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1.
Neuroscience Bulletin ; (6): 143-155, 2018.
Article in English | WPRIM | ID: wpr-777067

ABSTRACT

Most attempts at rational development of new analgesics have failed, in part because chronic pain involves multiple processes that remain poorly understood. To improve translational success, one strategy is to select novel targets for which there is proof of clinical relevance, either genetically through heritable traits, or pharmacologically. Such an approach by definition yields targets with high clinical validity. The biology of these targets can be elucidated in animal models before returning to the patients with a refined therapeutic. For optimal treatment, having biomarkers of drug action available is also a plus. Here we describe a case study in rational drug design: the use of controlled inhibition of peripheral tetrahydrobiopterin (BH4) synthesis to reduce abnormal chronic pain states without altering nociceptive-protective pain. Initially identified in a population of patients with low back pain, the association between BH4 production and chronic pain has been confirmed in more than 12 independent cohorts, through a common haplotype (present in 25% of Caucasians) of the rate-limiting enzyme for BH4 synthesis, GTP cyclohydrolase 1 (GCH1). Genetic tools in mice have demonstrated that both injured sensory neurons and activated macrophages engage increased BH4 synthesis to cause chronic pain. GCH1 is an obligate enzyme for de novo BH4 production. Therefore, inhibiting GCH1 activity eliminates all BH4 production, affecting the synthesis of multiple neurotransmitters and signaling molecules and interfering with physiological function. In contrast, targeting the last enzyme of the BH4 synthesis pathway, sepiapterin reductase (SPR), allows reduction of pathological BH4 production without completely blocking physiological BH4 synthesis. Systemic SPR inhibition in mice has not revealed any safety concerns to date, and available genetic and pharmacologic data suggest similar responses in humans. Finally, because it is present in vivo only when SPR is inhibited, sepiapterin serves as a reliable biomarker of target engagement, allowing potential quantification of drug efficacy. The emerging development of therapeutics that target BH4 synthesis to treat chronic pain illustrates the power of combining human and mouse genetics: human genetic studies for clinical selection of relevant targets, coupled with causality studies in mice, allowing the rational engineering of new analgesics.


Subject(s)
Analgesics , Therapeutic Uses , Animals , Biopterin , Metabolism , Chronic Pain , Drug Therapy , Genetics , Disease Models, Animal , Drug Discovery , GTP Cyclohydrolase , Genetics , Metabolism , Humans , Rodentia , Signal Transduction , Genetics
2.
Article in Chinese | WPRIM | ID: wpr-335155

ABSTRACT

<p><b>OBJECTIVE</b>To explore genetic mutations and clinical features of a pedigree affected with dopa-responsive dystonia.</p><p><b>METHODS</b>PCR and Sanger sequencing were applied to detect mutations of the GCH1 gene among 7 members from the pedigree.</p><p><b>RESULTS</b>The family was detected to have a known heterozygous mutation of the GCH1 gene (c.550C>T). For the 7 members from the pedigree, the age of onset has ranged from 13 to 60 years. The mother of the proband has carried the same mutation but was still healthy at 80. The symptoms of the other three patients were in slow progression, with diurnal fluctuation which can be improved with sleeping, dystonias of lower limbs, and tremor of both hands. Treatment with small dose of levodopa has resulted in significant improvement of clinical symptoms. By database analysis, the c.550C>T mutation was predicted as probably pathological.</p><p><b>CONCLUSION</b>The c.550C>T mutation probably underlies the disease in this pedigree. The clinical phenotypes of family members may be variable for their ages of onset. Some may even be symptom free.</p>


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Base Sequence , DNA Mutational Analysis , Dystonic Disorders , Genetics , Female , GTP Cyclohydrolase , Genetics , Heterozygote , Humans , Male , Middle Aged , Molecular Sequence Data , Mutation , Pedigree , Phenotype , Young Adult
3.
Article in Chinese | WPRIM | ID: wpr-291761

