Heterogeneous expression of DOPA decarboxylase to improve the production of dopamine in Escherichia coli / 生物工程学报

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.

Age related interaction of dopamine and serotonin synthesis in striatal synaptosomes

Tyrosine hydroxylase and tryptophan hydroxylase are key rate limiting enzymes in the biosynthesis of dopamine and serotonin, respectively. Since both enzymes are active in striatum, and affected by age, this study was undertaken to investigate interaction between dopamine and serotonin synthesis in brain striatal synaptosomes of aging rat. Male Wistar rats (3 and 30 month old) were killed by decapitation and brain striatal synaptosomes were prepared by discontinuous Ficoll/sucrose gradient technique. Synaptosomes were incubated in the presence of added pargiline (monoamineoxidase inhibitor), dopamine or serotonin synthesized during 25 min was measured by HPLC, employing electrochemical detection. Dopamine synthesis in synaptosomes prepared from young animals was markedly inhibited by addition of 5 microM serotonin concentrations (30%) and increasing serotonin concentrations up to 50 microM caused only a smaller additional inhibition. Dopamine synthesis in synaptosomes obtained from old rats was significantly lower than that of youg animals and addition of serotonin concentrations up to 50 microM had little effect on these preparations. In case of serotonin synthesis, exogenously added 5 microM dopamine inhibited serotonin synthesis in the synaptosomes of both ages by about 40%, whereas with higher concentration of dopamine (10-50 microM) the rate of inhibition was highly pronounced in old rats as compared to that of young animals. It is concluded that dopamine and serotonin interaction may be significant, and that these should be considered in long-term treatments of Parkinson's disease with L-DOPA.

El impacto de tener las piernas inquietas: avances en el diagnóstico y tratamiento de una patología prevalente, pero subdiagnosticada / The impact to have the restless legs: advances in the diagnosis and treatment of a prevalent, but under-diagnosed pathology

El Síndrome de Piernas Inquietas (SPI) se caracteriza por una sensación desagradable en las piernas que lleva a la imperiosa necesidad de moverlas. Ocurre en reposo y es de preferencia nocturna. Su frecuencia varía entre 2-15 por ciento de la población general adulta y 20-30 por ciento en pacientes urémicos en diálisis. A nivel nacional se da una frecuencia estimativa de 13 por ciento en la población general adulta y 27 por ciento en los pacientes urémicos en diálisis.

Circadian time-dependent effects of fencamfamine on inhibition of dopamine uptake and release in rat striatal slices

Fencamfamine (FCF) is a central stimulant that facilitates central dopaminergic transmission through inhibition of dopamine uptake and enhanced release of the transmitter. We evaluated the changes in the inhibition of uptake and the release of striatal [3H]-dopamine at 9:00 and 21:00 h, times corresponding to maximal and minimal behavioral responses to FCF, respectively. Adult male Wistar rats (200-250 g) maintained on a 12-h light/12-h dark cycle (lights on at 7:00 h) were used. In the behavioral experiments the rats (N = 8 for each group) received FCF (3.5 mg/kg, ip) or saline at 9:00 or 21:00 h. Fifteen minutes after treatment the duration of activity (sniffing, rearing and locomotion) was recorded for 120 min. The basal motor activity was higher (28.6 + 4.2 vs 8.4 + 3.5 s) after saline administration at 21:00 h than at 9:00 h. FCF at a sigle dose significantly enhanced the basal motor activity (38.3 + 4.5 vs 8.4 + 3.5 s) and increased the duration of exploratory activity (38.3 + 4.5 vs 32.1 + 4.6 s) during the light, but not the dark phase. Two other groups of rats (N = 6 for each group) were decapitated at 9:00 and 21:00 h and striata were dissected for dopamine uptake and release assays. The inhibition of uptake and release of [3H]-dopamine were higher at 9:00 than at 21:00 h, suggesting that uptake inhibition and the release properties of FCF undergo daily variation. These data suggest that the circadian time-dependent effects of FCF might be related to a higher susceptibility of dopamine presynaptic terminals to the action of FCF during the light phase which corresponds to the rats' resting period.

Actualización sobre receptores dopaminérgicos centrales / Current concepts of central dopaminergic receptors

El presente trabajo tiene como objetivo revisar los avances en el estudio de la dopamina, como neurotransmisor a nivel del sistema nervioso central y su posible relación funcional con diversos desórdenes, en especial las esquizofrenias. Se revisan los conocimientos del sistema dopaminérgico central, en lo que respecta a anatomía de las vías dopaminérgicas y a la bioquímica de la transmisión, desde su síntesis hasta su inactivación enzimática. Se analisan los diversos métodos y estudios, tanto in vivo como in vitro, que se emplean para reconocer los diversos tipos de receptores dopaminérgicos centrales. Así, desde que se reconoce un adenil ciclasa sensible a dopamina, se obtienen 2 tipos de receptores dopaminérgicos, el D-1 que aumenta el AMPc y el D-2, que no están asociados a la enzima. Posteriormente, usando técnicas de radioligandos, se logra reconocer a 3 subtipos de receptores dopaminérgicos con localización y características bien definidas. Un mayor estudio de la dopamina y en especial de los receptores dopaminérgicos centrales, permitirá comprender las bases neurofisiológicas y farmacológicas de algunos desórdenes tales com las esquizofrenias, la enfermedad de Parkinson, el corea de Huntington, entre otras, permitiendo el desarrollo de medicamentos más selectivos y con mayor acción

