyoshi


    In mammals, it is established that histamine is a neurotransmitter and/or neuromodulator in the central nervous system. It is produced by the enzyme histidine decarboxylase (HDC) in the tuberomammillary nucleus of the posterior hypothalamus. However, HDC as well as histaminergic neurons have not yet been characterized in the avian brain. We have cloned the cDNA for HDC from the chicken hypothalamus and demonstrated that the chicken HDC sequence is highly homologous to the mammalian counterpart, and that the expressed protein shows high enzymatic activity. The expression of HDC mRNA at various sites in the brain was investigated using quantitative RT-PCR. The results showed that the HDC mRNA was highly expressed in the hypothalamic infundibulum. In situ hybridization analyses revealed that the cells containing HDC mRNA were localized in the medial mammillary nucleus of the hypothalamic infundibulum. Intracerebroventricular injection of histamine in chicks resulted in inhibition of feeding behavior. This is the first report of the characterization of histaminergic neurons in the avian brain, and our findings indicate that neuronal histamine exerts anorexigenic effects in chicks.

  13. Molecular characterization of Mtb-OMP decarboxylase by modeling, docking and dynamic studies.

    Madhusudana, P; Babajan, B; Chaitanya, M; Anuradha, C M; Shobharani, C; Chikati, Rajasekar; Kumar, Chitta Suresh; Rao, K R S Sambasiva; Poda, Sudhakar


    Tuberculosis (TB), the second most deadly disease in the world is caused by Mycobacterium tuberculosis (Mtb). In the present work a unique enzyme of Mtb orotidine 5' monophosphate decarboxylase (Mtb-OMP Decase) is selected as drug target due to its indispensible role in biosynthesis of pyrimidines. The present work is focused on understanding the structural and functional aspects of Mtb-OMP Decase at molecular level. Due to absence of crystal structure, the 3D structure of Mtb-OMP Decase was predicted by MODELLER9V7 using a known structural template 3L52. Energy minimization and refinement of the developed 3D model was carried out with Gromacs 3.2.1 and the optimized homology model was validated by PROCHECK,WHAT-IF and PROSA2003. Further, the surface active site amino acids were quantified by WHAT-IF pocket. The exact binding interactions of the ligands, 6-idiouridine 5' monophosphate and its designed analogues with the receptor Mtb-OMP Decase were predicted by docking analysis with AUTODOCK 4.0. This would be helpful in understanding the blockade mechanism of OMP Decase and provide a candidate lead for the discovery of Mtb-OMP Decase inhibitors, which may bring insights into outcome new therapy to treat drug resistant Mtb.

  14. Characterization of striatal neurons expressing high levels of glutamic acid decarboxylase messenger RNA.

    Chesselet, M F; Robbins, E


    Two types of labelled cells are detected in sections of rat and mouse striata processed for in situ hybridization histochemistry with 35S-radiolabelled RNA probes complementary to the messenger RNA (mRNA) encoding glutamic acid decarboxylase (GAD), the synthesis enzyme for gamma-aminobutyric acid (GABA): numerous lightly, and fewer very densely labelled neurons. In order to determine whether the densely labelled cells correspond to the striatal somatostatinergic neurons with which they share morphological characteristics, the presence of GAD mRNA was examined in brain sections processed successively for dihydronicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry, a marker of striatal somatostatinergic neurons, and in situ hybridization histochemistry. In addition, the distribution of GABAergic interneurons was analyzed with regard to striatal compartments (striosomes) indicated by patches of dense opiate binding sites. The results show that NADPH diaphorase activity and GAD mRNA do not co-exist in striatal neurons. Furthermore, in contrast to the somatostatinergic neurons which are almost exclusively located in the extrastriosomal matrix, densely labelled GAD cells were present both in the striosomes and the matrix, further suggesting that GABAergic and somatostatinergic neurons form two distinct interneuronal systems in the striatum of rats and mice.

  15. Regulation of human ornithine decarboxylase expression by the c-Myc.Max protein complex.

    Peña, A; Reddy, C D; Wu, S; Hickok, N J; Reddy, E P; Yumet, G; Soprano, D R; Soprano, K J


    The presence of a CACGTG element within a region of the human ornithine decarboxylase (ODC) promoter located at -491 to -474 base pairs 5' to the start site of transcription suggested that the c-Myc.Max protein complex may play a role in the regulation of ODC expression during growth. Electrophoretic mobility shift assays and methylation interference analysis showed that the nuclei of WI-38 cells expressing ODC contained proteins that bound to this region of the ODC gene in a manner that correlated with growth-associated ODC expression. Also, use of antibodies against c-Myc and Max and purified recombinant c-Myc and Max protein in the electrophoretic mobility shift assay confirmed that these proteins can specifically bind this portion of the human ODC promoter. Transient transfection studies showed that increase in the level of c-Myc and/or Max led to a significant enhancement of expression of a human ODC promoter-CAT reporter construct. Moreover, treatment of actively growing WI-38 cells with an antisense oligomer to c-Myc reduced the amount of endogenous protein complex formed and the amount of endogenous ODC mRNA expressed. These studies show that the c-Myc.Max protein complex plays a role in the transcriptional regulation of human ODC in vivo.

  16. Ornithine decarboxylase expression in the small intestine of broilers submitted to feed restriction and glutamine supplementation

    AV Fischer da Silva


    Full Text Available Six hundred and forty one-day-old Cobb male broilers were used to evaluate ornithine decarboxylase (ODC expression in the mucosa of the small intestine. Birds were submitted to early feed restriction from 7 to 14 days of age. The provided feed was supplemented with glutamine. A completely randomized design with a 2 x 2 factorial arrangement was used (with or without glutamine, with or without feed restriction. Restricted-fed birds were fed at 30% the amount of the ad libitum fed group from 7 to 14 days of age. Glutamine was added at the level of 1% in the diet supplied from 1 to 28 days of age. Protein concentration in the small intestine mucosa was determined, and ODC expression at 7, 14, 21, and 28 days of age was evaluated by dot blotting. ODC was present in the mucosa of broilers, and the presence of glutamine in the diet increased ODC activation. Glutamine prevented mucosa atrophy by stimulating protein synthesis, and was effective against the effects of feed restriction. Dot blotting can be used to quantify ODC expression in the intestinal mucosa of broilers.

  17. Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2

    Bruno Ramos-Molina


    Full Text Available Ornithine decarboxylase (ODC is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs, small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2 are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well-characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme-binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.

  18. Cell wall peroxidases in the liverwort Dumortiera hirsuta are responsible for extracellular superoxide production, and can display tyrosinase activity.

    Li, Jackson L Y; Sulaiman, Mariam; Beckett, Richard P; Minibayeva, Farida V


    In our earlier work, we showed that the liverwort Dumortiera hirsuta produces an extracellular oxidative burst of superoxide radicals during rehydration following desiccation stress. The oxidative burst is a common early response of organisms to biotic and abiotic stresses, with suggested roles in signal transduction, formation of protective substances such as suberin, melanin and lignin and defense against pathogens. To discover which enzymes are responsible for the extracellular superoxide production, we isolated apoplastic fractions from D. hirsuta, surveyed for the presence of potential redox enzymes, and performed non-denaturing polyacrylamide gel electrophoresis activity stains. Various isoforms of peroxidase (EC and tyrosinase (o-diphenolase) (EC were present at significant levels in the apoplast. In-gel activity staining revealed that some peroxidases isoforms could produce superoxide, while tryosinases could readily metabolize 3,4-dihydroxy phenyl l-alanine (l-dopa) into melanins. Interestingly, some peroxidase isoforms could oxidize the native tyrosinase substrate l-dopa at significant levels, even in the absence of hydrogen peroxide, while others could do so only in the presence of hydrogen peroxide. In D. hirsuta, peroxidases may play an important role in melanin formation. Possible functions for these diverse oxidases in liverwort biology are discussed.

  19. Chilling Tolerance of Cucumber During Germination is Related to Expression of Lysine Decarboxylase Gene

    LU Ming-hui; LI Xiao-ming; CHEN Jin-feng; CHEN Long-zheng; QIAN Chun-tao


    Using cDNA-AFLP technique, a specific fragment was isolated from cucumber cultivar Changchun mici possessing chilling tolerance induced at low temperature (15℃). This fragment, named cctr 132, could not be induced in the chilling sensitive cucumber cultivar Beijing jietou. After recovering the fragment, sequencing and translating, the results of blastx and blastp in GenBank of NCBI indicated that CCTR132 had 88.37% identities and 100% positives with Oryza sativa putative lysine decarboxylase-like protein respectively, and PGGXGTXXE, the putative conserved domain of lysine decarboxylase family, was detected from CCTR132, suggesting the cucumber chilling tolerance during germination is related to the expression of the lysine decarboxylase gene.

  20. Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius.

    Van Zyl, L J; Taylor, M P; Eley, K; Tuffin, M; Cowan, D A


    This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75 × 10(5) M(-1) s(-1) at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35 ± 0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.


    N. V. Piven


    Full Text Available Abstract. A new method of enzyme-linked immunosorbent assay (in solid-phase ELISA format has been developed to determine concentrations of autoantibodies to glutamic acid decarboxylase, as well as an evidencebased methodology is proposed for its medical implications, as a quantitative pathogenetic predictive marker of autoimmune diagnostics in type 1 diabetes mellitus. This technique could be implied for serial production of diagnostic reagent kits, aimed for detection of autoantibodies to glutamic acid decarboxylase by means of ELISA approach. (Med. Immunol., 2011, vol. 13, N 2-3, pp 257-260

  2. [Antioxidant and antibacterial activities of dimeric phenol compounds].

    Ogata, Masahiro


    We studied the antioxidant and antibacterial activities of monomeric and dimeric phenol compounds. Dimeric compounds had higher antioxidant activities than monomeric compounds. Electron spin resonance spin-trapping experiments showed that phenol compounds with an allyl substituent on their aromatic rings directly scavenged superoxide, and that only eugenol trapped hydroxyl radicals. We developed a generation system of the hydroxyl radical without using any metals by adding L-DOPA and DMPO to PBS or MiliQ water in vitro. We found that eugenol trapped hydroxyl radicals directly and is metabolized to a dimer. On the other hand, dipropofol, a dimer of propofol, has strong antibacterial activity against Gram-positive bacteria. However, it lacks solubility in water and this property is assumed to limit its efficacy. We tried to improve the solubility and found a new solubilization method of dipropofol in water with the addition of a monosaccharide or ascorbic acid.

  3. Cloning of tomato (Lycopersicon esculentum Mill.) arginine decarboxylase gene and its expression during fruit ripening.

    Rastogi, R; Dulson, J; Rothstein, S J


    Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of putrescine biosynthesis in plants. The genes encoding ADC have previously been cloned from oat and Escherichia coli. Degenerate oligonucleotides corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction amplification of tomato (Lycopersicon esculentum Mill.) genomic DNA, and a 1.05-kb fragment was obtained. This genomic DNA fragment encodes an open reading frame of 350 amino acids showing about 50% identity with the oat ADC protein. Using this fragment as a probe, we isolated several partial ADC cDNA clones from a tomato pericarp cDNA library. The 5' end of the coding region was subsequently obtained from a genomic clone containing the entire ADC gene. The tomato ADC gene contains an open reading frame encoding a polypeptide of 502 amino acids and a predicted molecular mass of about 55 kD. The predicted amino acid sequence exhibits 47 and 38% identify with oat and E. coli ADCs, respectively. Gel blot hybridization experiments show that, in tomato, ADC is encoded by a single gene and is expressed as a transcript of approximately 2.2 kb in the fruit pericarp and leaf tissues. During fruit ripening the amount of ADC transcript appeared to peak at the breaker stage. No significant differences were seen when steady-state ADC mRNA levels were compared between normal versus long-keeping Alcobaca (alc) fruit, although alc fruit contain elevated putrescine levels and ADC activity at the ripe stage. The lack of correlation between ADC activity and steady-state mRNA levels in alc fruit suggests a translational and/or posttranslational regulation of ADC gene expression during tomato fruit ripening.

  4. Effects of Sipgeondaebo-tang Pharmacopuncture Extracts on the Collagenase Activity and Procollagen Synthesis in HS68 Human Fibroblasts and Tyrosinase Activity Original Articles

    Lee Sena


    Full Text Available Objectives: This study was designed to investigate the collagen metabolism and tyrosinase activity of Sipgeondaebo-tang Pharmacopuncture extracts (SP. Methods: The effect of SP on type I procollagen production and collagenase activity in human normal fibroblasts HS68 after UVB (312 nm irradiation was measured by ELISA method. The tyrosinase activity after treatment of SP was measured as well. Results: Type I procollagen production was recovered by SP in UVB damaged HS68 cells. The increased collagenase activity after UVB damage was significantly recovered by SP. The tyrosinase activity was significantly reduced as well. However, the L-DOPA oxidation was not changed. Conclusion: SP showed the anti-wrinkle effects and whitening effects in vitro. These results suggest that SP may be a potential pharmacopuncture as an anti-aging pharmacopuncture treatments.

  5. The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases.

    Docimo, Teresa; Reichelt, Michael; Schneider, Bernd; Kai, Marco; Kunert, Grit; Gershenzon, Jonathan; D'Auria, John C


    Despite the long history of cocaine use among humans and its social and economic significance today, little information is available about the biochemical and molecular aspects of cocaine biosynthesis in coca (Erythroxylum coca) in comparison to what is known about the formation of other pharmacologically-important tropane alkaloids in species of the Solanaceae. In this work, we investigated the site of cocaine biosynthesis in E. coca and the nature of the first step. The two principal tropane alkaloids of E. coca, cocaine and cinnamoyl cocaine, were present in highest concentrations in buds and rolled leaves. These are also the organs in which the rate of alkaloid biosynthesis was the highest based on the incorporation of ¹³CO₂. In contrast, tropane alkaloids in the Solanaceae are biosynthesized in the roots and translocated to the leaves. A collection of EST sequences from a cDNA library made from young E. coca leaves was employed to search for genes encoding the first step in tropane alkaloid biosynthesis. Full-length cDNA clones were identified encoding two candidate enzymes, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), and the enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli and complementation of a yeast mutant. The transcript levels of both ODC and ADC genes were highest in buds and rolled leaves and lower in other organs. The levels of both ornithine and arginine themselves showed a similar pattern, so it was not possible to assign a preferential role in cocaine biosynthesis to one of these proteins.

  6. Hemocyanin-derived phenoloxidase activity is dependent on dodecameric structure in shrimp Litopenaeus vannamei

    Wang Ke-Zhou


    Full Text Available Hemocyanin (Hc is a multifunctional protein in both mollusks and arthropods. Phenoloxidase (PO activities are the most important physiological functions for Hcs after conversion. In shrimp, Hc occurs as two oligomer forms, dodecamers and hexamers. Differences in the transport oxygen capacity and agglutination activity between the two oligomers of shrimp Hc have been found. In the present study, we investigated the differences in the Hc-derived PO activity between the dodecameric and hexameric Hc forms of the shrimp Litopenaeus vannamei. The two oligomers were separated by non-denaturing polyacrylamide gel electrophoresis, converted by trypsin cleavage and their PO activities were determined by oxidation of L-DOPA. The dodecamers exhibited PO activity after enzymatic conversion while the hexamers did not exhibit PO activity. This result provides new insight into the structural/functional relationships of Hcs.

  7. Quantitative analysis of histidine decarboxylase gene (hdcA) transcription and histamine production by Streptococcus thermophilus PRI60 under conditions relevant to cheese making.