ABSTRACT

<p><b>OBJECTIVE</b>To identify potential mutation of the GCH1 gene in a Chinese family affected with dopa-responsive dystonia.</p><p><b>METHODS</b>Genomic DNA of patients was extracted from peripheral blood samples. The 6 exons of the GCH1 gene and at least 100 bp of flanking intronic sequences were amplified with PCR. Potential mutations were screened by direct sequencing. Identified mutation was verified with denaturing high performance liquid chromatography (DHPLC) in 100 healthy controls.</p><p><b>RESULTS</b>All patients were found to be heterozygous for a novel c.597delT (p.Ala200LeufsX5) deletion in the exon 5 of the GCH1 gene. The deletion of T has resulted in formation of a shorter (203 amino acids) truncated non-functional guanosine triphosphate cyclohydrolase I. The same mutation was not found in the 100 controls.</p><p><b>CONCLUSION</b>A novel GCH1 gene frameshifing mutation probably underlies the dopa-responsive dystonia in this Chinese family.</p>


Subject(s)
Adolescent , Adult , Base Sequence , Child , Dystonic Disorders , Genetics , Exons , Female , Frameshift Mutation , GTP Cyclohydrolase , Genetics , Humans , Male , Middle Aged , Molecular Sequence Data , Pedigree , Young Adult
4.
Article in English | WPRIM | ID: wpr-29139

ABSTRACT

Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia and a dramatic response to relatively low doses of levodopa. However, patients with DRD can be misdiagnosed as cerebral palsy or spastic diplegia due to phenotypic variation. Here we report a young woman with DRD who were severely disabled and misdiagnosed as cerebral palsy for over 10 yr. A small dose of levodopa restored wheelchair-bound state to normality. However, thoracolumbar scoliosis has remained as a sequel due to late detection of DRD. Genetic analysis by using PCR-direct sequencing revealed a novel initiation codon mutation (c.1A>T; p.Met1Leu) in GTP cyclohydrolase 1 (GCH1) gene. Although it is known that DRD can be misdiagnosed as cerebral palsy, this case reinforces the importance of differential diagnosis of DRD from cerebral palsy.


Subject(s)
Adult , Cerebral Palsy/diagnosis , Codon, Initiator , Diagnosis, Differential , Dystonic Disorders/diagnosis , Female , GTP Cyclohydrolase/genetics , Humans , Levodopa/therapeutic use , Mutation , Sequence Analysis, DNA
5.
Article in English | WPRIM | ID: wpr-349759

ABSTRACT

Tetrahydrobiopterin (BH4) is an essential cofactor for all three nitric oxide synthase (NOS isoforms), which plays an important role in vascular diseases. GTP cyclohydrolase 1 (GCH 1) is the first-step and rate-limiting enzyme for BH4 biosynthesis in its de novo pathway. Common GCH1 gene variant C+243T in the 3'-untranslated region predicts NO excretion. The present study examined the predictive role of GCH 1 gene 3'-UTR C+243T variant in the long-term outcome of ischemic stroke. A total of 142 patients with first-onset ischemic stroke were recruited and detected for genotype of GCH1 3'-UTR C+243T by a TaqMan SNP Genotyping assay. Subsequent vascular events and death were determined over a 5-year follow-up period. The frequency of GCH1 3'-UTR +243 C/T or T/T genotype was significantly increased in patients with endpoint events as compared with those without events (74% vs 57.8%, P=0.06). Cox regression survival analysis indicated that an increased probability of death or new vascular events was found in patients with GCH1 3'-UTR +243 C/T or T/T genotype compared with those with GCH1 3'-UTR C/C genotype (40.6% vs 25.5%), GCH1 3'-UTR +243 C/T or T/T genotype relative to GCH1 3'-UTR C/C genotype was associated with the increased risk of death or vascular events even after adjustment for other risk factors (OR=2.171, 95% CI: 1.066-4.424, P=0.033). It was concluded that GCH1 3'-UTR C+243T variant was an independent predictor of worsening long-term outcomes in patients with first-onset ischemic stroke.


