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.
Animals ; Dopa Decarboxylase/genetics* ; Dopamine/biosynthesis* ; Drosophila melanogaster/genetics* ; Escherichia coli/metabolism* ; Humans ; Metabolic Engineering

OBJECTIVE: To investigate the expression of dopamine D2 receptors (D2R) and dopamine transportors (DAT) located in the medial prefrontal contex (mPFC) in high and low conditioned place preference (CPP) rats, and to unveil the possible mechanism leading to different CPP susceptibilities. METHODS: One hundred and sixty male Sprague-Dawley rats were randomly assigned into an experiment group (n = 130) and a control group (n = 30). The experiment group was re-classified into 2 groups according to CPP values:high preference group (HP group) and low preference group (LP group). According to the execution time-points after the last administration, the HP and LP group was classified into a 3-hour group (3 h), a 72-hour group (J3d), and a 14-day group (J14d), respectively. At 3 hours, 72 hours, and 14 days after the final injection, rats were killed and cardio-perfused, and the brains were removed and sliced up coronarily. The mRNA levels of D2R and DAT in mPFC were determined with in situ hybridization. RESULTS: There were no significant differences of pretest scores staying at the non-preference chamber among the groups(P = 0.470). However, the test scores of the CPP time stayed at pretest natural preference in the HP group were significantly higher than those of the LP group(P = 0.000). In 3h, J3d, and J14d groups,the expressions of D2R mRNA in the HP group (125.43 +/- 2.90 approximately 142.92 +/- 3.32) were lower than those of LP group (122.25 +/- 2.20 approximately 136.67 +/-5.39) (P = 0.000). In 3h and J3d,the expressions of DAT mRNA in the HP group (157.00 +/- 3.55 approximately 145.15 +/- 3.69) were significantly lower than those of the LP group (150.69 +/- 3.12 approximately 138.84 +/- 3.99) (P = 0.000). In J14d, there were no differences among 3 groups in mPFC (P = 0.458). CONCLUSION D2R and DAT may be correlated closely and underlie the different susceptibilities to morphine induced CPP.
Animals ; Conditioning, Psychological ; drug effects ; Disease Susceptibility ; metabolism ; Dopamine Plasma Membrane Transport Proteins ; biosynthesis ; genetics ; Male ; Morphine Dependence ; metabolism ; Prefrontal Cortex ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Dopamine D2 ; biosynthesis ; genetics

Gerbil forebrain ischemia/reperfusion(I/R) injury model was used to study the effects of D(1) and D(2) receptor agonists and antagonists on neuronal apoptosis of hippocampal CA1 area. All animals were tested for habituation deficits in an open field test on the 1st, 3rd and 7th days after reperfusion. The animals were then killed, and brains underwent paraffin embedding for hematoxylin-eosin staining, in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) staining and immunohistochemistry (bax, bcl-2). The result of open field test showed that the I/R group was significantly impaired (higher activity scores) when compared with the control group. Pretreatment with pergolide significantly reduced this habituation impairment. Forebrain ischemia for 5 min resulted in extensive CA1 apoptosis on the 3rd and 7th days after I/R injury. About 95% neurons in hippocampal CA1 area entered apoptosis and only 2%-7% pyramidal neurons stayed alive due to an inhibition of bcl-2 expression and an increase in bax expression. Pretreatment of pergolide attenuated neuronal damage caused by transient ischemia. Infusion of pergolide could induce the expression of bcl-2 and reduce the expression of bax. Pretreatment with SKF38393, SCH23390 and spiperone had no effects on these changes in this transient I/R injury model. All these results indicate that pergolide plays an important role in the protection of hippocampal neurons from apotosis through upregulating the expression of bcl-2 protein and reducing the expression of bax protein.
2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine ; pharmacology ; Animals ; Apoptosis ; Brain ; physiopathology ; Brain Ischemia ; physiopathology ; Dopamine Agonists ; pharmacology ; Dopamine Antagonists ; pharmacology ; Gerbillinae ; Hippocampus ; physiopathology ; Ischemic Attack, Transient ; physiopathology ; Male ; Neurons ; physiology ; Neuroprotective Agents ; pharmacology ; Pergolide ; pharmacology ; Prosencephalon ; physiopathology ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Receptors, Dopamine D1 ; Receptors, Dopamine D2 ; Reperfusion Injury ; physiopathology ; bcl-2-Associated X Protein