    Rossi, Franca; Gardini, Fausto; Rizzotti, Lucia; La Gioia, Federica; Tabanelli, Giulia; Torriani, Sandra


    This study evaluated the influence of parameters relevant for cheese making on histamine formation by Streptococcus thermophilus. Strains possessing a histidine decarboxylase (hdcA) gene represented 6% of the dairy isolates screened. The most histaminogenic, S. thermophilus PRI60, exhibited in skim milk a high basal level of expression of hdcA, upregulation in the presence of free histidine and salt, and repression after thermization. HdcA activity persisted in cell extracts, indicating that histamine might accumulate after cell lysis in cheese.

  8. Quantitative Analysis of Histidine Decarboxylase Gene (hdcA) Transcription and Histamine Production by Streptococcus thermophilus PRI60 under Conditions Relevant to Cheese Making▿†

    Rossi, Franca; Gardini, Fausto; Rizzotti, Lucia; La Gioia, Federica; Tabanelli, Giulia; Torriani, Sandra


    This study evaluated the influence of parameters relevant for cheese making on histamine formation by Streptococcus thermophilus. Strains possessing a histidine decarboxylase (hdcA) gene represented 6% of the dairy isolates screened. The most histaminogenic, S. thermophilus PRI60, exhibited in skim milk a high basal level of expression of hdcA, upregulation in the presence of free histidine and salt, and repression after thermization. HdcA activity persisted in cell extracts, indicating that histamine might accumulate after cell lysis in cheese. PMID:21378060

  9. Production of dopamine by aromatic L-amino acid decarboxylase cells after spinal cord injury

    Ren, Liqun; Wienecke, Jacob; Hultborn, Hans;


    Aromatic L-amino acid decarboxylase (AADC) cells are widely distributed in the spinal cord and their functions are largely unknown. We have previously found that AADC cells in the spinal cord could increase their ability to produce serotonin from 5-hydroxytryptophan after spinal cord injury (SCI)...

  10. The Degradation of 14C-Glutamic Acid by L-Glutamic Acid Decarboxylase.

    Dougherty, Charles M; Dayan, Jean


    Describes procedures and semi-micro reaction apparatus (carbon dioxide trap) to demonstrate how a particular enzyme (L-Glutamic acid decarboxylase) may be used to determine the site or sites of labeling in its substrate (carbon-14 labeled glutamic acid). Includes calculations, solutions, and reagents used. (Author/SK)

  11. DPD epitope-specific glutamic acid decarboxylase GAD)65 autoantibodies in children with Type 1 diabetes

    To study whether DPD epitope-specific glutamate decarboxylase autoantibodies are found more frequently in children with milder forms of Type 1 diabetes. We prospectively evaluated 75 children with new-onset autoimmune Type 1 diabetes, in whom we collected demographic, anthropometric and clinical dat...

  12. Clinical and biochemical features of aromatic L-amino acid decarboxylase deficiency.

    Brun, L.; Ngu, L.H.; Keng, W.T.; Ch'ng, G.S.; Choy, Y.S.; Hwu, W.L.; Lee, W.T.; Willemsen, M.A.A.P.; Verbeek, M.M.; Wassenberg, T.; Regal, L.; Orcesi, S.; Tonduti, D.; Accorsi, P.; Testard, H.; Abdenur, J.E.; Tay, S.; Allen, G.F.; Heales, S.; Kern, I.; Kato, M.; Burlina, A.; Manegold, C.; Hoffmann, G.F.; Blau, N.


    OBJECTIVE: To describe the current treatment; clinical, biochemical, and molecular findings; and clinical follow-up of patients with aromatic l-amino acid decarboxylase (AADC) deficiency. METHOD: Clinical and biochemical data of 78 patients with AADC deficiency were tabulated in a database of pediat

  13. Detection and transfer of the glutamate decarboxylase gene in Streptococcus thermophilus

    GABA (gamma-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermen...

  14. Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

    GABA ('-aminobutyric acid) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented da...

  15. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)


    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  16. [Catabolism of carnitine: products of carnitine decarboxylase and carnitine dehydrogenase in vivo].

    Seim, H; Löster, H; Strack, E


    1) Rats and mice were given large oral or subcutaneous doses of (-)-L-, (+)-D- and DL-carnitine (5 mg/g body weight). The carnitine metabolites, beta-methylcholine and acetonyltrimethylammonium, were isolated from the urine by special methods, and determined as their characteristic derivatives (2,4-dinitrophenylhydrazone and butyric ester) by thin-layer chromatography or photometry. 2) beta-Methylcholine, the product of carnitine decarboxylase, was not excreted, even when animals were heavily dosed with both carnitine isomers, with or without starvation. 3) After the administration of (+)-D- and DL-carnitine, both species excreted acetonyltrimethylammonium, which is already known as the spontaneous decarboxylation product of dehydrocarnitine (product of carnitine dehydrogenase) in bacteria. Injection of 0.71 mmol (+)-D-carnitine resulted in the excretion of 5.0 mumol (average) acetonyltrimethylammonium per mouse during the 48 h post injection. Under the same conditions, rats produced up to 40 mumol acetonyltrimethylammonium. The ratio of excreted acetonyltrimethylammonium to injected (+)-D-carnitine depended on the method of administration and the dose. 4) Production of the pharmacologically active (+)-acetyl-L-beta-methylcholine is not to be expected, following high exogenous doses of (-)-L-carnitine or (-)-acetyl-L-carnitine. The chief metabolites are trimethylamine, trimethylamine oxide and gamma-butyrobetaine (this journal 361, 1059), and both the (-)-L-carnitine pool and exogenous (-)-L-carnitine are dehydrogenated or decarboxylated only to a very small extent, if at all. When DL-carnitine is used therapeutically, the formation of acetonyltrimethylammonium must be taken into account.

  17. Disease-specific monoclonal antibodies targeting glutamate decarboxylase impair GABAergic neurotransmission and affect motor learning and behavioral functions

    Mario U Manto


    Full Text Available Autoantibodies to the smaller isoform of glutamate decarboxylase can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The detection of disease-specific autoantibody epitopes led to the hypothesis that distinct glutamate decarboxylase autoantibodies may elicit specific neurological phenotypes. We explored the in vitro/in vivo effects of well-characterized monoclonal glutamate decarboxylase antibodies. We found that glutamate decarboxylase autoantibodies present in patients with stiff person syndrome (n = 7 and cerebellar ataxia (n = 15 recognized an epitope distinct from that recognized by glutamate decarboxylase autoantibodies present in patients with type 1 diabetes mellitus (n = 10 or limbic encephalitis (n = 4. We demonstrated that the administration of a monoclonal glutamate decarboxylase antibody representing this epitope specificity (1 disrupted in vitro the association of glutamate decarboxylase with γ-Aminobutyric acid containing synaptic vesicles, (2 depressed the inhibitory synaptic transmission in cerebellar slices with a gradual time course and a lasting suppressive effect, (3 significantly decreased conditioned eyelid responses evoked in mice, with no modification of learning curves in the classical eyeblink-conditioning task, (4 markedly impaired the facilitatory effect exerted by the premotor cortex over the motor cortex in a paired-pulse stimulation paradigm, and (5 induced decreased exploratory behavior and impaired locomotor function in rats. These findings support the specific targeting of glutamate decarboxylase by its autoantibodies in the pathogenesis of stiff-person syndrome and cerebellar ataxia. Therapies of these disorders based on selective removal of such glutamate decarboxylase antibodies could be envisioned.

  18. Real-Time kinetic studies of Bacillus subtilis oxalate decarboxylase and Ceriporiopsis subvermispora oxalate oxidase using a luminescent oxygen sensor

    Laura Molina


    Full Text Available Oxalate decarboxylase (OxDC, an enzyme of the bicupinsuperfamily, catalyzes the decomposition of oxalate into carbondioxide and formate at an optimal pH of 4.3 in the presence ofoxygen. However, about 0.2% of all reactions occur through anoxidase mechanism that consumes oxygen while producing twoequivalents of carbon dioxide and one equivalent of hydrogenperoxide. The kinetics of oxidase activity were studied bymeasuring the consumption of dissolved oxygen over time using a luminescent oxygen sensor. We describe the implementation of and improvements to the oxygen consumption assay. The oxidase activity of wild type OxDC was compared to that of the T165V OxDC mutant, which contains an impaired flexible loop covering the active site. The effects of various carboxylic acid-based buffers on the rate of oxidase activity were also studied. These results were compared to the oxidase activity of oxalate oxidase (OxOx, a similar bicupin enzyme that only carries out oxalate oxidation. Thetemperature dependence of oxidase activity was analyzed, andpreliminary results offer an estimate for the overall activationenergy of the oxidase reaction within OxDC. The data reported here thus provide insights into the mechanism of the oxidase activity of OxDC.

  19. Crystal Structures of Apo and Liganded 4-Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β-Keto Acid.

    Guimarães, Samuel L; Coitinho, Juliana B; Costa, Débora M A; Araújo, Simara S; Whitman, Christian P; Nagem, Ronaldo A P


    The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria Pseudomonas putida G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg(2+) as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD.

  20. Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.

    Torriani, Sandra; Gatto, Veronica; Sembeni, Silvia; Tofalo, Rosanna; Suzzi, Giovanna; Belletti, Nicoletta; Gardini, Fausto; Bover-Cid, Sara


    In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.

  1. The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89

    Lei Yuwen; Feng-Li Zhang; Qi-Hua Chen; Shuang-Jun Lin; Yi-Lei Zhao; Zhi-Yong Li


    For biosynthesis of bacillamide C by Bacillus atrophaeus C89 associated with South China sea sponge Dysidea avara, it is hypothesized that decarboxylation from L-tryptophan to tryptamine could be performed before amidation by the downstream aromatic L-amino acid decarboxylase (AADC) to the non-ribosomal peptide synthetases (NRPS) gene cluster for biosynthesizing bacillamide C. The structural analysis of decarboxylases' known substrates in KEGG database and alignment analysis of amino acid seq...

  2. Molecular Docking Studies and Anti-Tyrosinase Activity of Thai Mango Seed Kernel Extract

    Patchreenart Saparpakorn


    Full Text Available The alcoholic extract from seed kernels of Thai mango (Mangifera indica L. cv. ‘Fahlun’ (Anacardiaceae and its major phenolic principle (pentagalloylglucopyranose exhibited potent, dose-dependent inhibitory effects on tyrosinase with respect to L-DOPA. Molecular docking studies revealed that the binding orientations of the phenolic principles were in the tyrosinase binding pocket and their orientations were located in the hydrophobic binding pocket surrounding the binuclear copper active site. The results indicated a possible mechanism for their anti-tyrosinase activity which may involve an ability to chelate the copper atoms which are required for the catalytic activity of tyrosinase.

  3. Altered resting state cortico-striatal connectivity in mild to moderate stage Parkinson’s disease

    Youngbin Kwak


    Full Text Available Parkinson’s disease (PD is a progressive neurodegenerative disorder that is characterized by dopamine depletion in the striatum. One consistent pathophysiological hallmark of PD is an increase in spontaneous oscillatory activity in the basal ganglia thalamocortical networks. We evaluated these effects using resting state functional connectivity MRI (fcMRI in mild to moderate stage Parkinson’s patients on and off L-DOPA and age-matched controls using six different striatal seed regions. We observed an overall increase in the strength of cortico-striatal functional connectivity in PD patients off L-DOPA compared to controls. This enhanced connectivity was down-regulated by L-DOPA as shown by an overall decrease in connectivity strength, particularly within motor cortical regions. We also performed a frequency content analysis of the BOLD signal time course extracted from the six striatal seed regions. PD off L-DOPA exhibited increased power in the frequency band 0.02 – 0.05 Hz compared to controls and to PD on L-DOPA. The L-DOPA associated decrease in the power of this frequency range modulated the L-DOPA associated decrease in connectivity strength between striatal seeds and the thalamus. In addition, the L-DOPA associated decrease in power in this frequency band also correlated with the L-DOPA associated improvement in cognitive performance. Our results demonstrate that PD and L-DOPA modulate striatal resting state BOLD signal oscillations and corticostriatal network coherence.

  4. A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin.

    Furuya, Toshiki; Miura, Misa; Kino, Kuniki


    Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 g L(-1) ) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

  5. Characterization of a Novel Putative S-Adenosylmethionine Decarboxylase-Like Protein from Leishmania donovani.

    Saurabh Pratap Singh

    Full Text Available In addition to the S-adenosylmethionine decarboxylase (AD present in all organisms, trypanosomatids including Leishmania spp. possess an additional copy, annotated as the putative S-adenosylmethionine decarboxylase-like proenzyme (ADL. Phylogenetic analysis confirms that ADL is unique to trypanosomatids and has several unique features such as lack of autocatalytic cleavage and a distinct evolutionary lineage, even from trypanosomatid ADs. In Trypanosoma ADL was found to be enzymaticaly dead but plays an essential regulatory role by forming a heterodimer complex with AD. However, no structural or functional information is available about ADL from Leishmania spp. Here, in this study, we report the cloning, expression, purification, structural and functional characterization of Leishmania donovani (L. donovani ADL using biophysical, biochemical and computational techniques. Biophysical studies show that, L. donovani ADL binds S-adenosylmethionine (SAM and putrescine which are natural substrates of AD. Computational modeling and docking studies showed that in comparison to the ADs of other organisms including human, residues involved in putrescine binding are partially conserved while the SAM binding residues are significantly different. In silico protein-protein interaction study reveals that L. donovani ADL can interact with AD. These results indicate that L. donovani ADL posses a novel substrate binding property and may play an essential role in polyamine biosynthesis with a different mode of function from known proteins of the S-adenosylmethionine decarboxylase super family.

  6. Sequencing, characterization, and gene expression analysis of the histidine decarboxylase gene cluster of Morganella morganii.

    Ferrario, Chiara; Borgo, Francesca; de Las Rivas, Blanca; Muñoz, Rosario; Ricci, Giovanni; Fortina, Maria Grazia


    The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.

  7. Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

    Eram, Mohammad S; Wong, Alton; Oduaran, Erica; Ma, Kesen


    Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

  8. Melanogenesis and antityrosinase activity of selected South african plants.

    Mapunya, Manyatja Brenda; Nikolova, Roumiana Vassileva; Lall, Namrita


    Melanin is the pigment that is responsible for the colour of eyes, hair, and skin in humans. Tyrosinase is known to be the key enzyme in melanin biosynthesis. Overactivity of this enzyme leads to dermatological disorders such as age spots, melanoma and sites of actinic damage. Ten plants belonging to four families (Asphodelaceae, Anacardiaceae, Oleaceae, and Rutaceae) were investigated for their effect on tyrosinase using both L-tyrosine and L-DOPA as substrates. Ethanol leaf extracts (500 μg/mL) of Aloe ferox, Aloe aculeata, Aloe pretoriensis, and Aloe sessiliflora showed 60%, 31%, 17%, and 13% inhibition of tyrosinase activity respectively, when L-tyrosine was used as a substrate. Harpephyllum caffrum (leaves) at a concentration of 500 μg/mL had an inhibitory effect of 70% on tyrosinase when L-DOPA was used as a substrate. The IC(50) of Harpephyllum caffrum (leaves and bark) were found to be 51 ± 0.002 and 40 ± 0.035 μg/mL, respectively. Following the results obtained from the tyrosinase assay, extracts from Harpephyllum caffrum were selected for further testing on their effect on melanin production and their cytotoxicity on melanocytes in vitro. The IC(50) of both extracts was found to be 6.25 μg/mL for melanocyte cells. Bark extract of Harpephyllum caffrum showed 26% reduction in melanin content of melanocyte cells at a concentration of 6.25 μg/mL. The leaf extract of this plant showed some toxicity on melanocyte cells. Therefore, the bark extract of Harpephyllum caffrum could be considered as an antityrosinase agent for dermatological disorders such as age spots and melasoma.