Subject(s)
3' Untranslated Regions , Genetics , Aged , Brain Ischemia , Genetics , Female , GTP Cyclohydrolase , Genetics , Humans , Male , Middle Aged , Nitric Oxide , Metabolism , Prognosis , Risk Factors , Stroke , Diagnosis , Genetics
6.
Article in English | WPRIM | ID: wpr-205430

ABSTRACT

Recently it was shown that single nucleotide polymorphisms (SNPs) can explain individual variation because of the small changes of the gene expression level and that the 50% decreased expression of an allele might even lead to predisposition to cancer. In this study, we found that a decreased expression of an allele might cause predisposition to genetic disease. Dopa responsive dystonia (DRD) is a dominant disease caused by mutations in GCH1 gene. The sequence analysis of the GCH1 in a patient with typical DRD symptoms revealed two novel missense mutations instead of a single dominant mutation. Family members with either of the mutations did not have any symptoms of DRD. The expression level of a R198W mutant allele decreased to about 50%, suggesting that modestly decreased expression caused by an SNP should lead to predisposition of a genetic disease in susceptible individuals.


Subject(s)
Child , Clubfoot/genetics , Dopamine/deficiency , Dystonic Disorders/drug therapy , GTP Cyclohydrolase/genetics , Genes, Recessive , Genetic Predisposition to Disease , Humans , Levodopa/administration & dosage , Male , Mutation, Missense , Pedigree , Polymorphism, Genetic
7.
Arq. neuropsiquiatr ; 65(4b): 1224-1227, dez. 2007. ilus, tab
Article in English | LILACS | ID: lil-477776

ABSTRACT

Dopa-responsive dystonia (DRD) is an inherited metabolic disorder now classified as DYT5 with two different biochemical defects: autosomal dominant GTP cyclohydrolase 1 (GCH1) deficiency or autosomal recessive tyrosine hydroxylase deficiency. We report the case of a 10-years-old girl with progressive generalized dystonia and gait disorder who presented dramatic response to levodopa. The phenylalanine to tyrosine ratio was significantly higher after phenylalanine loading test. This condition had two different heterozygous mutations in the GCH1 gene: the previously reported P23L mutation and a new Q182E mutation. The characteristics of the DRD and the molecular genetic findings are discussed.


Distonia dopa-responsiva (DRD), classificada como DYT5, é um erro inato do metabolismo que pode ser causado por dois diferentes tipos de defeito bioquímico: deficiência de GTP ciclo-hidrolase 1 (GCH1) (autossômica dominante) ou de tirosina hidroxilase (autossômica recessiva). Descrevemos o caso de menina de 10 anos com distonia generalizada progressiva e alteração da marcha com importante melhora após uso de levodopa. A relação fenilalanina/tirosina estava aumentada após teste de sobrecarga com fenilalanina. O estudo molecular mostrou que o paciente apresenta uma combinação hererozigótica de mutação no gene GCH1: a já conhecida mutação P23L e uma nova mutação Q182E. Discutem-se as características da DRD e as alterações genéticas possíveis.


Subject(s)
Child , Female , Humans , Dopamine Agents/therapeutic use , Dystonia/drug therapy , Dystonia/genetics , GTP Cyclohydrolase/genetics , Levodopa/therapeutic use , Mutation, Missense/genetics , Dystonia/blood , Heterozygote , Phenylalanine/blood , Tyrosine/blood
8.
Article in Chinese | WPRIM | ID: wpr-247350