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) and by abnormal aggregation of alpha-synuclein. Previous studies have suggested that DA can interact with alpha-synuclein, thus modulating the aggregation process of this protein; this interaction may account for the selective vulnerability of DA neurons in patients with PD. However, the relationship between DA and alpha-synuclein, and the role in progressive degeneration of DA neurons remains elusive. We have shown that in the presence of DA, recombinant human alpha-synuclein produces non-fibrillar, SDS-resistant oligomers, while beta-sheet-rich fibril formation is inhibited. Pharmacologic elevation of the cytoplasmic DA level increased the formation of SDS-resistant oligomers in DA-producing neuronal cells. DA promoted alpha-synuclein oligomerization in intracellular vesicles, but not in the cytosol. Furthermore, elevation of DA levels increased secretion of alpha-synuclein oligomers to the extracellular space, but the secretion of monomers was not changed. DA-induced secretion of alpha-synuclein oligomers may contribute to the progressive loss of the dopaminergic neuronal population and the pronounced neuroinflammation observed in the SNpc in patients with PD.
Blotting, Western ; Cell Line, Tumor ; Dopamine/*metabolism ; Humans ; Levodopa/pharmacology ; Neurons/*metabolism/pathology/*secretion ; Parkinson Disease/metabolism/pathology ; Substantia Nigra/metabolism/pathology ; alpha-Synuclein/*biosynthesis/*secretion

The characteristic pathological changes of Parkinson s disease (PD) include a severe loss of dopamine neurons in the substantia nigra and a severe decrease in dopamine in the striatum. Since the expression of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) in the biosynthetic pathway for dopamine are low, a promising approach to the gene therapy of PD is to augment the gene expression of the enzymes in the biosynthetic pathway for dopamine. In the present study, human TH and AADC genes were reconstructed into retrovirous vectors pLHCX and pLNCX(2) respectively. Then pLHCX/TH and pLNCX(2)/AADC were transfected into packaging cell line PA317 with liposome. PA317/TH and PA317/AADC were selected by different antibiotics. Gene expression was examined by methods of immunohistochemistry and in situ hybridization. The catalytic activity of two cloned gene enzymes was assessed in vitro by HPLC-EC. Immunocytochemical staining showed that TH and AADC were expressed efficiently in vitro. Both TH and AADC mRNA were transcripted in PA317 cell lines by using in situ hybridazation. HPLC-EC experiments revealed that the transfected cells produced a significantly higher level of dopamine and L-dopa than the untransfected cells. The two genetically modified cells could improve the production of L-dopa and dopamine in response to suitable substrate. The present results suggest that not only recombinant TH and AADC genes are successfully expressed in vitro, but also the enzymes have respective functional activities. These results have set up a way for in vivo gene therapy of PD with TH and AADC genes.
Aromatic-L-Amino-Acid Decarboxylases ; biosynthesis ; genetics ; metabolism ; Cell Line ; Corpus Striatum ; enzymology ; Dopamine ; biosynthesis ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Humans ; Levodopa ; biosynthesis ; Parkinson Disease ; enzymology ; genetics ; RNA, Messenger ; biosynthesis ; Substantia Nigra ; metabolism ; Transfection ; Tyrosine 3-Monooxygenase ; biosynthesis ; genetics ; metabolism

Gonadotropin-releasing hormone (GnRH) and dopamine (DA) can stimulate growth hormone (GH) release, but their effects on GH mRNA synthesis are controversial and deficient in fish. Orange-spotted grouper (Epinephelus coioides) is a hermaphroditic marine fish with sex reversal. Few data are available concerning the regulation of GH in grouper. In the present study, the effects of GnRH and DA on GH release and GH mRNA expression were determined using pituitary fragments of orange-spotted grouper under static culture conditions. After incubation from 1 h to 24 h, salmon GnRH (sGnRH, 100 nmol/L) stimulated the release of GH and increased the level of GH mRNA time-dependently. The minimum duration of sGnRH effect was 1 h. Both of sGnRH and mammalian GnRH (mGnRH) augmented the release of GH and the level of GH mRNA in a dose-dependent manner. The potency of sGnRH on both GH release and GH mRNA level was more pronounced than that of mGnRH. The effects of 1 micromol/L APO (Apomorphine), an agonist of D(1)/ D(2) dopamine receptors, significantly stimulated GH release and GH mRNA synthesis after incubation for 12 h. APO stimulated GH release and GH mRNA abundance in a dose-dependent manner. These results demonstrate that both GnRH and DA directly stimulate GH release and GH mRNA expression at the pituitary level, the actions of GnRH are more potent than that of DA in orange-spotted grouper.
Animals ; Dopamine ; pharmacology ; Gene Expression Regulation ; Gonadotropin-Releasing Hormone ; pharmacology ; Gonadotropins, Pituitary ; metabolism ; Growth Hormone ; biosynthesis ; genetics ; secretion ; Perciformes ; genetics ; metabolism ; Pituitary Gland ; cytology ; metabolism ; Pituitary Hormone-Releasing Hormones ; secretion ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo approach the associated mechanism by which alpha-synuclein (alpha-Syn) might regulate the metabolism of dopamine.