  9. Melanogenesis and Antityrosinase Activity of Selected South African Plants

    Manyatja Brenda Mapunya


    Full Text Available Melanin is the pigment that is responsible for the colour of eyes, hair, and skin in humans. Tyrosinase is known to be the key enzyme in melanin biosynthesis. Overactivity of this enzyme leads to dermatological disorders such as age spots, melanoma and sites of actinic damage. Ten plants belonging to four families (Asphodelaceae, Anacardiaceae, Oleaceae, and Rutaceae were investigated for their effect on tyrosinase using both L-tyrosine and L-DOPA as substrates. Ethanol leaf extracts (500 μg/mL of Aloe ferox, Aloe aculeata, Aloe pretoriensis, and Aloe sessiliflora showed 60%, 31%, 17%, and 13% inhibition of tyrosinase activity respectively, when L-tyrosine was used as a substrate. Harpephyllum caffrum (leaves at a concentration of 500 μg/mL had an inhibitory effect of 70% on tyrosinase when L-DOPA was used as a substrate. The IC50 of Harpephyllum caffrum (leaves and bark were found to be 51±0.002 and 40±0.035 μg/mL, respectively. Following the results obtained from the tyrosinase assay, extracts from Harpephyllum caffrum were selected for further testing on their effect on melanin production and their cytotoxicity on melanocytes in vitro. The IC50 of both extracts was found to be 6.25 μg/mL for melanocyte cells. Bark extract of Harpephyllum caffrum showed 26% reduction in melanin content of melanocyte cells at a concentration of 6.25 μg/mL. The leaf extract of this plant showed some toxicity on melanocyte cells. Therefore, the bark extract of Harpephyllum caffrum could be considered as an antityrosinase agent for dermatological disorders such as age spots and melasoma.

  10. Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

    Soriano, Erika V. [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850-1301 (United States); McCloskey, Diane E. [Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, PA 17033 (United States); Kinsland, Cynthia [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850-1301 (United States); Pegg, Anthony E. [Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, PA 17033 (United States); Ealick, Steven E., E-mail: [Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850-1301 (United States)


    The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.

  11. Impact of Cell-free Supernatant of Lactic Acid Bacteria on Putrescine and Other Polyamine Formation by Foodborne Pathogens in Ornithine Decarboxylase Broth.

    Ozogul, Fatih; Tabanelli, Giulia; Toy, Nurten; Gardini, Fausto


    Conversion of ornithine to putrescine by Salmonella Paratyphi A, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli was investigated in ornithine decarboxylase broth (ODB) using cell-free supernatants (CFSs) obtained from Leuconostoc mesenterodies subsp. cremoris, Pediococcus acidilactici, Lactococcus lactis subsp. lactis, Streptococcus thermophilus. Two groups of cell-free supernatants (25 or 50%) and control (only ODB) were prepared to investigate putrescine (PUT) and other polyamine formation by foodborne pathogens (FBPs). Significant differences (p < 0.05) were observed among the species for each amine. All of the CFSs reduced the formation of PUT by ≥65%. The production of cadaverine (CAD) was scarcely affected by the presence of CFSs, with the exception of the samples inoculated with L. monocytogenes. The variation in polyamine was found with respect to the control samples. Spermidine (SPD) was produced in lower amount in many samples, especially in Gram-negative FBPs, whereas spermine (SPN) increased drastically in the major part of the samples concerning the control. Histamine (HIS) was characterized by a marked concentration decrease in all of the samples, and tyramine (TYR) was accumulated in very low concentrations in the controls. Therefore, the ability of bacteria to produce certain biogenic amines such as HIS, TYR, PUT, and CAD has been studied to assess their risk and prevent their formation in food products. The results obtained from this study concluded that the lactic acid bacteria (LAB) strains with non-decarboxylase activity are capable of avoiding or limiting biogenic amine formation by FBP.

  12. Elimination of islet cell antibodies and glutamic acid decarboxylase antibodies II in a patient with newly diagnosed insulin-dependent diabetes mellitus.

    Richter, W O; Donner, M G; Schwandt, P


    Islet cell antibodies and glutamic acid decarboxylase II (GAD II) antibodies have been discussed in the autoimmune pathogenesis of insulin-dependent diabetes mellitus (IDDM). Hence, immunosuppressants, intravenous immunoglobulins, and plasmapheresis have been used in an effort to modulate autoimmune activity and thereby prevent the destruction of pancreatic beta-cells. We describe the autoantibody (islet cell antibody and GAD II) kinetics and clinical course in a patient with newly diagnosed IDDM treated with a specific immunoglobulin apheresis technique. Five days after the initial diagnosis a 37-year-old patient with IDDM underwent a series of seven immunoglobulin aphereses. Immunoglobulin (IgG, IgA, IgM), islet cell antibody, GAD II, and C-peptide concentrations were monitored for a time course of 74 days. Daily insulin requirements were recorded. One single immunoglobulin apheresis decreased IgG by 66.2 +/- 9.1%, IgA by 66.8 +/- 8.7%, and IgM by 57.7 +/- 12.9%. GAD II antibodies were reduced by 61.9 +/- 12.4%. The islet cell antibody titer declined from 1:32 to 1:4 after the treatment series. There were no relevant changes in the safety parameters determined nor were there any clinical side effects. The efficient decrease in islet cell antibodies and glutamic acid decarboxylase II antibodies in a patient with IDDM encourages further investigations into the impact of this treatment on the clinical course of this autoimmune disorder.

  13. Convulsions and inhibition of glutamate decarboxylase by pyridoxal phosphate-gamma-glutamyl hydrazone in the developing rat.

    Massieu, L; Rivera, A; Tapia, R


    We have previously shown that in the adult rat the inhibition of brain glutamate decarboxylase (GAD) activity by pyridoxal phosphate-gamma-glutamyl hydrazone (PLPGH) administration does not result in convulsions, whereas in the adult mouse intense convulsions invariably occur. In the present study we report that, surprisingly, immature rats from 2 to 20 days of age treated with PLPGH (80 mg/kg) showed generalized tonic-clonic convulsions, whereas no convulsions at all were present in 30 days-old or older rats. GAD activity, measured by enzymic determination of GABA formed in forebrain homogenates, was inhibited by about 60% at the time of convulsions in 15 days-old and younger rats, whereas the inhibition was between 40 and 50% in older animals. The addition of the coenzyme pyridoxal 5'-phosphate to the incubation medium completely reversed this inhibition. In all treated animals GABA levels were lower compared to controls. The results indicate that the susceptibility of GAD in vivo to a diminished cofactor concentration decreases with age. It seems possible that changes in the expression of enzyme forms are reflected in developmental variations in the susceptibility to seizures induced by vitamin B6 depletion, but alterations of other B6-dependent biochemical pathways cannot be discarded.

  14. Enhanced production of recombinant Escherichia coli glutamate decarboxylase through optimization of induction strategy and addition of pyridoxine.

    Su, Lingqia; Huang, Yan; Wu, Jing


    This report describes the optimization of recombinant Escherichia coli glutamate decarboxylase (GAD) production from engineered E. coli BL21(DE3) in a 3-L fermentor. Investigation of different induction strategies revealed that induction was optimal when the temperature was maintained at 30°C, the inducer (lactose) was fed at a rate of 0.2 g L(-1)h(-1), and protein expression was induced when the cell density (OD600) reached 50. Under these conditions, the GAD activity of 1273.8 U mL(-1) was achieved. Because GAD is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, the effect of supplementing the medium with pyridoxine hydrochloride (PN), a cheap and stable PLP precursor, on GAD production was also investigated. When the culture medium was supplemented with PN to a concentration of 2mM at the initiation of protein expression, and then again 10h later, the GAD activity reached 3193.4 U mL(-1), which represented the highest GAD production ever reported.

  15. Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli.

    Kang, Taek Jin; Ho, Ngoc Anh Thu; Pack, Seung Pil


    Escherichia coli glutamate decarboxylase (GAD) converts glutamate into γ-aminobutyric acid (GABA) through decarboxylation using proton as a co-substrate. Since GAD is active only at acidic conditions even though pH increases as the reaction proceeds, the conventional practice of using this enzyme involved the use of relatively high concentration of buffers, which might complicate the downstream purification steps. Here we show by simulation and experiments that the free acid substrate, glutamic acid, rather than its monosodium salt can act as a substrate and buffer at the same time. This yielded the buffer- and salt-free synthesis of GABA conveniently in a batch mode. Furthermore, we engineered GAD to hyper active ones by extending or reducing the length of the enzyme by just one residue at its C-terminus. Through the buffer-free reaction with engineered GAD, we could synthesize 1M GABA in 3h, which can be translated into a space-time yield of 34.3g/L/h. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding.

    Jetten, A M; Ganong, B R; Vandenbark, G R; Shirley, J E; Bell, R M


    Tumor-promoting phorbol esters induce ornithine decarboxylase (ODCase) activity and reduce epidermal growth factor (EGF) binding in rat tracheal epithelial 2C5 cells. Phorbol esters activate protein kinase C by interacting at the same site as sn-1,2-diacylglycerols, the presumed physiological regulators. The effects of added sn-1,2-diacylglycerols and those generated by phospholipase C treatment of 2C5 cells on ODCase induction and EGF binding were investigated to establish a role for protein kinase C in these cellular responses. Treatment of 2C5 cells with phospholipase C induced ODCase activity and reduced EGF binding, whereas phospholipases A2 and D were inactive. When sn-1,2-diacylglycerols containing fatty acids 3-10 carbons in length were added to 2C5 cells, those diacylglycerols containing fatty acids 5-10 carbons in length caused ODCase induction and reduction in EGF binding. sn-1,2-Dioctanoylglycerol was one of the most active compounds tested. It induced ODCase in a dose- (50-500 microM) and time-dependent manner. The reduction of binding of 125I-labeled EGF by sn-1,2-dioctanoylglycerol was also time and dose dependent and appeared to result from a change in EGF affinity and not the number of receptor sites. This series of sn-1,2-diacylglycerols showed similar structure-function relationships in their ability to induce ODCase activity, to decrease EGF binding, to stimulate protein kinase C, and to inhibit [3H]phorbol dibutyrate binding to the phorbol ester receptor. These data demonstrate biological activities for a number of diacylglycerols and indicate that protein kinase C activation is implicated in ODCase induction and decreased EGF binding. PMID:3157191

  17. Substrate Binding Induces Domain Movements in Orotidine 5'-Monophosphate Decarboxylase

    Harris, Pernille Hanne; Poulsen, Jens-Christian Navarro; Jensen, Kaj Frank;


    ); here we present the 2.5 Å structure of the uncomplexed apo enzyme, determined from twinned crystals. A structural analysis and comparison of the two structures of the E. coli enzyme show that binding of the inhibitor is accompanied by significant domain movements of approximately 12° around a hinge...... that crosses the active site. Hence, the ODCase dimer, which contains two active sites, may be divided in three domains: a central domain that is fixed, and two lids which independently move 12° upon binding. Corresponding analyses, presented herein, of the two Saccharomyces cerevisiae ODCase structures (with...

  18. Active Theater as a Complementary Therapy for Parkinson's Disease Rehabilitation: A Pilot Study

    Nicola Modugno


    Full Text Available Most medical treatments of Parkinson's disease (PD are aimed at the reduction of motor symptoms. However, even when motor improvements are evident, patients often report a deterioration of their daily lives. Thus, to achieve a global improvement in personal well-being, not only drugs, but also complementary therapies, such as physical exercise, occupational and speech therapy, and active music therapy, have been used. We hypothesized that theater could reduce clinical disability and improve the quality of life of PD patients (primary end points more efficiently than other complementary therapies because (1 in order to impersonate a character, patients are forced to regain the control of their bodies; and (2 while being part of a group, patients have a high degree of social interaction. The need to regain the control of their bodies and their social functioning is very likely to deeply motivate patients. To assess this hypothesis, we ran a randomized, controlled, and single-blinded study that lasted 3 years, on 20 subjects affected by a moderate form of idiopathic PD, in stable treatment with L-dopa and L-dopa agonists, and without severe sensory deficits. Ten patients were randomly assigned to an active theater program (in which patients were required to participate, while the others underwent physiotherapy (control group, the most common nonpharmacological treatment for PD rehabilitation. Patients of both groups were evaluated at the beginning of each year, using five clinical rating scales (Unified ParkinsonParkinson'ss Disease Rating Scale [UPDRS], Schwab and England Scale, ParkinsonParkinson'ss Disease Quality of Life [PDQ39] Scale, Epworth Sleepiness Scale, and Hamilton Depression Rating Scale. The theater patients showed progressive improvements and, at the end of the third year, they showed significant improvements in all clinical scales. Conversely, the control patients did not exhibit significant ameliorations with time. Thus, the

  19. Active theater as a complementary therapy for Parkinson's disease rehabilitation: a pilot study.

    Modugno, Nicola; Iaconelli, Sara; Fiorlli, Mariagrazia; Lena, Francesco; Kusch, Imogen; Mirabella, Giovanni


    Most medical treatments of Parkinson's disease (PD) are aimed at the reduction of motor symptoms. However, even when motor improvements are evident, patients often report a deterioration of their daily lives. Thus, to achieve a global improvement in personal well-being, not only drugs, but also complementary therapies, such as physical exercise, occupational and speech therapy, and active music therapy, have been used. We hypothesized that theater could reduce clinical disability and improve the quality of life of PD patients (primary end points) more efficiently than other complementary therapies because (1) in order to impersonate a character, patients are forced to regain the control of their bodies; and (2) while being part of a group, patients have a high degree of social interaction. The need to regain the control of their bodies and their social functioning is very likely to deeply motivate patients. To assess this hypothesis, we ran a randomized, controlled, and single-blinded study that lasted 3 years, on 20 subjects affected by a moderate form of idiopathic PD, in stable treatment with L-dopa and L-dopa agonists, and without severe sensory deficits. Ten patients were randomly assigned to an active theater program (in which patients were required to participate), while the others underwent physiotherapy (control group), the most common nonpharmacological treatment for PD rehabilitation. Patients of both groups were evaluated at the beginning of each year, using five clinical rating scales (Unified Parkinson's Disease Rating Scale [UPDRS], Schwab and England Scale, Parkinson's Disease Quality of Life [PDQ39] Scale, Epworth Sleepiness Scale, and Hamilton Depression Rating Scale). The theater patients showed progressive improvements and, at the end of the third year, they showed significant improvements in all clinical scales. Conversely, the control patients did not exhibit significant ameliorations with time. Thus, the present study provides the first

  20. Study on the antioxidant activity and membrane interaction of a multiple antioxidant

    Jodko-Jodko-Piórecka, Kasia; Klösgen, Beate; Litwinienko, Grzegorz

    Catecholamines, including dopamine, have a role in the transduction of nervous stimuli. Apart from that, they might behave as endogenous phenolic antioxidants protecting the neuronal tissue from deleterious effects of oxidative stress. Results from our preliminary study1 even indicate a synergistic...... effect: an interplay of catecholamines with other molecules seems to enhance their antioxidant activity (e.g. 3,4-dihydroxyphenylalanine - L-DOPA -, and 2,2,5,7,8-pentamethyl-6-hydroxychroman - PMHC -, an analogue of α-tocopherol). Addition of small amounts of catecholamines suppresses or largely...... inhibits the peroxidation of lipids2. The understanding of the mechanism of antioxidant action of catecholamines and their interplay with other antioxidants in lipid membranes requires interdisciplinary research on the kinetics and thermodynamics of antioxidant(s)/membrane interactions. Here we report...

  1. Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum (L.).