ABSTRACT

<p><b>OBJECTIVE</b>To study the incidence of various enzyme deficiency in tetrahydrobiopterin (BH4) metabolism and the related gene mutation among the patients with motor disturbance and mental retardation.</p><p><b>METHODS</b>One hundred patients with unknown motor disturbance and mental retardation were referred to this study. All patients were performed by phenylalanine (Phe) and BH4 loading test, urinary pterin analysis and dihydropteridine reductase (DHPR) activity. Some patients received the dopa treatment for diagnosis of dopa-responsive dystonia (DRD). The analysis of GTP cyclohydrolase 1 gene (GCH1) mutation for DRD patients and the analysis of 6-pyruvoyl tetrahydropterin synthase (PTS) gene mutations for PTS deficient patients were done under the consent from their parents.</p><p><b>RESULTS</b>Seventy of 100 patients had normal basic blood Phe levels, six (6%) patients were diagnosed as DRD. Thirty patients had hyperphenylalaninemia (HPA), eight (8%) were diagnosed as PTS deficiency and 22(22%) were diagnosed as phenylalanine hydroxylase (PAH) deficiency. All patients had normal DHPR activity. The mutation IVS5+3insT of GCH1 was found in 2 patients with DRD. Seven kinds of PTS mutations were found in 8 patients with PTS deficiency, and 75% of the mutations were 259C-->T,286G-->A and 155A-->G.</p><p><b>CONCLUSION</b>Some patients with unknown motor disturbance and mental retardation may suffer from BH4 metabolism related diseases. Theses patients are necessary to be screened for such kind of diseases in order to confirm the diagnosis.</p>


Subject(s)
Adolescent , Biopterin , Metabolism , Child , Child, Preschool , Dihydropteridine Reductase , Genetics , Metabolism , Dystonia , Genetics , Metabolism , Female , GTP Cyclohydrolase , Genetics , Metabolism , Humans , Infant , Intellectual Disability , Genetics , Metabolism , Male , Mutation , Phenylalanine Hydroxylase , Genetics , Metabolism , Phosphorus-Oxygen Lyases , Genetics , Metabolism
9.
Article in Chinese | WPRIM | ID: wpr-247330

ABSTRACT

<p><b>OBJECTIVE</b>To detect mutations of guanosine triphosphate cyclohydrolase I (GCH1) gene in Chinese patients with dopa responsive dystonia (DRD).</p><p><b>METHODS</b>Six sporadic patients with DRD were examined. GCH1 gene mutations were detected using polymerase chain reaction (PCR), DNA sequence analysis and restriction enzyme digestion analysis. One hundred normal people were detected using PCR and restriction enzyme digestion analysis.</p><p><b>RESULTS</b>A new point mutation, 151(G-->A) in exon one was found in a patient. It lead to substitution of a methionine for isoleucine at amino acid 1(M1I). This mutation was not found in normal control people.</p><p><b>CONCLUSION</b>The authors report a new heterozygotic point mutation 151(G-->A) in GCH1 gene. There are GCH1 gene mutations in Chinese sporadic patients with DRD.</p>


Subject(s)
Asians , Genetics , Case-Control Studies , DNA , Genetics , DNA Mutational Analysis , Dihydroxyphenylalanine , Therapeutic Uses , Dystonia , Drug Therapy , Genetics , Exons , Genetics , Female , GTP Cyclohydrolase , Genetics , Humans , Male , Point Mutation , Genetics , Polymerase Chain Reaction
10.
Chinese Journal of Pediatrics ; (12): 492-495, 2006.
Article in Chinese | WPRIM | ID: wpr-278673

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the clinical characteristics and GCH I gene mutations in patients with dopa-responsive dystonia (DRD).</p><p><b>METHODS</b>The clinical features of 3 families with 6 affected members and 8 sporadic cases were analyzed to determine the clinical characteristics, and 2 families with 4 affected members and 2 sporadic cases were screened for mutations of the GCH I gene.</p><p><b>RESULTS</b>Age at onset was (10 +/- 3) years. Onset occurred earlier in female (9 +/- 4) years than in male (12 +/- 1) years. The initial symptom was a gait disorder, dystonia or tremor in most patients and nine patients (64%) presented with diurnal fluctuation. Thirteen patients (93%) were cured and one was improved after administration of low doses of levodopa for 3 months and no long-term side effects of levodopa had occurred. Two independent mutations were found in three patients. Gln161Pro, a new missense mutation, was found in a sporadic case, leading to a relatively severe phenotype. The two patients with mild phenotype in one family were found to have Lys224Arg mutation, as previously described.</p><p><b>CONCLUSIONS</b>DRD patients have diverse phenotypes and diurnal fluctuation is an important feature. They have dramatic and sustained response to levodopa. There may be a correlation between genotype and phenotype. The detection of GCH I mutations is helpful in early diagnosis of non-typical cases.</p>