Regulación de la liberación de dopamina de neuronas mesocorticales / Regulation of dopamine release from mesocortical neurons

The mechanism involved in the regulation of dopamine (DA) rease from mesocortical dopaminergic neurons were studied. Prefrontal cortical slices (PFC) were incubated with 3H-Da in the presence of 0.3 uM desipramine. Under these conditions,6 to 10% of 3H-norepinephrine was formed. After incubation, the slices were superfused (2 slices/chamber). At 45 minutes of washout, a constant rate of efflux of 3H- norepinephrine was obtained; 1.46+/- 0.1 % of the 3H present in the slices was released every 5 minutes. Electrical stimulation (1Hz, 120 pulses) produced a calcium dependent increase in the outflow of 3H- products above prestimulation levels (3.4+/_ 0.1%) Since the release of a neurotransmitter may be modulated by presynaptic receptors others than autoreceptors, we investigated the pharmacology of presynaptic receptors monitoring the effects of DA, norepinephrine, serotonine, acetylcholine, Gaba and CCK agonists and antagonists on the electrically evoked relase of DA. Apomorphine, LY171555 and bromocriptine inhibited Da realese. Maximun inhibition of release values for these drugs were 66 and 59% (EMAX) and those for EC50 were 25, 29 and 20 nM, respectively. Haloperidol antagonized the inhibitory effect of DA agonist whereas phentolamine hsd no effect. Haloperidol produced singnificant increase in the stimulation- evoked release of 3H (76%). The other agents did not modify DA release from PFC slices. These results suggest that DA release is modulated by auyoreceptors whereas the presynaptic noradrenergic, cholinergic serotoninergic, Gabaergic and CCK receptors do not appear to play a significant role in the regulation of DA

Estudios de los mecanismos de captación y liberación de 3H-Dopamina en retina de Eugerres plumieri / Studies on the uptake and release mechanisms of 3H-Dopamine in retina of Eugerres plumieri

En el presente trabajo se realizaron estudios de captación y liberación de 3H-Dopamina en la retina de Eugerres plumieri. Se pudo demostrar que la captación es dependiente del tiempo y de la concentración de DA exógena, presentando cinética de saturación, calculándose el Km en 0,41 ñ 0,08 uM y el Vmax en 33 ñ 2 pmol/mgr proteína/minuto. Esta captación demostró ser dependiente de la temperatura y de la concentración externa del ion Nañ, bloqueándose en un 75 por ciento a bajas temperaturas y en un 66 por ciento al eliminar el Na externo. Por otro lado, la captación de 3H-Dopamina no parece depender del Ca++ externo ni de las condiciones de luz u oscuridad. Utilizando diferentes aminas biógenas y antagonistas, se pudo demostrar la especificidad del proceso de captación por la DA. Así, el antagonista para DA (la clorimipramina) inhibe en un 60 por ciento la captación de DA, mientras que la imipramina, un bloqueador más específico de la 5 HT, sólo bloqueó la captación en un 45 por ciento. En otro grupo de experimentos se demostró que la liberación de 3 H-Dopamina depende de las concentraciones externas de K+ (25-50-75 nM), siendo la liberación también dependiente del Ca++ externo. 2 mM Co++, 25 mM Mg++ y 4 uM D-600 fueron todos capaces de inhibir significativamente la liberación estimulada por 25 mM, K+. Al mantener la retina en oscuridad se favorece la liberación de 3H-Dopamina estimulada por 25mM K+. Estos resultados demuestran la existencia, en la retina del pez Eugerres plumieri, de mecanismos específicos de captación y liberación de 3H-Dopamina, apoyando la hipótesis de que la DA es un neurotransmisor en la retina de ..

Effect of alpha-methyl-p-tyrosine on neuroleptic-induced catalepsy in rat.

Pretreatment with alpha-methyl-p-tyrosine, a tyrosine hydroxylase inhibitor, was found to increase the intensity of catalepsy induced by haloperidol, chlorpromazine and molindone. The drug probably decreases the synthesis of dopamine and makes less dopamine available for release and to compete with the neuroleptic for the postsynaptic striatal dopamine receptor sites with resultant potentiation of the neuroleptic-induced catalepsy.

Teoría dopaminérgica de la esquizofrenia una revisión crítica / Dopaminergic theory of the schizophrenia. A critical review

Se hace una revisión de la literatura acerca de la llamada hipótesis dopaminérgica de la esquizofrenia. Las evidencias a favor y en contra de los mecanismos dopaminérgicos en la esquizofrenia son analizadas separadamente en dos hipótesis potencialmente relacionadas, la del bloqueo de la dopamina y la de la sobreactividad de las acciones dopaminérgicas. En adición se discuten otras hipótesis alternativas derivadas de las anteriores. Se concluye en que aparentemente no existe, a pesar de las cuidadosas investigaciones descritas, un agente etiológico que en forma individual pueda causar la esquizofrenia