    Pérez-Amador, M A; Carbonell, J; Granell, A


    A cDNA coding for arginine decarboxylase (ADC, EC has been isolated from a cDNA library of parthenocarpic young fruits of Pisum sativum (L.). The deduced aminoacid sequence is 74%, 46% and 35% identical to ADCs from tomato, oat and Escherichia coli, respectively. When the pea ADC cDNA was put under the control of the galactose inducible yeast promoter CYC1-GAL10 and introduced into Saccharomyces cerevisiae, it conferred galactose-regulated expression of the ADC activity. The ADC activity expressed in S. cerevisiae was inhibited 99% by alpha-DL-difluoromethylarginine (DFMA), a specific inhibitor of ADC activity. No activity was detected in the untransformed S. cerevisiae, nor when it was transformed with an antisense ADC construct. This provides direct evidence that the ADC cDNA from pea encoded a functional, specific ADC activity and that S. cerevisiae is able to process correctly the protein. In the pea plant, gene expression of the ADC is high in young developing tissues like shoot tips, young leaflets and flower buds. Fully expanded leaflets and roots have much lower, but still detectable, levels of the ADC transcript. In the ovary and fruit, they are developmentally regulated, showing high levels of expression during the early stages of fruit growth, which in pea is mainly due to cell expansion. The observed changes in the steady-state levels of ADC mRNA alone, however, cannot account for the differences in ADC activity suggesting that other regulatory mechanisms must be acting.

  2. Walther Birkmayer, Co-describer of L-Dopa, and his Nazi connections: victim or perpetrator?

    Czech, Herwig; Zeidman, Lawrence A


    Walther Birkmayer, an Austrian neurologist, codiscovered the efficacy of levodopa therapy for Parkinsonism in 1961. However, little has been published regarding Birkmayer's ties to National Socialism. Through documentary review, we have determined that he was an early illegal member of the SS and the Nazi party, taking part in the "de-Jewification" of the Vienna University Clinic of Psychiatry and Neurology. He also was a leader in the Nazi racial policy office and was praised for his dedication and fanaticism despite being forced to later resign from the SS. He sought support from leading Viennese Nazis, and was able to maintain his professional status for the war's remainder. Postwar, he succeeded at reintegration personally and professionally into Austrian society, all but erasing any obvious ties to his Nazi past. His story reflects ethical transgressions regarding professional and personal behavior in response to a tyrannical regime and provides lessons for today's neuroscientists.

  3. Cerebrospinal fluid biomarkers for Parkinson's disease and L-DOPA-induced dyskinesia

    Dammann Andersen, Andreas


    Parkinson’s disease (PD) is a common disease, affecting 1% of the population older than 60 years of age[1]. Neuropathological characteristics of the disease include the presence of alpha-synuclein containing Lewy bodies in the brain stem and loss of nigrostriatal dopaminergic neurons, which result...... in motor deficits that are used as diagnostic criteria for PD[2]. So far there is no treatment available slowing the progression of PD. The otherwise effective dopamine replacement therapy not only loses its effectiveness during the natural course of the disease but also increases the risk of developing...... of patients, who are also clinically rated using the Unified Parkinson’s Disease rating scale (UPDRS)[5] and the Unified Dyskinesia Rating Scale (UDysRS). 1. de Lau LM, Breteler MM. Epidemiology of Parkinson's disease. Lancet neurology. 2006;5(6):525-35. 2. Rodriguez-Oroz MC, Jahanshahi M, Krack P, et al...

  4. PSD-95 expression controls l-DOPA dyskinesia through dopamine D1 receptor trafficking

    Porras, Gregory; Berthet, Amandine; Dehay, Benjamin; Li, Qin; Ladepeche, Laurent; Normand, Elisabeth; Dovero, Sandra; Martinez, Audrey; Doudnikoff, Evelyne; Martin-Négrier, Marie-Laure; Chuan, Qin; Bloch, Bertrand; Choquet, Daniel; Boué-Grabot, Eric; Groc, Laurent; Bezard, Erwan


    l-DOPA–induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson’s disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson’s patients. PMID:23041629

  5. Highly Efficient Diastereoselective Synthesis of Tetrahydro-isoquinoline-3- carboxylate Ester Analogs from L-DOPA

    WANG Ye; LIU Zhan-Zhu; CHEN Shi-Zhi; LIANG Xiao-Tian


    @@ Tetrahydroisoquinoline-3-carboxylate esters are an important motif of naturally occurring bioactive alkaloids and pharmacophores. They are generally regarded as neurotoxic compounds and are putatively involved in a variety of pathologic conditions of central nervous system, including alcoholism, phenylketonuria, and neurodegenerative disorders such as Parkinson's disease.

  6. Anti-Inflammatory and Antihyperalgesic Activities of Ethanolic Extract and Fruticulin A from Salvia lachnostachys Leaves in Mice

    Ana Claudia Piccinelli


    Full Text Available The anti-inflammatory and analgesic effects of the ethanolic extract (SLEE and fruticulin A from the leaves of Salvia lachnostachys were evaluated in mice, using experimental models of inflammation (paw oedema and pleurisy induced by carrageenan injection and hyperalgesia (electronic Von Frey. Oral administration of SLEE (30, 100, and 300 mg/kg and fruticulin A (0.3 and 3.0 mg/kg decreased the total leucocytes number in pleural lavage, protein extravasation, and paw oedema. SLEE (100 mg/kg and fruticulin A (3 mg/kg also exhibited antihyperalgesic activity in carrageenan induced mechanical hyperalgesia. In addition, fruticulin A (3 mg/kg prevented mechanical hyperalgesia, inhibiting TNF but not L-DOPA-induced mechanical hyperalgesia. In conclusion, SLEE and fruticulin A display anti-inflammatory and analgesic properties. Therefore, fruticulin A is at least partially responsible for the activity observed in the ethanolic extract of Salvia lachnostachys.

  7. Spinal cord hemisection facilitates aromatic L-amino acid decarboxylase cells to produce serotonin in the subchronic but not the chronic phase

    Azam, Bushra; Wienecke, Jacob; Jensen, Dennis Bo;


    12) were used with a postoperation interval at 5 days or 60 days. Using immunohistochemistry, first, we observed a significant reduction in the density of 5-HT-immunoreactive fibers in the spinal cord below the lesion on the injured side for both groups. Second, we found that the AADC cells were......Neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT) and noradrenalin, play an essential role in regulating the motor and sensory functions in the spinal cord. We have previously shown that in the rat spinal cord the activity of aromatic L-amino acid decarboxylase (AADC) cells to produce...... 5-HT from its precursor (5-hydroxytryptophan, 5-HTP) is dramatically increased following complete spinal cord transection. In this study, we investigated whether a partial loss of 5-HT innervation could similarly increase AADC activity. Adult rats with spinal cord hemisected at thoracic level (T11/T...

  8. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  9. Thiol Redox Sensitivity of Two Key Enzymes of Heme Biosynthesis and Pentose Phosphate Pathways: Uroporphyrinogen Decarboxylase and Transketolase

    Brian McDonagh


    Full Text Available Uroporphyrinogen decarboxylase (Hem12p and transketolase (Tkl1p are key mediators of two critical processes within the cell, heme biosynthesis, and the nonoxidative part of the pentose phosphate pathway (PPP. The redox properties of both Hem12p and Tkl1p from Saccharomyces cerevisiae were investigated using proteomic techniques (SRM and label-free quantification and biochemical assays in cell extracts and in vitro with recombinant proteins. The in vivo analysis revealed an increase in oxidized Cys-peptides in the absence of Grx2p, and also after treatment with H2O2 in the case of Tkl1p, without corresponding changes in total protein, demonstrating a true redox response. Out of three detectable Cys residues in Hem12p, only the conserved residue Cys52 could be modified by glutathione and efficiently deglutathionylated by Grx2p, suggesting a possible redox control mechanism for heme biosynthesis. On the other hand, Tkl1p activity was sensitive to thiol redox modification and although Cys622 could be glutathionylated to a limited extent, it was not a natural substrate of Grx2p. The human orthologues of both enzymes have been involved in certain cancers and possess Cys residues equivalent to those identified as redox sensitive in yeast. The possible implication for redox regulation in the context of tumour progression is put forward.

  10. QSAR study of malonyl-CoA decarboxylase inhibitors using GA-MLR and a new strategy of consensus modeling.

    Li, Jiazhong; Lei, Beilei; Liu, Huanxiang; Li, Shuyan; Yao, Xiaojun; Liu, Mancang; Gramatica, Paola


    Quantitative structure-activity relationship (QSAR) of a series of structural diverse malonyl-CoA decarboxylase (MCD) inhibitors have been investigated by using the predictive single model as well as the consensus analysis based on a new strategy proposed by us. Self-organizing map (SOM) neural network was employed to divide the whole data set into representative training set and test set. Then a multiple linear regressions (MLR) model population was built based on the theoretical molecular descriptors selected by Genetic Algorithm using the training set. In order to analyze the diversity of these models, multidimensional scaling (MDS) was employed to explore the model space based on the Hamming distance matrix calculated from each two models. In this space, Q(2) (cross-validated R(2)) guided model selection (QGMS) strategy was performed to select submodels. Then consensus modeling was built by two strategies, average consensus model (ACM) and weighted consensus model (WCM), where each submodel had a different weight according to the contribution of model expressed by MLR regression coefficients. The obtained results prove that QGMS is a reliable and practical method to guide the submodel selection in consensus modeling building and our weighted consensus model (WCM) strategy is superior to the simple ACM. 2008 Wiley Periodicals, Inc.

  11. Apraxia in anti-glutamic acid decarboxylase-associated stiff person syndrome: link to corticobasal degeneration?

    Bowen, Lauren N; Subramony, S H; Heilman, Kenneth M


    Corticobasal syndrome (CBS) is associated with asymmetrical rigidity as well as asymmetrical limb-kinetic and ideomotor apraxia. Stiff person syndrome (SPS) is characterized by muscle stiffness and gait difficulties. Whereas patients with CBS have several forms of pathology, many patients with SPS have glutamic acid decarboxylase antibodies (GAD-ab), but these 2 disorders have not been reported to coexist. We report 2 patients with GAD-ab-positive SPS who also had signs suggestive of CBS, including asymmetrical limb rigidity associated with both asymmetrical limb-kinetic and ideomotor apraxia. Future studies should evaluate patients with CBS for GAD-ab and people with SPS for signs of CBS.

  12. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    Christiansen, L; Ged, C; Hombrados, I


    The two porphyrias, familial porphyria cutanea tarda (fPCT) and hepatoerythropoietic porphyria (HEP), are associated with mutations in the gene encoding the enzyme uroporphyrinogen decarboxylase (UROD). Several mutations, most of which are private, have been identified in HEP and fPCT patients......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

  13. Glutamic acid decarboxylase antibody-positive paraneoplastic stiff limb syndrome associated with carcinoma of the breast


    Stiff limb syndrome (SLS) is a rare "focal" variant of stiff person syndrome which presents with rigidity and painful spasms of a distal limb, and abnormal fixed foot or hand postures. Anti-glutamic acid decarboxylase antibodies (GAD-Ab) are variably present in most cases. Most reported cases of SLS are unassociated with cancer. We describe a patient with SLS as a paraneoplastic manifestation of breast carcinoma, in whom GAD-Ab was present. The patient responded very well to oral diazepam, ba...

  14. Glutamic acid decarboxylase antibody-positive paraneoplastic stiff limb syndrome associated with carcinoma of the breast.

    Agarwal, Pankaj A; Ichaporia, Nasli R


    Stiff limb syndrome (SLS) is a rare "focal" variant of stiff person syndrome which presents with rigidity and painful spasms of a distal limb, and abnormal fixed foot or hand postures. Anti-glutamic acid decarboxylase antibodies (GAD-Ab) are variably present in most cases. Most reported cases of SLS are unassociated with cancer. We describe a patient with SLS as a paraneoplastic manifestation of breast carcinoma, in whom GAD-Ab was present. The patient responded very well to oral diazepam, baclofen and steroids.This is the third reported case of SLS as a paraneoplastic accompaniment to cancer.

  15. Fluorimetric assay for ornithine decarboxylase by high-performance liquid chromatography.

    Haraguchi, K; Kai, M; Kohashi, K; Ohkura, Y


    A highly sensitive method for the assay of ornithine decarboxylase in sample solutions prepared from rat tissue homogenate is described which employs high-performance liquid chromatography with fluorescence detection. Putrescine formed from ornithine under the optimal conditions for the enzyme reaction is treated by Cellex P column chromatography for clean-up and converted into the fluorescamine derivative in the presence of cupric ion which inhibits the reaction of interfering amines with fluorescamine. The derivative is separated by reversed-phase chromatography on LiChrosorb RP-18 with linear gradient elution. The lower limit of detection for putrescine formed enzymatically is 5 pmol.

  16. Structural basis for the catalytic mechanism of a proficient enzyme: Orotidine 5'-Monophosphate Decarboxylase

    Harris, Pernille Hanne; Poulsen, Jens-Christian Navarro; Jensen, Kaj Frank


    Orotidine 5‘-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5‘-monophosphate, the last step in the de novo synthesis of uridine 5‘-monophosphate. ODCase is a very proficient enzyme [Radzicka, A., and Wolfenden, R. (1995) Science 267, 90-93], enhancing the reaction...... rate by a factor of 1017. This proficiency has been enigmatic, since it is achieved without metal ions or cofactors. Here we present a 2.5 Å resolution structure of ODCase complexed with the inhibitor 1-(5‘-phospho-ß-d-ribofuranosyl)barbituric acid. It shows a closely packed dimer composed of two a...

  17. Glutamic acid decarboxylase antibody-positive paraneoplastic stiff limb syndrome associated with carcinoma of the breast

    Agarwal Pankaj


    Full Text Available Stiff limb syndrome (SLS is a rare "focal" variant of stiff person syndrome which presents with rigidity and painful spasms of a distal limb, and abnormal fixed foot or hand postures. Anti-glutamic acid decarboxylase antibodies (GAD-Ab are variably present in most cases. Most reported cases of SLS are unassociated with cancer. We describe a patient with SLS as a paraneoplastic manifestation of breast carcinoma, in whom GAD-Ab was present. The patient responded very well to oral diazepam, baclofen and steroids.This is the third reported case of SLS as a paraneoplastic accompaniment to cancer.

  18. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli.

    Le Vo, Tam Dinh; Kim, Tae Wan; Hong, Soon Ho


    Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.

  19. Immunohistochemical Expression of Ornithine Decarboxylase, Diamine Oxidase, Putrescine, and Spermine in Normal Canine Enterocolic Mucosa, in Chronic Colitis, and in Colorectal Cancer

    Giacomo Rossi


    Full Text Available We compared the immunohistochemical expression of putrescine (PUT, spermine (SPM, ornithine decarboxylase (ODC, and diamine oxidase (DAO in bioptic samples of canine colonic mucosa with chronic inflammation (i.e., granulomatous colitis and lymphoplasmacytic colitis or neoplasia. Single and total polyamines levels were significantly higher in neoplastic tissue than in normal samples. Samples with different degrees of inflammation showed a general decrease expression of ODC if compared to controls; SPM was practically not expressed in control samples and very low in samples with chronic-granulomatous inflammation. In carcinomatous samples, the ODC activity was higher with respect to controls and samples with inflammation. This is the first description of polyamines expression in dog colonic mucosa in normal and in different pathological conditions, suggesting that the balance between polyamine degradation and biosynthesis is evidently disengaged during neoplasia.

  20. Immunohistochemical expression of ornithine decarboxylase, diamine oxidase, putrescine, and spermine in normal canine enterocolic mucosa, in chronic colitis, and in colorectal cancer.