METHODSA DNA fragment, located at -495 to +25 of the human tyrosine hydroxylase (TH) gene, was amplified by PCR and inserted into the pGL(3)-Basic luciferase reporter vector. The recombinant plasmid pGL(3)-THprom was transfected into a dopaminergic cell line MES23.5 or a alpha-Syn over-expressed MES23.5 (named MES23.5/halpha-Syn(+)). The promoter activity was detected by the Dual Luciferase Assay System.

RESULTSThe luciferase activities in the MES23.5 cells transfected with pGL(3)-Basic, pGL(3)-THprom, and pGL(3)-Control vectors were 5.60+/-0.67, 26.80+/-4.11, and 32.90+/-4.75, respectively. On the other hand, the luciferase activity of pGL(3)-THprom in the MES23.5 (26.80+/-4.11) was significantly higher than that in the MES23.5/halpha-Syn(+) (14.40+/-0.61) (P<0.01).

CONCLUSIONThese results indicate that the - 495 to +25 region in the TH gene possesses promoter activity for controlling the gene expression, and that alpha-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.

Animals ; Cell Line, Tumor ; Dopamine ; biosynthesis ; Down-Regulation ; genetics ; Gene Expression Regulation, Enzymologic ; genetics ; Genes, Reporter ; genetics ; Genetic Vectors ; genetics ; Hybridomas ; Luciferases ; genetics ; Mice ; Neurons ; metabolism ; Parkinson Disease ; genetics ; metabolism ; physiopathology ; Promoter Regions, Genetic ; genetics ; Rats ; Regulatory Elements, Transcriptional ; genetics ; Substantia Nigra ; metabolism ; physiopathology ; Transfection ; Tyrosine 3-Monooxygenase ; genetics ; alpha-Synuclein ; genetics

OBJECTIVETo analyze the clinical characteristics of the patient with tyrosine hydroxylase deficiency, and investigate it's molecular mechanism.

METHODThe clinical characteristics of a patient with tyrosine hydroxylase deficiency were summarized and analyzed, his and his family's peripheral blood specimens were collected after informed consent was signed. All exons and the intron-exon boundaries of guanosine triphosphate hydroxylase I gene, tyrosine hydroxylase gene and sepiapterin reductase gene were examined by DNA-PCR, bi-directional sequencing.

RESULTThe patient was a 3-year-old boy, presented with unexplained dystonia for 3 years, without significant impairment of intelligence. Physical examination showed limb muscle strength grade V, rigidity of extremities, hypertonicity, brisk deep tendon reflexes in limbs, without obvious abnormalities in auxiliary examination, such as brain MRI, hepatic biochemical panel, creatine kinase, and ceruloplasmin. He dramatically responded to small doses of levodopa in the follow-up for half a year. A homozygous missense change in exon 5 of TH gene, c.605G > A (p.R202H), which was a known pathogenic mutation, was found in the patient. His parents were heterozygous for the R202H mutation.

CONCLUSIONThe age of onset in tyrosine hydroxylase deficiency patients is usually within the first year of life. Unexplained dystonia and hypokinesia were the main clinical features of tyrosine hydroxylase deficiency. The dopa-responsive effects for some patients are so obvious that we should strengthen awareness of the disease. TH gene c.605G > A (p.R202H) may be a common type of causative mutations for the mild form at home and abroad.

Brain ; metabolism ; pathology ; Catecholamines ; biosynthesis ; Child, Preschool ; DNA ; genetics ; DNA Mutational Analysis ; Dopamine Agents ; administration & dosage ; therapeutic use ; Dystonic Disorders ; drug therapy ; genetics ; metabolism ; Homozygote ; Humans ; Hypokinesia ; drug therapy ; genetics ; metabolism ; Levodopa ; administration & dosage ; therapeutic use ; Male ; Muscle Rigidity ; drug therapy ; genetics ; metabolism ; Mutation, Missense ; Polymerase Chain Reaction ; Tyrosine 3-Monooxygenase ; deficiency ; genetics ; metabolism

OBJECTIVETo investigate the toxicity of copper on MES23.5 dopaminergic cells and the probable mechanisms involved in this process.