Subject(s)
Age of Onset , Child , China , DNA Mutational Analysis , Dopamine Agents , Therapeutic Uses , Dystonia , Diagnosis , Drug Therapy , Genetics , Early Diagnosis , Female , GTP Cyclohydrolase , Genetics , Genotype , Humans , Levodopa , Therapeutic Uses , Male , Molecular Sequence Data , Mutation , Mutation, Missense , Pedigree , Phenotype , Polymerase Chain Reaction , Sex Factors , Treatment Outcome
11.
Chinese Journal of Surgery ; (12): 1127-1131, 2005.
Article in Chinese | WPRIM | ID: wpr-306173

ABSTRACT

<p><b>OBJECTIVE</b>To observe the influence of treatment with the inhibitor of extracellular-signal regulated protein kinase (ERK) signal transduction pathway on the expression of biopterin/nitric oxide (NO) as well as the activation of nuclear factor-kappaB (NF-kappaB), and to clarify the potential cross-talk regulation mechanisms between ERK and NF-kappaB pathway in biopterin-mediated NO induction in rats with endotoxic shock.</p><p><b>METHODS</b>Using an endotoxic shock model, 60 male Wistar rats were randomly divided into normal controls (n = 8), endotoxic shock group (n = 32) and PD98059 treatment group (n = 20). At serial time points animals in each group were sacrificed, and tissue samples from liver, lungs as well as kidneys were harvested to detect NF-kappaB activity, guanosine triphosphate-cyclohydrolase (GTP-CHI) and inducible nitric oxide synthase (iNOS) mRNA expression. Biopterin and NO levels in plasma and tissues were also assayed.</p><p><b>RESULTS</b>It was found that after lipopolysaccharide (LPS) challenge, GTP-CHI mRNA expression and biopterin levels significantly elevated in liver, lungs and kidneys, keeping at high values up to 24 h, so did the values of iNOS mRNA expression and NO levels. NF-kappaB DNA binding activity was enhanced rapidly in various tissues, peaking at 2 h after LPS challenge. Treatment with PD98059, an inhibitor of ERK signal transduction pathway, could significantly inhibit GTP-CHI mRNA expression in kidneys, and GTP-CHI mRNA expression in liver and lungs showed certain down-regulation tendency. At the same time, biopterin level was significantly decreased in plasma, liver and kidneys at 12 h. Similarly, iNOS/NO induction at early stage markedly decreased in various tissues. In addition, treatment with PD98059 reduced NF-kappaB DNA binding activity in liver, lungs, as well as kidneys at 2-6 h, 2 h, 24 h and 24 h after LPS challenge, respectively.</p><p><b>CONCLUSIONS</b>Inhibition of ERK pathway could partially inhibit the production of biopterin/NO as well as the activation of NF-kappaB pathway, which indicated that cross-talk regulation seems to be existed between ERK and NF-kappaB pathway, and they might be involved in the regulatory process of biopterin-mediated nitric oxide induction in rats with endotoxic shock.</p>


Subject(s)
Animals , Biopterin , Metabolism , Physiology , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases , Physiology , GTP Cyclohydrolase , Genetics , Male , NF-kappa B , Metabolism , Nitric Oxide , Nitric Oxide Synthase Type II , Genetics , Random Allocation , Rats , Rats, Wistar , Shock, Septic , Signal Transduction
12.
Article in Korean | WPRIM | ID: wpr-207296