    Rossi, Giacomo; Cerquetella, Matteo; Pengo, Graziano; Mari, Subeide; Balint, Emilia; Bassotti, Gabrio; Manolescu, Nicolae


    We compared the immunohistochemical expression of putrescine (PUT), spermine (SPM), ornithine decarboxylase (ODC), and diamine oxidase (DAO) in bioptic samples of canine colonic mucosa with chronic inflammation (i.e., granulomatous colitis and lymphoplasmacytic colitis) or neoplasia. Single and total polyamines levels were significantly higher in neoplastic tissue than in normal samples. Samples with different degrees of inflammation showed a general decrease expression of ODC if compared to controls; SPM was practically not expressed in control samples and very low in samples with chronic-granulomatous inflammation. In carcinomatous samples, the ODC activity was higher with respect to controls and samples with inflammation. This is the first description of polyamines expression in dog colonic mucosa in normal and in different pathological conditions, suggesting that the balance between polyamine degradation and biosynthesis is evidently disengaged during neoplasia.

  1. Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase.

    Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang


    γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca(2+)-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca(2+) increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.

  2. Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

    Tajabadi, Naser; Baradaran, Ali; Ebrahimpour, Afshin; Rahim, Raha A; Bakar, Fatimah A; Manap, Mohd Yazid A; Mohammed, Abdulkarim S; Saari, Nazamid


    Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.

  3. The role of hypusine depletion in cytostasis induced by S-adenosyl-L-methionine decarboxylase inhibition: new evidence provided by 1-methylspermidine and 1,12-dimethylspermine.

    Byers, T L; Lakanen, J R; Coward, J K; Pegg, A E


    The abilities of the natural polyamines, spermidine and spermine, and of the synthetic analogues, 1-methylspermidine and 1,12-dimethylspermine, to reverse the effects of the S-adenosyl-L-methionine decarboxylase inhibitor 5'-([(Z)-4-aminobut-2-enyl]methylamino)-5'-deoxyadenosine (AbeAdo) on L1210-cell growth were studied. L1210 cells were exposed to AbeAdo for 12 days to induce cytostasis and then exposed to spermidine, spermine, 1-methylspermidine or 1,12-dimethylspermine in the continued presence of AbeAdo. AbeAdo-induced cytostasis was overcome by the natural polyamines, spermidine and spermine. The cytostasis was also reversed by 1-methylspermidine. 1,12-Dimethylspermine had no effect on the AbeAdo-induced cytostasis of chronically treated cells, although it was active in permitting growth of cells treated with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The initial 12-day exposure to AbeAdo elevated intracellular putrescine levels, depleted intracellular spermidine and spermine, and resulted in the accumulation of unmodified eukaryotic translation initiation factor 5A (eIF-5A). Exposure of these cells to exogenous spermidine, which is the natural substrate for deoxyhypusine synthase, resulted in a decrease in the unmodified eIF-5A content. 1-Methylspermidine, which was found to be a substrate of deoxyhypusine synthase in vitro, also decreased the levels of unmodified eIF-5A in the AbeAdo-treated cells. Although spermine is not a substrate of deoxyhypusine synthase, spermine was converted into spermidine in the L1210 cells, and spermine addition to AbeAdo-treated cells resulted in the appearance of both intracellular spermine and spermidine and in the decrease in unmodified eIF-5A. Exogenous 1,12-dimethylspermine, which was not metabolized to spermine or to 1-methylspermidine and was not a substrate of deoxyhypusine synthase in vitro, did not decrease levels of unmodified eIF-5A. The finding that AbeAdo-induced cytostasis was only

  4. Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis.

    Goupil-Feuillerat, N; Cocaign-Bousquet, M; Godon, J J; Ehrlich, S D; Renault, P


    The alpha-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 microM leucine, the alpha-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the alpha-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.

  5. Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

    Lee, Won-Heong; Seo, Seung-Oh; Bae, Yi-Hyun; Nan, Hong; Jin, Yong-Su; Seo, Jin-Ho


    Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

  6. Hypothalamic L-Histidine Decarboxylase Is Up-Regulated During Chronic REM Sleep Deprivation of Rats

    Hoffman, Gloria E.; Koban, Michael


    A competition of neurobehavioral drives of sleep and wakefulness occurs during sleep deprivation. When enforced chronically, subjects must remain awake. This study examines histaminergic neurons of the tuberomammillary nucleus of the posterior hypothalamus in response to enforced wakefulness in rats. We tested the hypothesis that the rate-limiting enzyme for histamine biosynthesis, L-histidine decarboxylase (HDC), would be up-regulated during chronic rapid eye movement sleep deprivation (REM-SD) because histamine plays a major role in maintaining wakefulness. Archived brain tissues of male Sprague Dawley rats from a previous study were used. Rats had been subjected to REM-SD by the flowerpot paradigm for 5, 10, or 15 days. For immunocytochemistry, rats were transcardially perfused with acrolein-paraformaldehyde for immunodetection of L-HDC; separate controls used carbodiimide-paraformaldehyde for immunodetection of histamine. Immunolocalization of histamine within the tuberomammillary nucleus was validated using carbodiimide. Because HDC antiserum has cross-reactivity with other decarboxylases at high antibody concentrations, titrations localized L-HDC to only tuberomammillary nucleus at a dilution of ≥ 1:300,000. REM-SD increased immunoreactive HDC by day 5 and it remained elevated in both dorsal and ventral aspects of the tuberomammillary complex. Our results suggest that up-regulation of L-HDC within the tuberomammillary complex during chronic REM-SD may be responsible for maintaining wakefulness. PMID:27997552

  7. Production of pyruvate from mannitol by mannitol-assimilating pyruvate decarboxylase-negative Saccharomyces cerevisiae.

    Yoshida, Shiori; Tanaka, Hideki; Hirayama, Makoto; Murata, Kousaku; Kawai, Shigeyuki


    Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.

  8. Evolutionary trails of plant group II Pyridoxal phosphate-dependent decarboxylase genes

    Rahul Kumar


    Full Text Available Type II pyridoxal phosphate-dependent decarboxylase (PLP_deC enzymes play important metabolic roles during nitrogen metabolism. Recent evolutionary profiling of these genes revealed a sharp expansion of histidine decarboxylase (HDC genes in the members of Solanaceae family. In spite of the high sequence homology shared by PLP_deC orthologs, these enzymes display remarkable differences in their substrate specificities. Currently, limited information is available on the gene repertoires and substrate specificities of PLP_deCs which renders their precise annotation challenging and offers technical challenges in the immediate identification and biochemical characterization of their full gene complements in plants. Herein, we explored their evolutionary trails in a comprehensive manner by taking advantage of high-throughput data accessibility and computational approaches. We discussed the premise that has enabled an improved reconstruction of their evolutionary lineage and evaluated the factors offering constraints in their rapid functional characterization, till date. We envisage that the synthesized information herein would act as a catalyst for the rapid exploration of their biochemical specificity and physiological roles in more plant species.

  9. Crystallization and preliminary X-ray diffraction experiments of arylmalonate decarboxylase from Alcaligenes bronchisepticus

    Nakasako, Masayoshi, E-mail: [Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan); The RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan); Obata, Rika; Okubo, Ryosuke; Nakayama, Shyuichi [Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan); Miyamoto, Kenji; Ohta, Hiromichi [Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan); Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Kanagawa 223-8522 (Japan)


    Crystals of arylmalonate decarboxylase from A. bronchisepticus were obtained which diffracted X-rays to a resolution of at least 3.0 Å. Arylmalonate decarboxylase catalyses the enantioselective decarboxylation of α-aryl-α-methylmalonates to produce optically pure α-arylpropionates. The enzyme was crystallized with ammonium sulfate under alkaline pH conditions with the aim of understanding the mechanism of the enantioselective reaction. X-ray diffraction data collected to a resolution of 3.0 Å at cryogenic temperature showed that the crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 83.13, b = 99.62, c = 139.64 Å. This suggested that the asymmetric unit would contain between four and six molecules. Small-angle X-ray scattering revealed that the enzyme exists as a monomer in solution. Thus, the assembly of molecules in the asymmetric unit was likely to have been induced during the crystallization process.

  10. Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

    Abell, L.M.; O' Leary, M.H.


    The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k/sup 14//k/sup 15/ = 0.9770 +/- 0.0021, a carbon isotope effect k/sup 12//k/sup 13/ = 1.0308 +/- 0.0006, and a carbon isotope effect for L-(..cap alpha..-/sup 2/H)histidine of 1.0333 +/- 0.0001 at pH 6.3, 37/sup 0/C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli, the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.

  11. Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells

    Zhu, Qingsong; Jin, Lihua; Casero, Robert A.


    Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N1-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer. PMID:22976807

  12. MDMA decreases glutamic acid decarboxylase (GAD) 67-immunoreactive neurons in the hippocampus and increases seizure susceptibility: Role for glutamate.

    Huff, Courtney L; Morano, Rachel L; Herman, James P; Yamamoto, Bryan K; Gudelsky, Gary A


    3,4-Methylenedioxy-methamphetamine (MDMA) is a unique psychostimulant that continues to be a popular drug of abuse. It has been well documented that MDMA reduces markers of 5-HT axon terminals in rodents, as well as humans. A loss of parvalbumin-immunoreactive (IR) interneurons in the hippocampus following MDMA treatment has only been documented recently. In the present study, we tested the hypothesis that MDMA reduces glutamic acid decarboxylase (GAD) 67-IR, another biochemical marker of GABA neurons, in the hippocampus and that this reduction in GAD67-IR neurons and an accompanying increase in seizure susceptibility involve glutamate receptor activation. Repeated exposure to MDMA (3×10mg/kg, ip) resulted in a reduction of 37-58% of GAD67-IR cells in the dentate gyrus (DG), CA1, and CA3 regions, as well as an increased susceptibility to kainic acid-induced seizures, both of which persisted for at least 30days following MDMA treatment. Administration of the NMDA antagonist MK-801 or the glutamate transporter type 1 (GLT-1) inducer ceftriaxone prevented both the MDMA-induced loss of GAD67-IR neurons and the increased vulnerability to kainic acid-induced seizures. The MDMA-induced increase in the extracellular concentration of glutamate in the hippocampus was significantly diminished in rats treated with ceftriaxone, thereby implicating a glutamatergic mechanism in the neuroprotective effects of ceftriaxone. In summary, the present findings support a role for increased extracellular glutamate and NMDA receptor activation in the MDMA-induced loss of hippocampal GAD67-IR neurons and the subsequent increased susceptibility to evoked seizures.

  13. 21 CFR 173.115 - Alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation derived from a recombinant Bacillus...


    ... preparation derived from a recombinant Bacillus subtilis. 173.115 Section 173.115 Food and Drugs FOOD AND DRUG... Bacillus subtilis. The food additive alpha-acetolactate decarboxylase (α-ALDC) enzyme preparation, may be... derived from a modified Bacillus subtilis strain that contains the gene coding for α-ALDC from...

  14. An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

    Oud, B.; Flores, C.L.; Gancedo, C.; Zhang, X.; Trueheart, J.; Daran, J.M.; Pronk, J.T.; Van Maris, A.J.A.


    Background Pyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards


    Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and B-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E.coli, three major...

  16. A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Taurine Distribution in Various Tissues With and Without Taurine Supplementation.

    Park, Eunkyue; Park, Seung Yong; Cho, In Soo; Kim, Bo Sook; Schuller-Levis, Georgia


    Taurine, a sulfur containing amino acid, has various physiological functions including development of the eye and brain, immune function, reproduction, osmo-regulatory function as well as anti-oxidant and anti-inflammatory activities. In order to understand the physiological role, we developed taurine deficient mice deleting a rate-liming enzyme, cysteine sulfinic acid decarboxylase (CSAD) for biosynthesis of taurine. Taurine was measured in various tissues including the liver, brain, lung, spleen, thymus, pancreas, heart, muscle and kidney as well as plasma from CSAD knock-out mice (CSAD KO) with and without treatment of taurine in the drinking water at the age of 2 months (2 M). Taurine was determined using HPLC as a phenylisothiocyanate derivative of taurine at 254 nm. Taurine concentrations in the liver and kidney from homozygotes of CSAD KO (HO), in which CSAD level is high, were 90% and 70% lower than WT, respectively. Taurine concentrations in the brain, spleen and lung, where CSAD level is low, were 21%, 20% and 28% lower than WT, respectively. At 2 M, 1% taurine treatment of HO restored taurine concentrations in all tissues compared to that of WT. To select an appropriate taurine treatment, HO were treated with various concentrations (0.05, 0.2, 1%) of taurine for 4 months (4 M). Restoration of taurine in all tissues except the liver, kidney and lung requires 0.05% taurine to be restored to that of WT. The liver and kidney restore taurine back to WT with 0.2% taurine. To examine which enzymes influence taurine concentrations in various tissues from WT and HO at 2 M, expression of five taurine-related enzymes, two antioxidant enzymes as well as lactoferrin (Lft) and prolactin receptor (Prlr) was determined using RT(2) qPCR. The expression of taurine transporter in the liver, brain, muscle and kidney from HO was increased except in the lung. Our data showed expression of glutamate decarboxylase-like 1(Gadl-1) was increased in the brain and muscle in HO

  17. Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica.


    Full Text Available BACKGROUND: Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC. ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC is reported to exist in a pentameric state. METHODOLOGY AND RESULTS: In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer. CONCLUSION: To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer

  18. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)


    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  19. Monoclonal antibodies to 65kDa glutamate decarboxylase induce epitope specific effects on motor and cognitive functions in rats.

    Hampe, Christiane S; Petrosini, Laura; De Bartolo, Paola; Caporali, Paola; Cutuli, Debora; Laricchiuta, Daniela; Foti, Francesca; Radtke, Jared R; Vidova, Veronika; Honnorat, Jérôme; Manto, Mario


    Stiff Person Syndrome (SPS) is a rare autoimmune movement disorder characterized by the presence of autoantibodies specific to the smaller isoform of glutamate decarboxylase (GAD65). A pathological role of these antibodies has been suggested by their capacity to inhibit GAD65 enzyme activity and by the observation that rats receiving cerebellar injections of GAD65Ab showed cerebellar motor hyperexcitability. To assess the effect of epitope-specific GAD65Ab on cognitive and motor functions, we conducted behavioral experiments in rats that received cerebellar injections with two distinct monoclonal GAD65Ab (b96.11 and b78). Rats received three injections of GAD65Ab b96.11 (5 or 7 μg), GAD65Ab b78 (5 or 7 μg), or saline at the level of three cerebellar nuclei. Animals were submitted to neurological evaluation and Morris Water Maze (MWM) test. Cellular internalization of GAD65Ab was analyzed by Flow Cytometry, Fluorescence and Bright Field microscopy. Monoclonal GAD65Ab induced dose-dependent and epitope-specific effects on motor and cognitive functions. Injections of the higher dose altered motor and spatial procedural behaviors, while the lower dose induced only modest cerebellar motor symptoms and did not affect MWM performances. While b96.11 provoked immediate severe effects, which rapidly decreased, b78 induced moderate but prolonged effects. Both GAD65Ab were taken up by live cells in a dose-dependent manner. Our findings support the hypothesis that epitope-specific GAD65Ab induce cerebellar dysfunction impairing motor and procedural abilities. This is the first demonstration of a critical role of cerebellar nuclei GAD65 enzyme in procedural spatial functions.

  20. Lower Expression of Glutamic Acid Decarboxylase 67 in the Prefrontal Cortex in Schizophrenia: Contribution of Altered Regulation by Zif268

    Kimoto, Sohei; Bazmi, H. Holly; Lewis, David A.