METHODSMES23.5 dopaminergic cells were selected as our experimental model. [3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide] (MTT) assay was used to detect the influence of copper on the cell viability. The semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting and the high performance liquid chromatography-electrochemical detection (HPLC-ECD) have been used to detect the tyrosine hydroxlase (TH) mRNA and protein expression and the dopamine content in MES23.5 cells. The flow cytometry have been used to detect the changes of mitochondrial transmembrane potential.

RESULTS100 and 200 mumol/L copper had no effect on the MES23.5 cell viability, whereas 400 and 800 mumol/L of copper could decrease the cell viability (P < 0.01). Treating cells with 200 mumol/L copper for 24 h decreased the TH mRNA expression, the TH expression and the dopamine content compared with the control (P < 0.01, P < 0.01, P < 0.05, respectively). Besides, the mitochondrial transmembrane potential also decreased with the treatment of 200 mumol/L copper for 24 h (P < 0.01).

CONCLUSIONCopper could exert the toxic effects on MES23.5 dopaminergic cells and decrease the cell function. The dysfunction of mitochondria may be the mechanism of this toxicity effect.

Animals ; Cell Survival ; drug effects ; genetics ; Cells, Cultured ; Copper ; metabolism ; toxicity ; Dopamine ; biosynthesis ; Dose-Response Relationship, Drug ; Hybridomas ; Membrane Potential, Mitochondrial ; drug effects ; genetics ; Mice ; Mitochondria ; drug effects ; metabolism ; pathology ; Nerve Degeneration ; chemically induced ; metabolism ; physiopathology ; Neurons ; drug effects ; metabolism ; pathology ; Neurotoxins ; toxicity ; Oxidative Stress ; drug effects ; physiology ; Parkinson Disease ; etiology ; metabolism ; physiopathology ; RNA, Messenger ; drug effects ; metabolism ; Rats ; Tyrosine 3-Monooxygenase ; drug effects ; genetics ; metabolism

OBJECTIVETo evaluate the role of thrombin-activated microglia in the neurodegeneration of nigral dopaminergic neurons in the rat substantia nigra (SN) in vivo.

METHODSAfter stereotaxic thrombin injection into unilateral SN of rats, immunostaining, reverse transcription polymerase chain reaction (RT-PCR) and biochemical methods were used to observe tyrosine hydroxylase (TH) immunoreactive positive cells, microglia activation, nitric oxide (NO) amount and inducible nitric-oxide synthase (iNOS) expression.

RESULTS(1) Selective damage to dopaminergic neurons was produced after thrombin injection, which was evidenced by loss of TH immunostaining in time-dependent manner; (2) Strong microglial activation was observed in the SN; (3) RT-PCR demonstrated the early and transient expression of neurotoxic factors iNOS mRNA in the SN. Immunofluorescence results found that thrombin induced expression of iNOS in microglia. The NO production in the thrombin-injected rats was significantly higher than that of controls (P < 0.05).

CONCLUSIONThrombin intranigral injection can injure the dopaminergic neurons in the SN. Thrombin-induced microglia activation precedes dopaminergic neuron degeneration, which suggest that activation of microglia and release of NO may play important roles in dopaminergic neuronal death in the SN.

Animals ; Disease Progression ; Dopamine ; biosynthesis ; Encephalitis ; chemically induced ; metabolism ; physiopathology ; Female ; Gliosis ; chemically induced ; metabolism ; physiopathology ; Immunohistochemistry ; Inflammation Mediators ; toxicity ; Injections ; Microglia ; drug effects ; metabolism ; Nerve Degeneration ; chemically induced ; metabolism ; physiopathology ; Neurons ; drug effects ; metabolism ; pathology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; physiology ; Parkinsonian Disorders ; chemically induced ; metabolism ; physiopathology ; RNA, Messenger ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Substantia Nigra ; drug effects ; metabolism ; physiopathology ; Thrombin ; toxicity ; Time Factors ; Tyrosine 3-Monooxygenase ; drug effects ; genetics ; metabolism ; Up-Regulation ; drug effects ; physiology