ABSTRACT

Segawa disease, hereditary progressive dystonia with marked diurnal fluctuations or defined dopa-responsive dystonia has age-dependent clinical courses, which are characterized with marked progression in the first one and half decades, its subsiding in the third decade and almost stationary courses after the fourth decade. Also, it has characteristic diurnally fluctuating symptoms, aggravated towards the evening and alleviated after sleep. This autosomally dominantly inherited dystonia is caused by abnormalities of the gene of GTP cyclohydrolase I. The heterozygotic gene's abnormality induces partial decrement of tetrahydrobiopterin and affects synthesis of tyrosine hydroxylase(TH) rather selectively. The reduction of TH induces decrement of dopamine and disfacilitates the D1 receptor-striatal direct pathway. The pathognomonic finding in biochemical examination is the decrease of neopterin in the cerebrospinal fluid(CSF). Levodopa, by replacing dopamine contents at the terminal, alleviates motor symptoms completely and the effects sustain without any side effects. We experienced a girl diagnosed as Segawa disease with typical clinical courses and a decrease of neopterin in the CSF.


Subject(s)
Dopamine , Dystonia , Female , Genetic Diseases, Inborn , GTP Cyclohydrolase , Humans , Levodopa , Neopterin , Tyrosine
13.
Chinese Journal of Burns ; (6): 84-87, 2002.
Article in Chinese | WPRIM | ID: wpr-289183

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the protective effect and its underlying mechanism of 2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP-cyclohydrolase I (GTP-CHI), on postburn Staphylococcus aureus (S. aureus) sepsis in rats.</p><p><b>METHODS</b>Fifty-six Wistar rats were randomly divided into four groups, i.e. normal control, scalding control, postburn sepsis group and DAHP treatment group. Tissue samples from liver, kidneys, lungs and heart were aseptically taken, and in which the GTP-CHI and inducible nitric oxide synthase (iNOS) contents and the mRNA expression of tumor necrosis factor-alpha (TNFalpha) were determined. Furthermore, biopterin (BH(4)) and nitric oxide (NO) levels in these tissue were also measured.</p><p><b>RESULTS</b>After the scalding injury followed by bacterial challenge, the GTP-CHI gene expression and biopterin levels were significantly increased in all tissue sampled, and so were iNOS mRNA expression and NO (P < 0.01), especially in liver and lungs. The expressions of GTP-CHI mRNA and iNOS mRNA and the production of BH(4) and NO in all tissue were evidently inhibited by the pretreatment with DAHP (P < 0.05 approximately 0.01). At the same time, the TNFalpha expression was also obviously decreased. In addition, The mortality at 6 hr in rats of DAHP treatment group was decreased.</p><p><b>CONCLUSION</b>The prognosis of the scalding rats complicated by sepsis caused by G(+) bacteria could be improved by DAHP pretreatment, which might be related to the inhibition of the production of BH(4) and NO by DAHP.</p>


Subject(s)
Animals , Biopterin , Metabolism , Burns , Genetics , Metabolism , Enzyme Inhibitors , Pharmacology , GTP Cyclohydrolase , Genetics , Gene Expression Regulation , Heart , Hypoxanthines , Pharmacology , Kidney , Metabolism , Liver , Metabolism , Lung , Metabolism , Male , Myocardium , Metabolism , Nitric Oxide , Metabolism , Nitric Oxide Synthase , Genetics , RNA, Messenger , Genetics , Metabolism , Rats , Rats, Wistar , Sepsis , Staphylococcal Infections , Staphylococcus aureus , Sugar Acids , Time Factors , Tumor Necrosis Factor-alpha , Genetics
14.
Article in Korean | WPRIM | ID: wpr-192401

ABSTRACT

BACKGROUND: Various vectors have been developed and tried for the delivery of tyrosine hydroxylase (TH) in order to supplement dopamine, which is severely deficient in Parkinson's disease, however, none of the protocols tried have yielded fruitful results that can be applied directly to humans. One of the problems revealed from previous trials was a short duration of expression of the delivered gene, that is, tyrosine hydroxylase. METHODS: To extend the stability and to improve the enzymatic characteristics of the protein, part of the regulatory domain was deleted via PCR technique. The cDNA for regulatory domain-deleted THs (dTH) were sub-cloned into a retroviral vector and the resulting recom-binant retrovirus was used to infect NIH-3T3. After selection, expression levels of TH were determined by Western blot analysis and the enzymatic characteristics were examined. RESULTS: The deletion increased steady state expression level of TH protein by 7-fold for d19TH (TH with amino acids #2-19 are deleted) and 3-fold for d31TH (TH with amino acids #2-31 are deleted. The elevated expression level of d19TH is likely due to the enhanced stability of the protein as determined by a treatment of cycloheximide. The activity of d19TH was also increased approximately by 3-fold but no increase of the L-dopa production was observed. However, the production of L-dopa was dramatically increased when GTP cyclohydrolase I (GTPCH I) was co-transfected suggesting that the activity of d19TH is dependent on the presence of cofactor. d19TH seem to be free of feedback inhibition at low concentration of dopamine (10 nM~1 nM) but more sensitive to the inhibition at high concentration of dopamine (10 mM). CONCLUSIONS: The deletion of 18 amino acids on the regulatory domain increases the stability of the protein, reduces the activity, and frees it from the feedback inhibi-tion by the end product.


Subject(s)
Amino Acids , Blotting, Western , Cycloheximide , DNA, Complementary , Dopamine , Fruit , GTP Cyclohydrolase , Humans , Levodopa , Parkinson Disease , Polymerase Chain Reaction , Retroviridae , Staphylococcal Protein A , Tyrosine 3-Monooxygenase , Tyrosine , Zidovudine
15.
Article in Korean | WPRIM | ID: wpr-191038

ABSTRACT

BACKGROUND: Parkinson's Disease (PD) is a progressive neurodegenerative disorder characterized by resting tremor, rigidity, and bradykinesia. L-3,4-dihydroxyphenylalanine (L-dopa) has been used for over last 3 decades to treat this disorder, however, its usage is limited due to the reducing effectiveness on time and severe side effects. The best strategy for treating this disorder without serious side effects would be to keep a constant level of dopamine in the brain. This could be achieved by gene or cell therapy using gene(S) involved in dopamine biosynthesis or cells from other individual. For Parkinson's gene therapy, however, there still are controversies on which gene In what combination will yield the best result. In this report, we propose a biochemical background for using GTP cyc]ohydrolase I (GTPCH I) in addition to TH for higher and/or more stable expression of TH. METHODS: TH and GTPCH I cDNA were subcloned into retroviral vectos and resulting recombinant retrovirus packaged in BOSC 23 cells were used to infect NIH-3T3. Confirming successful infections by westers blot analysls, the new cell lines were used to examine steady state TH expression level and TH activity. Furthermore, the effect of ectopic expression of BH4 to the proliferation of these cells were studied. RESULTS: NIH-3T3 cells expressing both TH and GTPCH I showed approximately 10 fold higher expression of TH protein than the cells expressing TH alone. The activity of KNTH2GC6 was approximately 4-6 fold higher than that of striatal tissue and 60 fold higher than KNTH2. Furthermore, growth rate of KNTH2GC6 was strikingly reduced by inhibiting the biosynthesis of BH4. CONCLUSIONS: We showed that the use of GTPCH I in addition to TH not only increased the stability and/or expression of TH protein but also the activity of the enzyme. These improved characteristics of TH protein are very likely due to the expression of BH4 and should be very seriously considered for Parkinson's gene therapy.


Subject(s)
Brain , Cell Line , Cell- and Tissue-Based Therapy , DNA, Complementary , Dopamine , Genetic Therapy , GTP Cyclohydrolase , Guanosine Triphosphate , Hypokinesia , Levodopa , Neurodegenerative Diseases , NIH 3T3 Cells , Parkinson Disease , Retroviridae , Tremor , Tyrosine 3-Monooxygenase , Tyrosine , Zidovudine
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