    Objective Cognitive deficits of schizophrenia may be due at least in part to lower expression of the 67-kDa isoform of glutamic acid decarboxylase (GAD67), a key enzyme for GABA synthesis, in the dorsolateral prefrontal cortex of individuals with schizophrenia. However, little is known about the molecular regulation of lower cortical GAD67 levels in schizophrenia. The GAD67 promoter region contains a conserved Zif268 binding site, and Zif268 activation is accompanied by increased GAD67 expression. Thus, altered expression of the immediate early gene Zif268 may contribute to lower levels of GAD67 mRNA in the dorsolateral prefrontal cortex in schizophrenia. Method The authors used polymerase chain reaction to quantify GAD67 and Zif268 mRNA levels in dorsolateral pre-frontal cortex area 9 from 62 matched pairs of schizophrenia and healthy comparison subjects, and in situ hybridization to assess Zif268 expression at laminar and cellular levels of resolution. The effects of potentially confounding variables were assessed in human subjects, and the effects of antipsychotic treatments were tested in antipsychotic-exposed monkeys. The specificity of the Zif268 findings was assessed by quantifying mRNA levels for other immediate early genes. Results GAD67 and Zif268 mRNA levels were significantly lower and were positively correlated in the schizophrenia subjects. Both Zif268 mRNA-positive neuron density and Zif268 mRNA levels per neuron were significantly lower in the schizophrenia subjects. These findings were robust to the effects of the confounding variables examined and differed from other immediate early genes. Conclusions Deficient Zif268 mRNA expression may contribute to lower cortical GAD67 levels in schizophrenia, suggesting a potential mechanistic basis for altered cortical GABA synthesis and impaired cognition in schizophrenia. PMID:24874453



    The preparation and characterization of an immobilized L-glutamic decarboxylase (GDC)were studied This work is to develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO2 electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The properties of the immobilized enzyme on CM-CADB were studied by investigating the initial rate of the enzyme reaction, the efffect of various parameters on the immobilized GDC activity and its stability. An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO2 electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamate acid. The limit of detection is 1.O ×1O-5 M. The linearity response is in the range of 5 × 1O -2-5 × 1O -5 M. The equation of linear regression of the calibration curve is y= 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamate acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

  2. [Glutamate decarboxylase (GAD)--an autoantigen in insulin-dependent diabetes mellitus (IDDM)].

    Tursky, T; Bandzuchova, E


    The number of antibodies to pancreatic beta-cell antigens in IDDM increased in the last years, involving antibodies to glutamic acid decarboxylase (GADab). A short review is given about the diagnostic and prognostic value of GADab determination in IDDM. The GAD plays an important, possibly a key role in the initial immunological events leading to the destruction of beta cells. The question is open whether the immunological reaction against GAD is a primary one, or if it is a result of mimicry of a part of an infectious protein antigen (Coxackie virus). The immunological reaction to GAD is associated with both humoral and cellular responses. The cellular response seems to be more important than the humoral one. The cellular response may be mediated through the HLA complex class I cells (cytotoxic lymphocytes) and the HLA complex class II cells (helper lymphocytes). There are arguments for both possibilities. The principles of GADab determination are shortly described. (Ref. 34.)

  3. The importance of SERINE DECARBOXYLASE1 (SDC1) and ethanolamine biosynthesis during embryogenesis of Arabidopsis thaliana.

    Yunus, Ian Sofian; Liu, Yu-Chi; Nakamura, Yuki


    In plants, ethanolamine is considered a precursor for the synthesis of choline, which is an essential dietary nutrient for animals. An enzyme serine decarboxylase (SDC) has been identified and characterized in Arabidopsis, which directly converts serine to ethanolamine, a precursor to phosphorylethanolamine and its subsequent metabolites in plants. However, the importance of SDC and ethanolamine production in plant growth and development remains unclear. Here, we show that SDC is required for ethanolamine biosynthesis in vivo and essential in plant embryogenesis in Arabidopsis. The knockout of SDC1 caused an embryonic lethal defect due to the developmental arrest of the embryos at the heart stage. During embryo development, the expression was observed at the later stages, at which developmental defect occurred in the knockout mutant. Overexpression of SDC1 in planta increased levels of ethanolamine, phosphatidylethanolamine, and phosphatidylcholine both in leaves and siliques. These results suggest that SDC1 plays an essential role in ethanolamine biosynthesis during the embryogenesis in Arabidopsis.

  4. Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast.

    Dyda, F; Furey, W; Swaminathan, S; Sax, M; Farrenkopf, B; Jordan, F


    Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.

  5. Immunotherapy-responsive limbic encephalitis with antibodies to glutamic acid decarboxylase.

    Markakis, Ioannis; Alexopoulos, Harry; Poulopoulou, Cornelia; Akrivou, Sofia; Papathanasiou, Athanasios; Katsiva, Vassiliki; Lyrakos, Georgios; Gekas, Georgios; Dalakas, Marinos C


    Glutamic acid decarboxylase (GAD) has been recently identified as a target of humoral autoimmunity in a small subgroup of patients with non-paraneoplastic limbic encephalitis (NPLE). We present a patient with NPLE and positive anti-GAD antibodies who showed significant improvement after long-term immunotherapy. A 48-year old female was admitted with a two-year history of anterograde amnesia and seizures. Brain MRI revealed bilateral lesions of medial temporal lobes. Screening for anti-neuronal antibodies showed high anti-GAD titers in both serum and cerebrospinal fluid (CSF) with strong evidence of intrathecal production. The patient received treatment with prednisolone and long-term plasma exchange. During a 12-month follow-up, she exhibited complete seizure remission and an improvement in memory and visuo-spatial skills. Anti-GAD antibodies may serve as a useful marker to identify a subset of NPLE patients that respond to immunoregulatory treatment.

  6. Histidine decarboxylase deficiency causes Tourette syndrome: parallel findings in humans and mice

    Baldan, Lissandra Castellan; Rapanelli, Maximiliano; Crowley, Michael; Anderson, George M.; Loring, Erin; Gorczyca, Roxanne; Billingslea, Eileen; Wasylink, Suzanne; Panza, Kaitlyn E.; Ercan-Sencicek, A. Gulhan; Krusong, Kuakarun; Leventhal, Bennett L.; Ohtsu, Hiroshi; Bloch, Michael H.; Hughes, Zoë A.; Krystal, John H.; Mayes, Linda; de Araujo, Ivan; Ding, Yu-Shin; State, Matthew W.; Pittenger, Christopher


    Tourette syndrome (TS) is characterized by tics, sensorimotor gating deficiencies, and abnormalities of cortico-basal ganglia circuits. A mutation in histidine decarboxylase (Hdc), the key enzyme for the biosynthesis of histamine (HA), has been implicated as a rare genetic cause. Hdc knockout mice exhibited potentiated tic-like stereotypies, recapitulating core phenomenology of TS; these were mitigated by the dopamine D2 antagonist haloperidol, a proven pharmacotherapy, and by HA infusion into the brain. Prepulse inhibition was impaired in both mice and humans carrying Hdc mutations. HA infusion reduced striatal dopamine (DA) levels; in Hdc knockout mice, striatal DA was increased and the DA-regulated immediate early gene Fos was upregulated. Dopamine D2/D3 receptor binding was altered both in mice and in humans carrying the Hdc mutation. These data confirm HDC deficiency as a rare cause of TS and identify histamine-dopamine interactions in the basal ganglia as an important locus of pathology. PMID:24411733

  7. In vitro Characterization of Phenylacetate Decarboxylase, a Novel Enzyme Catalyzing Toluene Biosynthesis in an Anaerobic Microbial Community

    Zargar, K.; Saville, R.; Phelan, R. M.;


    an anaerobic, sewage-derived enrichment culture that quantitatively produces toluene from phenylacetate; complementary metagenomic and metaproteomic analyses are also presented. Among the noteworthy findings is that this enzyme is not the well-characterized clostridial p-hydroxyphenylacetate decarboxylase (Csd...... similarly inhibited by an amide analog of p-hydroxyphenylacetate. Based upon these and other data, we hypothesize that the toluene synthase reaction involves a glycyl radical decarboxylase. This first-time study of the phenylacetate decarboxylase reaction constitutes an important step in understanding...

  8. Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

    Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.; Tomchick, Diana R.; Goldsmith, Elizabeth J.; Phillips, Margaret A. (Sungkyunkwan); (UTSMC)


    Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.

  9. Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast

    A.J. van Maris; J.M. Geertman; A. Vermeulen; M.K. Groothuizen; A.A. Winkler; M.D. Piper; J.P. van Dijken; J.T. Pronk


    textabstractThe absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects:

  10. Danish children born with glutamic acid decarboxylase-65 and islet antigen-2 autoantibodies at birth had an increased risk to develop type 1 diabetes

    Eising, Stefanie; Nilsson, Anita; Carstensen, Bendix


    A large, population-based case-control cohort was used to test the hypothesis that glutamic acid decarboxylase-65 (GAD65) and islet antigen-2 autoantibodies (IA-2A) at birth predict type 1 diabetes....

  11. Mechanism of the Orotidine 5’-Monophosphate Decarboxylase-Catalyzed Reaction: Importance of Residues in the Orotate Binding Site†

    Iiams, Vanessa; Desai, Bijoy J.; Fedorov, Alexander A.; Fedorov, Elena V.; Almo, Steven C.; Gerlt, John A.


    The reaction catalyzed by orotidine 5’-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for the rate enhancement [kcat/knon = 7.1 × 1016] and catalytic proficiency [(kcat/KM)/knon = 4.8 × 1022 M−1]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that “substrate destabilization” by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ~5 kcal/mol (contributing ~2.7 × 103 to the rate enhancement) [K. K. Chan, B. M. Wood, A. A. Fedorov, E. V. Fedorov, H. J. Imker, T. L. Amyes, J. P. Richard, S. C. Almo, and J. A. Gerlt (2009) Biochemistry 48, 5518–31]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of kcat by as much as 400-fold but have minimal effect on the value of kex for exchange of H6 of the FUMP product analog with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions for Ser 127 that is proximal to O4 of the orotate ring decrease the value of kcat/KM, with the S127P substitution that eliminates hydrogen-bonding interactions with O4 producing a 2.5 × 106-fold reduction in the value of kcat/KM; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 to produce an anionic carbene intermediate and thereby provide a structural strategy for stabilization of the intermediate. These observations provide additional information on the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the

  12. Immunofluorescently labeling glutamic acid decarboxylase 65 coupled with confocal imaging for identifying GABAergic somata in the rat dentate gyrus-A comparison with labeling glutamic acid decarboxylase 67.

    Wang, Xiaochen; Gao, Fei; Zhu, Jianchun; Guo, Enpu; Song, Xueying; Wang, Shuanglian; Zhan, Ren-Zhi


    As γ-aminobutyric acid (GABA) is synthesized by two isoforms of glutamic acid decarboxylase (GAD), namely, GAD65 and GAD67, immunohistochemically targeting either isoform of GAD is theoretically useful for identifying GABAergic cell bodies. In practice, targeting GAD67 remains to be a popular choice. However, identifying GABAergic cell bodies with GAD67 immunoreactivity in the hippocampal dentate gyrus, especially in the hilus, is not without pitfalls. In the present study, we compared the characteristics of GAD65 immunoreactivity to GAD67 immunoreactivity in the rat dentate gyrus and examined perikaryal expression of GAD65 in four neurochemically prevalent subgroups of interneurons in the hilus. Experiments were done in normal adult Sprague-Dawley rats and GAD67-GFP knock-in mice. Horizontal hippocampal slices cut from the ventral portion of hippocampi were immunofluorescently stained and scanned using a confocal microscope. Immunoreactivity for both GAD67 and GAD65 was visible throughout the dentate gyrus. Perikaryal GAD67 immunoreactivity was denser but variable in terms of distribution pattern and intensity among cells whereas perikaryal GAD65 immunoreactivity displayed similar distribution pattern and staining intensity. Among different layers of the dentate gyrus, GAD67 immunoreactivity was densest in the hilus despite GAD65 immunoreactivity being more intense in the granule cell layer. Co-localization experiments showed that GAD65, but not GAD67, was expressed in all hilar calretinin (CR)-, neuronal nitric oxide synthase (nNOS)-, parvalbumin (PV)- or somatostatin (SOM)-positive somata. Labeling CR, nNOS, PV, and SOM in sections obtained from GAD67-GFP knock-in mice revealed that a large portion of SOM-positive cells had weak GFP expression. In addition, double labeling of GAD65/GABA and GAD67/GABA showed that nearly all of GABA-immunoreactive cells had perikaryal GAD65 expression whereas more than one-tenth of GABA-immunoreactive cells lacked perikaryal GAD

  13. Uroporphyrinogen Decarboxylase as a Potential Target for Specific Components of Traditional Chinese Medicine: A Virtual Screening and Molecular Dynamics Study

    Yung-An Tsou; Kuan-Chung Chen; Hung-Che Lin; Su-Sen Chang; Calvin Yu-Chian Chen


    Uroporphyrinogen decarboxylase (UROD) has been suggested as a protectant against radiation for head and neck cancer (HNC). In this study, we employed traditional Chinese medicine (TCM) compounds from TCM Database@Taiwan ( to screen for drug-like candidates with potential UROD inhibition characteristics using virtual screening techniques. Isopraeroside IV, scopolin, and nodakenin exhibited the highest Dock Scores, and were predicted to have good Absorption, Distribution,...

  14. Aromatic Amino Acid Decarboxylase Deficiency Not Responding to Pyridoxine and Bromocriptine Therapy: Case Report and Review of Response to Treatment


    Aromatic L-amino acid decarboxylase (AADC) deficiency (MIM #608643) is an autosomal recessive inborn error of monoamines. It is caused by a mutation in the DDC gene that leads to a deficiency in the AADC enzyme. The clinical features of this condition include a combination of dopamine, noradrenaline, and serotonin deficiencies, and a patient may present with hypotonia, oculogyric crises, sweating, hypersalivation, autonomic dysfunction, and progressive encephalopathy with severe developmental...

  15. Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

    Dalton, Heidi L; Blomstedt, Cecilia K; Neale, Alan D; Gleadow, Ros; DeBoer, Kathleen D; Hamill, John D


    Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

  16. Assessment of the effects of glutamic acid decarboxylase antibodies and trace elements on cognitive performance in older adults

    Alghadir AH


    Full Text Available Ahmad H Alghadir,1 Sami A Gabr,1,2 Einas Al-Eisa11Department of Rehabilitation Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia; 2Department of Anatomy, Faculty of Medicine, Mansoura University, Mansoura, EgyptBackground: Homeostatic imbalance of trace elements such as iron (Fe, copper (Cu, and zinc (Zn demonstrated adverse effects on brain function among older adults.Objective: The present study aimed to investigate the effects of trace elements and the presence of anti-glutamic acid decarboxylase antibodies (GADAs in human cognitive abilities among healthy older adults.Methods: A total of 100 healthy subjects (65 males, 35 females; age range; 64–96 years were recruited for this study. Based on Loewenstein Occupational Therapy Cognitive Assessment (LOTCA score, the participants were classified according to cognitive performance into normal (n=45, moderate (n=30, and severe (n=25. Cognitive functioning, leisure-time physical activity (LTPA, serum trace elements – Fe, Cu, Zn, Zn/Cu, and GADAs were assessed using LOTCA battery, pre-validated physical activity (PA questionnaire, atomic absorption, and immunoassay techniques, respectively.Results: Approximately 45% of the study population (n=45 had normal distribution of cognitive function and 55% of the study population (n=55 had abnormal cognitive function; they were classified into moderate (score 62–92 and severe (score 31–62. There was a significant reduction in the level of Zn and Zn/Cu ratio along with an increase in the level of Fe, Cu, and anti-GADAs in subjects of severe (P=0.01 and moderate (P=0.01 cognitive performance. LOTCA-cognitive scores correlated positively with sex, HbA1c, Fe, Cu, Zn, and Zn/Cu ratio, and negatively with age, PA, body mass index, and anti-GADAs. Significant inter-correlation was reported between serum trace element concentrations and anti-GADAs which suggest producing a cognitive decline via oxidative and neural

  17. Role of the Proteasome in Excitotoxicity-Induced Cleavage of Glutamic Acid Decarboxylase in Cultured Hippocampal Neurons

    Armelão, Mário; Herrmann, Dennis; Pimentel, Diogo O.; Leal, Graciano; Caldeira, Margarida V.; Bahr, Ben A.; Bengtson, Mário; Almeida, Ramiro D.; Duarte, Carlos B.


    Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms—GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity

  18. Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocampal neurons.

    Márcio S Baptista

    Full Text Available Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during

  19. Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase.

    Thöny, Beat; Calvo, Ana C; Scherer, Tanja; Svebak, Randi M; Haavik, Jan; Blau, Nenad; Martinez, Aurora


    Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l-DOPA responsive and non-responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH(4) supplementation to mice increased TH activity and protein levels in brain extracts, while the Th-mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH(4) and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH(4) crosses the blood-brain barrier and therapeutic regimes based on BH(4) supplementation should thus consider the effect on TH. Furthermore, BH(4) supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

  20. Identification of polymorphisms and balancing selection in the male infertility candidate gene, ornithine decarboxylase antizyme 3

    Atkins John F


    Full Text Available Abstract Background The antizyme family is a group of small proteins that play a role in cell growth and division by regulating the biosynthesis of polyamines (putrescine, spermidine, spermine. Antizymes regulate polyamine levels primarily through binding ornithine decarboxylase (ODC, an enzyme key to polyamine production, and targeting ODC for destruction by the 26S proteosome. Ornithine decarboxylase antizyme 3 (OAZ3 is a testis-specific antizyme paralog and the only antizyme expressed in the mid to late stages of spermatogenesis. Methods To see if mutations in the OAZ3 gene are responsible for some cases of male infertility, we sequenced and evaluated the genomic DNA of 192 infertile men, 48 men of known paternity, and 34 African aborigines from the Mbuti tribe in the Democratic Republic of the Congo. The coding sequence of OAZ3 was further screened for polymorphisms by SSCP analysis in the infertile group and an additional 250 general population controls. Identified polymorphisms in the OAZ3 gene were further subjected to a haplotype analysis using PHASE 2.02 and Arlequin 2.0 software programs. Results A total of 23 polymorphisms were identified in the promoter, exons or intronic regions of OAZ3. The majority of these fell within a region of less than two kilobases. Two of the polymorphisms, -239 A/G in the promoter and 4280 C/T, a missense polymorphism in exon 5, may show evidence of association with male infertility. Haplotype analysis identified 15 different haplotypes, which can be separated into two divergent clusters. Conclusion Mutations in the OAZ3 gene are not a common cause of male infertility. However, the presence of the two divergent haplotypes at high frequencies in all three of our subsamples (infertile, control, African suggests that they have been maintained in the genome by balancing selection, which was supported by a test of Tajima's D statistic. Evidence for natural selection in this region implies that these haplotypes

  1. Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice.

    Shimizu, Tokiko; Hayashi, Yoshinori; Yamasaki, Ryo; Yamada, Jun; Zhang, Jian; Ukai, Kiyoharu; Koike, Masato; Mine, Kazunori; von Figura, Kurt; Peters, Christoph; Saftig, Paul; Fukuda, Takaichi; Uchiyama, Yasuo; Nakanishi, Hiroshi


    Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms

  2. Taurine homeostasis requires de novo synthesis via cysteine sulfinic acid decarboxylase during zebrafish early embryogenesis.

    Chang, Yen-Chia; Ding, Shih-Torng; Lee, Yen-Hua; Wang, Ya-Ching; Huang, Ming-Feng; Liu, I-Hsuan


    Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos.

  3. Histidine decarboxylase knockout mice, a genetic model of Tourette syndrome, show repetitive grooming after induced fear.

    Xu, Meiyu; Li, Lina; Ohtsu, Hiroshi; Pittenger, Christopher


    Tics, such as are seen in Tourette syndrome (TS), are common and can cause profound morbidity, but they are poorly understood. Tics are potentiated by psychostimulants, stress, and sleep deprivation. Mutations in the gene histidine decarboxylase (Hdc) have been implicated as a rare genetic cause of TS, and Hdc knockout mice have been validated as a genetic model that recapitulates phenomenological and pathophysiological aspects of the disorder. Tic-like stereotypies in this model have not been observed at baseline but emerge after acute challenge with the psychostimulant d-amphetamine. We tested the ability of an acute stressor to stimulate stereotypies in this model, using tone fear conditioning. Hdc knockout mice acquired conditioned fear normally, as manifested by freezing during the presentation of a tone 48h after it had been paired with a shock. During the 30min following tone presentation, knockout mice showed increased grooming. Heterozygotes exhibited normal freezing and intermediate grooming. These data validate a new paradigm for the examination of tic-like stereotypies in animals without pharmacological challenge and enhance the face validity of the Hdc knockout mouse as a pathophysiologically grounded model of tic disorders.

  4. Detection of histidine decarboxylase in rat aorta and cultured rat aortic smooth muscle cells.

    Tippens, A S; Davis, S V; Hayes, J R; Bryda, E C; Green, T L; Gruetter, C A


    Having previously demonstrated release of histamine from mast-cell-deficient rat aorta, the objective of this study was to determine and localize histamine synthesis capability in the aorta by detecting histidine decarboxylase (HDC), the enzyme that catalyzes histamine formation. Experiments were conducted with nested reverse transcription-polymerase chain reaction (nRT-PCR) to detect HDC mRNA and with immunofluorescence and western blot analysis to detect HDC protein in rat aorta, cultured rat aortic smooth muscle (RASMC) and endothelial cells (RAEC). Gel electrophoresis of nRT-PCR products indicated HDC mRNA in liver, aorta and RASMC but not in RAEC or kidney. Sequence analysis confirmed that the band observed in RASMC was the target HDC amplicon. Immunofluorescence indicated the presence of HDC protein in RASMC and not in RAEC. Western Blot analysis revealed HDC protein (55 kDa) in liver, aorta, RASMC but not in RAEC or kidney. The results of this study are the first to demonstrate the presence of HDC mRNA and protein in rat aorta and more specifically in RASMC, indicative of their capability to synthesize histamine. Copyright 2004 Birkhäuser Verlag, Basel

  5. Detection of histidine decarboxylase mRNA in human vascular smooth muscle and endothelial cells.

    Tippens, A S; Gruetter, C A


    The objective of this study was to investigate histamine synthesis capability of human vascular smooth muscle and endothelial cells by detecting histidine decarboxylase (HDC) mRNA. HDC catalyzes exclusively the formation of histamine in mammalian cells. Experiments utilizing nested reverse transcription-polymerase chain reaction (nRT-PCR) were conducted to detect the presence of HDC mRNA. Human aortic smooth muscle cells (HAoSMC) and human aortic endothelial cells (HAEC) were cultured and RNA was extracted and amplified using two sets of HDC-specific primers. Rat liver and kidney RNA were isolated and amplified to serve as positive and negative controls, respectively. Gel electrophoresis of HAoSMC, HAEC and liver mRNA revealed bands coinciding with an expected product size of 440 base pairs. Sequence analysis revealed that the observed bands were the appropriate HDC amplicons. These findings are the first to indicate the presence of HDC mRNA in vascular smooth muscle cells and confirm the presence of HDC mRNA in endothelial cells which is consistent with an ability of these cell types to synthesize histamine in the vascular wall.

  6. Inhibition of scratching behaviour caused by contact dermatitis in histidine decarboxylase gene knockout mice.

    Seike, M; Ikeda, M; Kodama, H; Terui, T; Ohtsu, H


    A neuronal system dedicated to itch consists of primary afferent and spinothalamic projection neurons. Histamine is thought to be one of the main mediators for the transmission of itch sensation. However, there are little available information on the role of histamine in scratching behaviour and sensory transmission of atopic dermatitis and chronic eczema. In the present study, the role of histamine in scratching behaviour and neural conduction of sensation in the chronic eczema model was investigated by using l-histidine decarboxylase (HDC) gene knockout mice lacking histamine. The chronic contact dermatitis was induced with daily application of diphenylcyclopropenone (DCP) on a hind paw of HDC (+/+) and HDC (-/-) mice for 2 months. The observation of scratching behaviour and the hot-plate test were performed in both mice. Histological studies were performed in the skin and spinal cord tissues. Histological examination revealed that both HDC (+/+) and HDC (-/-) mice displayed the similar extent of inflammatory cell infiltration, hyperplastic epidermis and newly spreading of neuronal processes in the skin tissue. Scratching behaviour was exclusively induced in HDC (+/+) mice, whereas it was barely observed in HDC (-/-) mice. The expression of c-Fos was specifically upregulated in HDC (+/+) mice in lamina I of the spinal dorsal horn following repeated DCP application. Scratching behaviour in chronic contact dermatitis in mice was thought mainly mediated with histamine. The afferent pathway of sensation in chronic contact dermatitis model may connect with the central nervous system through lamina I of the spinal dorsal horn.

  7. Evolution and expression analysis of the soybean glutamate decarboxylase gene family

    Tae Kyung Hyun; Seung Hee Eom; Xiao Han; Ju-Sung Kim


    Glutamate decarboxylase (GAD) is an enzyme that catalyses the conversion of L-glutamate into -aminobutyric acid (GABA), which is a four-carbon non-protein amino acid present in all organisms. Although plant GAD plays important roles in GABA biosynthesis, our knowledge concerning GAD gene family members and their evolutionary relationship remains limited. Therefore, in this study, we have analysed the evolutionary mechanisms of soybean GAD genes and suggested that these genes expanded in the soybean genome partly due to segmental duplication events. The approximate dates of duplication events were calculated using the synonymous substitution rate, and we suggested that the segmental duplication of GAD genes in soybean originated 9.47 to 11.84 million years ago (Mya). In addition, all segmental duplication pairs (GmGAD1/3 and GmGAD2/4) are subject to purifying selection. Furthermore, GmGAD genes displayed differential expression either in their transcript abundance or in their expression patterns under abiotic stress conditions like salt, drought, and cold. The expression pattern of paralogous pairs suggested that they might have undergone neofunctionalization during the subsequent evolution process. Taken together, our results provide valuable information for the evolution of the GAD gene family and represent the basis for future research on the functional characterization of GAD genes in higher plants.

  8. Enhanced production of butanol and acetoin by heterologous expression of an acetolactate decarboxylase in Clostridium acetobutylicum.

    Shen, Xiaoning; Liu, Dong; Liu, Jun; Wang, Yanyan; Xu, Jiahui; Yang, Zhengjiao; Guo, Ting; Niu, Huanqing; Ying, Hanjie


    Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum, acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9g/L), acetoin (6.5g/L), and ethanol (1.9g/L) were generated by the recombinant strain, with acetone no more than 1.8g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Solvent-derived protons in catalysis by brewers' yeast pyruvate decarboxylase.

    Harris, T K; Washabaugh, M W


    Catalysis of proton transfer to thiamin diphosphate (TDP) and 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC from Saccharomyces carlsbergensis was investigated by determining the solvent discrimination tritium isotope effect, (kH/kT)disc, on the reaction of pyruvate to form acetaldehyde in the presence of the nonsubstrate allosteric effector pyruvamide. The fractionation factors for TDP C(2)-L (phi C(2) = 0.98 +/- 0.06) and HETDP C(alpha)-L (phi C(alpha) = 1.01 +/- 0.07) (L = H or D) do not contribute significantly to observed enzymic isotopic discrimination. The value of (kH/kT)disc = 1.0 for reprotonation of TDP C(2)-L under single-turnover conditions ([E] > [S]) is consistent with C(2)-hydron transfer via a catalytic group (phi = 1) equilibrated with solvent. [1-L]Acetaldehyde formation under transient steady-state ([E] or = 1.2) provides significant intramolecular catalysis in the enzyme-bound coenzyme.

  10. Simultaneous silencing of two arginine decarboxylase genes alters development in Arabidopsis

    Diana eSánchez-Rangel


    Full Text Available Polyamines (PAs are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2 catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC. The generated transgenic lines (amiR:ADC-L1 and -L2 showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes.

  11. Polyamine metabolism and osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase.

    Tiburcio, A F; Kaur-Sawhney, R; Galston, A W


    We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with DL-alpha-difluoromethylarginine (DFMA), a specific 'suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.

  12. The impact of serotonergic stimulation on reelin and glutamate decarboxylase gene expression in adult female rats.

    Lakatosova, S; Celec, P; Schmidtova, E; Kubranska, A; Durdiakova, J; Ostatnikova, D


    Reelin plays an important role in the regulation of synaptic plasticity in adulthood. Administration of 5-metoxytryptamine (5MT), an agonist of serotonin receptors, during natal and neonatal periods results in decreased reelin expression. In adulthood, reelin is expressed by GABAergic neurons. The purpose of this study was to reveal the effect of elevated serotonergic stimulation on the expression of reelin and glutamate decarboxylase (GAD1) in adulthood as well as on depressive behavior and spatial cognitive abilities in adult female rats. Rats were injected with 5MT. A forced swimming test was used for evaluation of the depressive behavior and Morris water maze test was used for evaluation of spatial cognition. Brains were used for measuring the expression of reelin and GAD1. We found a significant decrease in reelin expression in the cerebellum and prefrontal cortex of 5MT-treated rats. GAD1 expression was decreased in the cerebellum of 5MT-treated rats. 5MT-treated rats reached a lower immobility score in the forced swimming test. The Morris water maze test did not reveal any significant differences. We have shown that administration of serotonin receptor agonist resulted in a decreased RELN and GAD1 expression in the cerebellum of adult female rats. We propose that this phenomenon might be relevant in the pathogenesis of autism (Fig. 3, Ref. 38). Full Text in free PDF

  13. Nucleotide variation at the dopa decarboxylase (Ddc) gene in natural populations of Drosophila melanogaster

    Andrey Tatarenkov; Francisco J. Ayala


    We studied nucleotide sequence variation at the gene coding for dopa decarboxylase (Ddc) in seven populations of Drosophila melanogaster. Strength and pattern of linkage disequilibrium are somewhat distinct in the extensively sampled Spanish and Raleigh populations. In the Spanish population, a few sites are in strong positive association, whereas a large number of sites in the Raleigh population are associated nonrandomly but the association is not strong. Linkage disequilibrium analysis shows presence of two groups of haplotypes in the populations, each of which is fairly diverged, suggesting epistasis or inversion polymorphism. There is evidence of two forms of natural selection acting on Ddc. The McDonald–Kreitman test indicates a deficit of fixed amino acid differences between D. melanogaster and D. simulans, which may be due to negative selection. An excess of derived alleles at high frequency, significant according to the -test, is consistent with the effect of hitchhiking. The hitchhiking may have been caused by directional selection downstream of the locus studied, as suggested by a gradual decrease of the polymorphism-to-divergence ratio. Altogether, the Ddc locus exhibits a complicated pattern of variation apparently due to several evolutionary forces. Such a complex pattern may be a result of an unusually high density of functionally important genes.

  14. Pathogenic Roles of Glutamic Acid Decarboxylase 65 Autoantibodies in Cerebellar Ataxias.

    Mitoma, Hiroshi; Manto, Mario; Hampe, Christiane S


    Reports suggesting a pathogenic role of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65Abs) in cerebellar ataxias (CAs) are reviewed, and debatable issues such as internalization of antibodies by neurons and roles of epitopes are discussed. GAD65 is one of two enzymes that catalyze the conversion of glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). A pathogenic role of GAD65Ab in CAs is suggested by in vivo and in vitro studies. (1) Intracerebellar administration of cerebrospinal fluid (CSF) immunoglobulins (IgGs) obtained from GAD65Ab-positive CA patients impairs cerebellar modulation of motor control in rats. (2) CSF IgGs act on terminals of GABAergic neurons and decrease the release of GABA in cerebellar slices from rats and mice. (3) Absorption of GAD65Ab by recombinant GAD65 diminishes the above effects, and monoclonal human GAD65Ab (b78) mimic the effects of CSF IgGs in vivo and in vitro. Studies using GAD65-KO mice confirm that the target molecule is GAD65. (4) Notably, the effects of GAD65Ab depend on the epitope specificity of the monoclonal GAD65Ab. Taken together, these results indicate that epitope-specific GAD65Ab-induced impairment of GABA release is involved in the pathogenesis of GAD65Ab-positive CA and support the early detection of GAD65Ab-associated CA to initiate immunotherapy before irreversible neuronal death in the cerebellum.

  15. Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

    Hui TIAN; Lin LI; Xian-Xi LIU; Yan ZHANG


    Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells, especially lung cancer cells. Some chemotherapeutic agents aimed at decreasing ODC gene expression showed inhibitory effects on cancer cells. In this study, we examined the effects of adenoviral transduced antisense ODC on lung cancer cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549. The 3-(4,5-me thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the effect on cell growth. Expression of ODC and concentration of polyamines in cells were determined by Western blot analysis and high performance liquid chromatography. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cell apoptosis. The expression of ODC in A-549 cells was reduced to 54%, and that of three polyamines was also decreased through the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3as could lead to cell apoptosis, with apoptosis index of 46%. This study suggests that rAd-ODC/Ex3as has an antitumor effect on the human lung cancer cells.

  16. Developmental changes of glutamate acid decarboxylase 67 in mouse brain after hypoxia ischemia

    Fa-Lin XU; Chang-Lian ZHU; Xiao-Yang WANG


    Objective To study the developmental changes of glutamic acid decarboxylase-67 ( GAD-67, a GABA synthetic enzyme) in normal and hypoxic ischemic (HI) brain. Methods C57/BL6 mice on postnatal day (P) 5, 9, 21and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD67 was up regulated with brain development and significant difference was seen between mature (P21, P60) and immature (P5, P9) brain. GAD67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia (HI) insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that (15.6 ±7.0)%TUNEL positive cells were GAD67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.

  17. Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells

    Giancarlo eRusso


    Full Text Available The function of the enzyme glutamate decarboxylase (GAD is to convert glutamate in -aminobutyric acid (GABA.GAD exists as two major isoforms, termed GAD65 and GAD67,.that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

  18. Pathogenic Roles of Glutamic Acid Decarboxylase 65 Autoantibodies in Cerebellar Ataxias

    Hiroshi Mitoma


    Full Text Available Reports suggesting a pathogenic role of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65Abs in cerebellar ataxias (CAs are reviewed, and debatable issues such as internalization of antibodies by neurons and roles of epitopes are discussed. GAD65 is one of two enzymes that catalyze the conversion of glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA. A pathogenic role of GAD65Ab in CAs is suggested by in vivo and in vitro studies. (1 Intracerebellar administration of cerebrospinal fluid (CSF immunoglobulins (IgGs obtained from GAD65Ab-positive CA patients impairs cerebellar modulation of motor control in rats. (2 CSF IgGs act on terminals of GABAergic neurons and decrease the release of GABA in cerebellar slices from rats and mice. (3 Absorption of GAD65Ab by recombinant GAD65 diminishes the above effects, and monoclonal human GAD65Ab (b78 mimic the effects of CSF IgGs in vivo and in vitro. Studies using GAD65-KO mice confirm that the target molecule is GAD65. (4 Notably, the effects of GAD65Ab depend on the epitope specificity of the monoclonal GAD65Ab. Taken together, these results indicate that epitope-specific GAD65Ab-induced impairment of GABA release is involved in the pathogenesis of GAD65Ab-positive CA and support the early detection of GAD65Ab-associated CA to initiate immunotherapy before irreversible neuronal death in the cerebellum.

  19. Pathogenic Roles of Glutamic Acid Decarboxylase 65 Autoantibodies in Cerebellar Ataxias

    Hampe, Christiane S.


    Reports suggesting a pathogenic role of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65Abs) in cerebellar ataxias (CAs) are reviewed, and debatable issues such as internalization of antibodies by neurons and roles of epitopes are discussed. GAD65 is one of two enzymes that catalyze the conversion of glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). A pathogenic role of GAD65Ab in CAs is suggested by in vivo and in vitro studies. (1) Intracerebellar administration of cerebrospinal fluid (CSF) immunoglobulins (IgGs) obtained from GAD65Ab-positive CA patients impairs cerebellar modulation of motor control in rats. (2) CSF IgGs act on terminals of GABAergic neurons and decrease the release of GABA in cerebellar slices from rats and mice. (3) Absorption of GAD65Ab by recombinant GAD65 diminishes the above effects, and monoclonal human GAD65Ab (b78) mimic the effects of CSF IgGs in vivo and in vitro. Studies using GAD65-KO mice confirm that the target molecule is GAD65. (4) Notably, the effects of GAD65Ab depend on the epitope specificity of the monoclonal GAD65Ab. Taken together, these results indicate that epitope-specific GAD65Ab-induced impairment of GABA release is involved in the pathogenesis of GAD65Ab-positive CA and support the early detection of GAD65Ab-associated CA to initiate immunotherapy before irreversible neuronal death in the cerebellum. PMID:28386570

  20. Role of the Sulfonium Center in Determining the Ligand Specificity of Human S-Adenosylmethionine Decarboxylase

    Bale, Shridhar; Brooks, Wesley; Hanes, Jeremiah W.; Mahesan, Arnold M.; Guida, Wayne C.; Ealick, Steven E.; (Moffitt); (Cornell)


    S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the polyamine biosynthetic pathway. Inhibition of this pathway and subsequent depletion of polyamine levels is a viable strategy for cancer chemotherapy and for the treatment of parasitic diseases. Substrate analogue inhibitors display an absolute requirement for a positive charge at the position equivalent to the sulfonium of S-adenosylmethionine. We investigated the ligand specificity of AdoMetDC through crystallography, quantum chemical calculations, and stopped-flow experiments. We determined crystal structures of the enzyme cocrystallized with 5{prime}-deoxy-5{prime}-dimethylthioadenosine and 5{prime}-deoxy-5{prime}-(N-dimethyl)amino-8-methyladenosine. The crystal structures revealed a favorable cation-{pi} interaction between the ligand and the aromatic side chains of Phe7 and Phe223. The estimated stabilization from this interaction is 4.5 kcal/mol as determined by quantum chemical calculations. Stopped-flow kinetic experiments showed that the rate of the substrate binding to the enzyme greatly depends on Phe7 and Phe223, thus supporting the importance of the cation-{pi} interaction.

  1. Aromatic L-amino acid decarboxylase deficiency diagnosed by clinical metabolomic profiling of plasma.

    Atwal, Paldeep S; Donti, Taraka R; Cardon, Aaron L; Bacino, C A; Sun, Qin; Emrick, L; Reid Sutton, V; Elsea, Sarah H


    Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.

  2. Dynamic regulation of glutamic acid decarboxylase 65 gene expression in rat testis

    Haixiong Liu; Shifeng Li; Yunbin Zhang; Yuanchang Yan; Yiping Li


    Glutamate decarboxylase 65 (GAD65) produces γ-amino-butyric acid,the main inhibitory neurotransmitter in adult mammalian brain.Previous experiments,per-formed in brain,showed that GAD65 gene possesses two TATA-less promoters,although the significance is unknown.Here,by rapid amplification of cDNA ends method,two distinct GAD65 mRNA isoforms transcribed from two independent clusters of transcription start sites were identified in post-natal rat testis.RT-PCR results revealed that the two mRNA isoforms had distinct expression patterns during post-natal testis maturation,suggesting that GAD65 gene expression was regulated by alternative promoters at the transcription level.By using GAD65-speciflc antibodies,western blotting analysis showed that the 58-kDa GAD65,N-terminal 69 amino acids truncated form of full-length GAD65 protein,was developmentally expressed during post-natal testis matu-ration,suggesting that GAD65 gene expression in testis may also be regulated by post-translational processing.Confocal immunofluorescence microscopy revealed that GAD65 protein was presented in Leydig cells of Day 1 testis,primary spermatocytes and spermatids of post-natal of Day 90 testis.The above results suggested that GAD65 gene expression is dynamically regulated at mul-tiple levels during post-natal testis maturation.

  3. Motor effects of broad beans (Vicia faba) in Parkinson's disease: single dose studies.

    Kempster, P A; Bogetic, Z; Secombei, J W; Martin, H D; Balazs, N D; Wahlqvist, M L


    Broad beans (Vicia faba) are a natural source of L-dopa. To investigate a possible role for this substance in the treatment of Parkinsonian motor oscillations, we carried out single dose studies of Vicia faba pod mixture plus carbidopa in six patients. Motor responses of equivalent magnitude to those of conventional L-dopa medication occurred in five cases with mean onset of 39 min and mean duration of 104 min. Vicia faba meals produced comparable L-dopa blood levels to fasting standard tablet doses and recovery studies yielded 0.25% L-dopa per weight of bean pod mixture. Vicia faba contains sufficient L-dopa to be pharmacologically active in patients with Parkinson's disease and can potentially be incorporated into dietary strategies to manage Parkinsonian motor oscillations.

  4. Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

    Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei


    We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

  5. Involvement of caspase-9 in execution of the maternal program of apoptosis in Xenopus late blastulae overexpressed with S-adenosylmethionine decarboxylase.

    Takayama, Eiji; Higo, Takayasu; Kai, Masatake; Fukasawa, Masashi; Nakajima, Keisuke; Hara, Hiroshi; Tadakuma, Takushi; Igarashi, Kazuei; Yaoita, Yoshio; Shiokawa, Koichiro


    We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [(35)S]procaspase-9, but not [(35)S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos.

  6. Spinal Cord Hemisection Facilitates Aromatic L-Amino Acid Decarboxylase Cells to Produce Serotonin in the Subchronic but Not the Chronic Phase

    Bushra Azam


    Full Text Available Neuromodulators, such as serotonin (5-hydroxytryptamine, 5-HT and noradrenalin, play an essential role in regulating the motor and sensory functions in the spinal cord. We have previously shown that in the rat spinal cord the activity of aromatic L-amino acid decarboxylase (AADC cells to produce 5-HT from its precursor (5-hydroxytryptophan, 5-HTP is dramatically increased following complete spinal cord transection. In this study, we investigated whether a partial loss of 5-HT innervation could similarly increase AADC activity. Adult rats with spinal cord hemisected at thoracic level (T11/T12 were used with a postoperation interval at 5 days or 60 days. Using immunohistochemistry, first, we observed a significant reduction in the density of 5-HT-immunoreactive fibers in the spinal cord below the lesion on the injured side for both groups. Second, we found that the AADC cells were similarly expressed on both injured and uninjured sides in both groups. Third, increased production of 5-HT in AADC cells following 5-HTP was seen in 5-day but not in 60-day postinjury group. These results suggest that plastic changes of the 5-HT system might happen primarily in the subchronic phase and for longer period its function could be compensated by plastic changes of other intrinsic and/or supraspinal modulation systems.

  7. Study of orotidine 5'-monophosphate decarboxylase in complex with the top three OMP, BMP, and PMP ligands by molecular dynamics simulation.

    Jamshidi, Shirin; Jalili, Seifollah; Rafii-Tabar, Hashem


    Catalytic mechanism of orotidine 5'-monophosphate decarboxylase (OMPDC), one of the nature most proficient enzymes which provides large rate enhancement, has not been fully understood yet. A series of 30 ns molecular dynamics (MD) simulations were run on X-ray structure of the OMPDC from Saccharomyces cerevisiae in its free form as well as in complex with different ligands, namely 1-(5'-phospho-D-ribofuranosyl) barbituric acid (BMP), orotidine 5'-monophosphate (OMP), and 6-phosphonouridine 5'-monophosphate (PMP). The importance of this biological system is justified both by its high rate enhancement and its potential use as a target in chemotherapy. This work focuses on comparing two physicochemical states of the enzyme (protonated and deprotonated Asp91) and three ligands (substrate OMP, inhibitor, and transition state analog BMP and substrate analog PMP). Detailed analysis of the active site geometry and its interactions is properly put in context by extensive comparison with relevant experimental works. Our overall results show that in terms of hydrogen bond occupancy, electrostatic interactions, dihedral angles, active site configuration, and movement of loops, notable differences among different complexes are observed. Comparison of the results obtained from these simulations provides some detailed structural data for the complexes, the enzyme, and the ligands, as well as useful insights into the inhibition mechanism of the OMPDC enzyme. Furthermore, these simulations are applied to clarify the ambiguous mechanism of the OMPDC enzyme, and imply that the substrate destabilization and transition state stabilization contribute to the mechanism of action of the most proficient enzyme, OMPDC.

  8. Elevation of arginine decarboxylase-dependent putrescine production enhances aluminum tolerance by decreasing aluminum retention in root cell walls of wheat.

    Yu, Yan; Jin, Chongwei; Sun, Chengliang; Wang, Jinghong; Ye, Yiquan; Lu, Lingli; Lin, Xianyong


    Aluminum (Al) stress induces putrescine (Put) accumulation in several plants and this response is proposed to alleviate Al toxicity. However, the mechanisms underlying this alleviation remain largely unknown. Here, we show that exposure to Al clearly increases Put accumulation in the roots of wheat plants (Triticum aestivum L. 'Xi Aimai-1') and that this was accompanied by significant increase in the activity of arginine decarboxylase (ADC), a Put producing enzyme. Application of an ADC inhibitor (d-arginine) terminated the Al-induced Put accumulation, indicating that increased ADC activity may be responsible for the increase in Put accumulation in response to Al. The d-arginine treatment also increased the Al-induced accumulation of cell wall polysaccharides and the degree of pectin demethylation in wheat roots. Thus, it elevated Al retention in cell walls and exacerbated Al accumulation in roots, both of which aggravate Al toxicity in wheat plants. The opposite effects were true for exogenous Put application. These results suggest that ADC-dependent Put accumulation plays important roles in providing protection against Al toxicity in wheat plants through decreasing cell wall polysaccharides and increasing the degree of pectin methylation, thus decreasing Al retention in the cell walls.

  9. N-ω-chloroacetyl-l-ornithine, a new competitive inhibitor of ornithine decarboxylase, induces selective growth inhibition and cytotoxicity on human cancer cells versus normal cells.

    Medina-Enríquez, Miriam Marlene; Alcántara-Farfán, Verónica; Aguilar-Faisal, Leopoldo; Trujillo-Ferrara, José Guadalupe; Rodríguez-Páez, Lorena; Vargas-Ramírez, Alba Laura


    Many cancer cells have high expression of ornithine decarboxylase (ODC) and there is a concerted effort to seek new inhibitors of this enzyme. The aim of the study was to initially characterize the inhibition properties, then to evaluate the cytotoxicity/antiproliferative cell based activity of N-ω-chloroacetyl-l-ornithine (NCAO) on three human cancer cell lines. Results showed NCAO to be a reversible competitive ODC inhibitor (Ki = 59 µM) with cytotoxic and antiproliferative effects, which were concentration- and time-dependent. The EC50,72h of NCAO was 15.8, 17.5 and 10.1 µM for HeLa, MCF-7 and HepG2 cells, respectively. NCAO at 500 µM completely inhibited growth of all cancer cells at 48 h treatment, with almost no effect on normal cells. Putrescine reversed NCAO effects on MCF-7 and HeLa cells, indicating that this antiproliferative activity is due to ODC inhibition.

  10. Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes.

    Bomser, J; Singletary, K; Meline, B


    Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis