ABSTRACT
OBJECTIVE@#To explore the genetic basis of a patient with early-onset Parkinson disease from a consanguineous family.@*METHODS@#Homozygosity mapping and Sanger sequencing of cDNA were used to identify the causative mutation.@*RESULTS@#A homozygous missense variation (c.56C>G, p.Thr19Arg) in the PARK7 gene was identified in the patient. In silico analysis suggested the c.56C>G variation to be pathogenic.@*CONCLUSION@#Homozygous c.56C>G variation of the PARK7 gene was the disease-causing variation in this family.
Subject(s)
Humans , Consanguinity , Homozygote , Mutation, Missense , Parkinson Disease , Genetics , Pedigree , Protein Deglycase DJ-1 , GeneticsABSTRACT
Parkinson's disease (PD) is a neurodegenerative movement disorder mainly due to degeneration of dopaminergic neurons in the substantia nigra. Most PD cases are sporadic and only 5%-10% of patients carry mutations with inheritance. Among them, the mutation of DJ-1 is related to the autosomal recessive early-onset parkinsonism. DJ-1, the Parkinson's disease-related protein, plays important roles in different physiopathological processes, including oxidative stress, cell translocation and regulation of transcription and translation. DJ-1 is known to be widely expressed in different areas of brain, including hippocampus, amygdala, substantia nigra and cortical areas. Several researchers have tried to demonstrate the clinical and neuroimaging features of DJ-1 related parkinsonism. The DJ-1 knockout mouse model was established to further explore the mechanisms of different functions. Moreover, the search for different forms of DJ-1 as potential biomarkers of PD also provides guidance for its accurate diagnosis and treatment in the future.
Subject(s)
Animals , Mice , Dopaminergic Neurons , Oncogene Proteins , Oxidative Stress , Parkinson Disease , Protein Deglycase DJ-1 , Substantia NigraABSTRACT
To investigate the effect of Parkinson's disease related protein DJ-1 on the cell proliferation, apoptosis, invasion and migration in human osteosarcoma cells and the underlying molecular mechanisms. Methods: The protein expression levels of DJ-1 were detected in human osteosarcoma cell lines (MG-63, Saos-2, and U2OS) and human osteoblast cell line hFOB1.19 with or without deficiency in phosphatase and tensin homolog deleted from chromosome 10 (PTEN) were detected by Western blot. Osteosarcoma cells were treated with DJ-1 siRNA, and then the protein expression levels of DJ-1 were detected by Western blot. Cell survival rate of osteosarcoma cells was detected by cell counting kit-8 (CCK-8) assay. Cell apoptosis of osteosarcoma cells was measured by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Cell invasive and migration ability of osteosarcoma cells were examined by transwell invasion and migration assay. Results: Compared with that of human osteoblast cell line (hFOB1.19), the protein expression level of DJ-1 was significantly upregulated in human osteosarcoma cell lines (MG-63, Saos-2, and U2OS) (all P<0.05), and U2OS had the highest level of DJ-1 when compared with the other three cell lines (P<0.01). DJ-1 siRNA could significantly down-regulate the DJ-1 protein expression in U2OS cells, and also diminish the cell survival rate. Moreover, DJ-1 down-regulation of DJ-1 could promote cell apoptosis, suppress the ability of cell invasion and migration, and increase the PTEN protein expression level (all P<0.05). In addition, the protein expression level of PTEN was markedly up-regulated in human osteosarcoma cell lines when compared with that in the hFOB1.19 cells (P<0.05). Conclusion: DJ-1 can promote the cell proliferation, inhibit cell apoptosis, and decrease the ability of cell invasion and migration, and the potential underlying mechanisms may be associated with the up-regulation of PTEN protein expression.
Subject(s)
Humans , Apoptosis , Genetics , Cell Line, Tumor , Cell Movement , Genetics , Cell Proliferation , Genetics , Gene Expression Regulation, Neoplastic , Neoplasm Invasiveness , Genetics , PTEN Phosphohydrolase , Genetics , Parkinson Disease , Protein Deglycase DJ-1 , Genetics , Metabolism , RNA, Small Interfering , GeneticsABSTRACT
To investigate the effect of silencing DJ-1 on xenografted human laryngeal squamous cell carcinoma (LSCC) Hep-2 cells in nude mice.Xenograft model of human LSCC was established by subcutaneous transplantation of Hep-2 cells in 24 nude mice. The LSCC-bearing nude mice were randomly divided into 3 groups (=8 in each):DJ-1 siRNA low dose group and DJ-1 siRNA high dose group were injected in tumors with 20 μg of DJ-1 siRNA or 40 μg of DJ-1 siRNA in 50 μL, respectively; control group was injected with 5% glucose solution in 50 μL, twice a week for 3 weeks. The weight and size of tumors were measured before injection. The animals were sacrificed 48 h after the final treatment, and the tumors were harvested and weighed. The apoptosis and proliferation of tumor cells were determined; the expressions of Caspase-3 and Ki-67 in tumor specimens were detected with immunohistochemistry. The expression of DJ-1, PTEN, survivin mRNA and protein in tumor tissues were detected by RT-PCR and Western blotting, respectively.Tumor weight in low dose group[(0.66±0.15)g] and high dose group[(0.48±0.11)g] were significantly lower than that in control group[(0.83±0.16)g, all<0.05]. The inhibition rates of low dose group and high dose group were (20.48±0.18)% and (42.16±0.13)%, respectively. Immunohistochemistry showed that the expression of Caspase-3 was increased and Ki-67 was reduced in tumor specimens, compared with the control group (all<0.05). RT-PCR and Western blot results showed that in low dose group and high dose group the mRNA and protein expression of DJ-1 and survivin significantly decreased (all<0.05), while PTEN mRNA and protein content increased (all<0.05).High dose DJ-1 siRNA can inhibit the tumor growth in human LSCC xenograft nude mouse model, which indicates that down-regulating DJ-1 and survivin, and up-regulating PTEN expression may lead to blockage of PI3K-PKB/Akt signaling pathway and promoting tumor cell apoptosis.
Subject(s)
Animals , Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Genetics , Carcinoma, Squamous Cell , Chemistry , Genetics , Caspase 3 , Cell Line, Tumor , Chemistry , Physiology , Transplantation , Cell Proliferation , Down-Regulation , Gene Expression Regulation , Genetics , Physiology , Head and Neck Neoplasms , Chemistry , Genetics , Heterografts , Physiology , Inhibitor of Apoptosis Proteins , Ki-67 Antigen , Laryngeal Neoplasms , Chemistry , Genetics , Mice, Nude , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases , Protein Deglycase DJ-1 , Pharmacology , Proto-Oncogene Proteins c-akt , RNA Interference , Physiology , RNA, Messenger , Pharmacology , RNA, Small Interfering , Physiology , Signal Transduction , Genetics , PhysiologyABSTRACT
OBJECTIVE@#To investigate the effect of the L10P mutation on the cellular mitochondrial disfunction.@*METHODS@#Spectrophotometer, flow cytometry and electron microscope was utilized to examine cell viability, reactive oxygen species (ROS), mitochondrial transmembrane potential, complex I activity and mitochondrial morphous of the HEK293 monoclone cell lines, in which wild-type and L10P mutant DJ-1 protein are stably expressed.@*RESULTS@#Compared with the cell lines expressing empty vector, we found the ROS levels were elevated, the cell viability, mitochondrial transmembrane potential, complex I activity were reduced in the cells expressing L10P mutant DJ-1 protein (P<0.05). We also found mitochondria in these cells were swelling and some mitochondria were vacuolar degeneration. These phenomena were more obvious when rotenone was used. But in the cells expressing wild-type DJ-1, ROS levels were lower, the cell viability, mitochondrial transmembrane potential, and complex I activity were higher than other cell lines (P<0.05), especially under the induction of rotenone. These results suggested that L10P mutant DJ-1 protein probably lost the ability of anti-oxidative stress and affect the normal function of mitochondria.@*CONCLUSION@#The L10P DJ-1 mutation results in a toxic protein, which lacks the protective function of wild-type protein on mitochondria due to the decrease in the ability of anti-oxidative stress.
Subject(s)
Humans , Cell Survival , HEK293 Cells , Intracellular Signaling Peptides and Proteins , Genetics , Membrane Potential, Mitochondrial , Mitochondria , Pathology , Mutation , Oncogene Proteins , Genetics , Oxidative Stress , Protein Deglycase DJ-1 , Reactive Oxygen Species , Metabolism , RotenoneABSTRACT
OBJECTIVE@#RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma SK-MES-1 cells, and the cell biological behaviors were investigated to explore the function of DJ-1 gene.@*METHODS@#A targeted DJ-1 siRNA lentiviral vector with a green fluorescent protein (GFP) as a reporter was constructed. The constructed DJ-1 siRNA and control-siRNA vectors were infected into SK-MES-1 cells as experimental (DJ-1 siRNA) and control (Control siRNA) groups, respectively. The DJ-1 protein expression was determined by Western blot. The cell proliferation capability was measured with methyl thiazolyl tetrazolium (MTT). The cell cycle was analyzed by flow cytometry. The capability of cell migration was determined by Transwell method.@*RESULTS@#Compared with control-siRNA and blank-control groups, the protein expression of DJ-1 gene was down-regulated, the capability of cell proliferation was obviously inhibited (P<0.01), the cell cycle was arrested with increased number of G1- and G2-phase cells and reduced number of S-phase cells, and the capability of cell migration was significantly decreased (P<0.01) in the DJ-1 siRNA-infected cells.@*CONCLUSION@#DJ-1 gene might play a role in promoting cell proliferation and cell migration capability in vitro in lung cancer SK-MES-1 cells.
Subject(s)
Humans , Base Sequence , Carcinoma, Squamous Cell , Genetics , Pathology , Cell Line, Tumor , Cell Movement , Genetics , Cell Proliferation , Genetic Vectors , Genetics , Intracellular Signaling Peptides and Proteins , Genetics , Metabolism , Lentivirus , Genetics , Metabolism , Lung Neoplasms , Genetics , Pathology , Molecular Sequence Data , Oncogene Proteins , Genetics , Metabolism , Protein Deglycase DJ-1 , RNA Interference , RNA, Small Interfering , GeneticsABSTRACT
OBJECTIVE@#To explore whether oncogenes DJ-1 and HSP27 are associated with invasiveness of human pituitary adenoma.@*METHODS@#Total proteins were extracted from samples of 20 invasive and 20 non-invasive pituitary adenomas and the expression of DJ-1 and HSP27 was analyzed by Western blot. The correlation of DJ-1and HSP27 with the invasiveness of pituitary adenoma was analyzed.@*RESULTS@#The strong positive rates of DJ-1 and HSP27 in the 20 invasive pituitary adenoma were 70% (14/20) and 80% (16/20), respectively. The invasive group had significantly higher expression of DJ-1 and HSP27 proteins than the noninvasive group [10% (2/20), 10% (2/20), respectively]. There was a positive correlation between the expression of DJ-1, HSP27 proteins and the invasiveness of pituitary adenoma as judged by the Spearman rank correlation test (P<0.05).@*CONCLUSION@#The proliferative activity and abnormal expression of oncogenes DJ-1 and HSP27 may play a significant role in tumorigenesis and progression of pituitary adenoma. There was a significant correlation between the expression of DJ-1 and HSP27 and the invasiveness of pituitary adenoma.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Young Adult , Adenoma , Metabolism , Pathology , Biomarkers, Tumor , Metabolism , Gene Expression Regulation, Neoplastic , HSP27 Heat-Shock Proteins , Genetics , Metabolism , Intracellular Signaling Peptides and Proteins , Genetics , Metabolism , Neoplasm Invasiveness , Oncogene Proteins , Genetics , Metabolism , Pituitary Neoplasms , Metabolism , Pathology , Protein Deglycase DJ-1 , Signal TransductionABSTRACT
OBJECTIVE@#To establish a technique platform of DJ-1 gene exon rearrangement using real-time PCR and to analyze DJ-1 gene exon rearrangement mutation in patients with autosomal recessive early-onset Parkinsonism(AREP).@*METHODS@#Real-time PCR was used to analyze DJ-1 gene exon rearrangement mutation in 22 probands with AREP from unrelated Chinese Han families and 30 normal controls.@*RESULTS@#We obtained satisfactory real-time PCR reaction conditions and primers of DJ-1 gene coding exons No exon rearrangement mutation in the DJ-1 gene is detected in this group.@*CONCLUSION@#We established platform of DJ-1 gene exon rearrangement using real-time PCR. Exon rearrangement mutation in the DJ-1 gene is rare in Chinese patients with AREP.
Subject(s)
Adolescent , Adult , Child , Child, Preschool , Female , Humans , Male , Young Adult , Age of Onset , China , Ethnology , DNA Mutational Analysis , Exons , Genetics , Gene Rearrangement , Intracellular Signaling Peptides and Proteins , Genetics , Mutation , Oncogene Proteins , Genetics , Parkinsonian Disorders , Genetics , Protein Deglycase DJ-1 , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To detect the expression of DJ-1 in laryngeal squamous cell carcinoma (LSCC) and to study the relationship between DJ-1 expression and clinical indexes of LSCC.</p><p><b>METHODS</b>The expressions of DJ-1 protein in 71 LSCC samples and 9 cases control samples from laryngeal mucosa tissues of non-LSCC patients were detected using streptavidin peroxidase immunohistochemistry staining and the relationships between DJ-1 protein expression and clinicopathologic characteristics were analyzed.</p><p><b>RESULTS</b>(1) The positive expression rate of DJ-1 protein in LSCC was 85.9%(61/71), which was significantly higher than the rate (55.5%, 5/9) in control laryngeal mucosa tissues (P < 0.05). (2) DJ-1 expression was related to tumor recurrence (P < 0.05), but not to sex, age, primary cancer position, T stage, clinical stage, lymph node metastasis and tumor differentiation. Tumor recurrence rate (53.3%) in the patients with higher expression of DJ-1 protein was higher than the rate (26.8%) in the patients with lower expression of DJ-1 protein (χ(2) = 5.164, P < 0.05). (3) With Kaplan-Meier curves and Cox regression analysis, the cumulative 5-year survival rates were correlated with DJ-1 expression levels in laryngeal cancer tissues or cervical lymph node metastasis (all P < 0.05), but not to sex, age, primary cancer position, T stage, clinical stage and tumor differentiation.</p><p><b>CONCLUSIONS</b>The expression of DJ-1 protein in LSCC is higher than that in control laryngeal mucous tissues. Overexpression of DJ-1 is associated with poor overall survival in LSCC patients.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Carcinoma, Squamous Cell , Metabolism , Pathology , Case-Control Studies , Intracellular Signaling Peptides and Proteins , Metabolism , Laryngeal Neoplasms , Metabolism , Pathology , Lymphatic Metastasis , Neoplasm Recurrence, Local , Neoplasm Staging , Oncogene Proteins , Metabolism , Protein Deglycase DJ-1ABSTRACT
<p><b>BACKGROUND AND OBJECTIVE</b>DJ-1, a suppressor of PTEN, promotes metastasis of different tumors, but its function and mechanisms in glioma metastasis remain unclear. This study aimed to investigate the effect of the DJ-1 protein on the migration and invasion of human glioma cells, and to explore potential mechanisms.</p><p><b>METHODS</b>The eukaryotic expression vector pEGFP/DJ-1 and small interfering RNA (siRNA) were constructed and transfected into human glioma SWO-38 cells. The expression of DJ-1 and PTEN in SWO-38 cells were detected by Western blot. Cell migration and invasion were detected by transwell assay.</p><p><b>RESULTS</b>After transfection of pEGFP/DJ-1, the expression of DJ-1 (1.28 ± 0.15 vs. 0.89 ± 0.04, P < 0.05) and focal adhesion kinase (FAK) phosphorylation (0.76 ± 0.12 vs. 0.51 ± 0.04, P < 0.05) were increased, whereas the expression of PTEN (0.74 ± 0.2 vs. 1.04 ± 0.14, P < 0.05) was suppressed. After transfection of DJ-1 siRNA, both DJ-1 (0.33 ± 0.04 vs. 0.88 ± 0.06, P < 0.05) and p-FAK levels (0.33 ± 0.01 vs. 0.44 ± 0.05, P < 0.05) were decreased, but PTEN expression (1.1 ± 0.06 vs. 0.81 ± 0.12, P < 0.05) was increased. Transwell assay data showed that pEGFP/DJ-1 transfection promoted SWO-38 cell migration (57.2 ± 6.50 vs. 40.4 ± 5.0, P < 0.05) and invasion (54.6 ± 4.9 vs. 27 ± 6.7, P < 0.05), whereas DJ-1 siRNA transfection inhibited SWO-38 cells migration (54.4 ± 6.9 vs. 73.4 ± 7.6, < 0.05) and invasion (44.6 ± 5.8 vs. 69.2 ± 9.2, P < 0.05).</p><p><b>CONCLUSION</b>Over-expression of DJ-1 promotes SWO-38 cell migration and invasion possibly through the DJ-1 and the PTEN/FAK pathway.</p>
Subject(s)
Humans , Cell Line, Tumor , Cell Movement , Down-Regulation , Focal Adhesion Protein-Tyrosine Kinases , Metabolism , Genetic Vectors , Glioma , Metabolism , Pathology , Neoplasm Invasiveness , Oncogene Proteins , Genetics , Metabolism , Physiology , PTEN Phosphohydrolase , Genetics , Metabolism , Peroxiredoxins , Phosphorylation , Plasmids , Protein Deglycase DJ-1 , RNA, Small Interfering , Signal Transduction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the expression of DJ-1 in human hepatocellular carcinoma and its relationship with tumor invasion and metastasis.</p><p><b>METHODS</b>Reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detect the expression of DJ-1 mRNA and protein in tumor tissue and para-tumor tissue of 46 cases of hepatocellular carcinoma. The relationship between the expression of DJ-1 and clinicopathologic parameters was analyzed.</p><p><b>RESULTS</b>DJ-1 mRNA and protein were expressed in 69.6% and 58.7% of the tumor tissues, which were significantly higher than those in para-tumor tissues (39.1% and 34.8%), chi2 = 8.587, P < 0.05. The increased expression of DJ-1 protein was not correlated with sex of patients, size of tumor, AFP, HBsAg, differentiation level of tumor and hepatocirrhosis (P > 0.05), but with the capsula of tumor, thrombus in the portal vein (P < 0.05).</p><p><b>CONCLUSIONS</b>DJ-1 gene expression may play an important role in the invasion and metastasis of HCC.</p>
Subject(s)
Humans , Biomarkers, Tumor , Genetics , Metabolism , Carcinoma, Hepatocellular , Genetics , Metabolism , Pathology , Gene Expression Regulation, Neoplastic , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Genetics , Metabolism , Liver Neoplasms , Genetics , Metabolism , Pathology , Neoplasm Invasiveness , Neoplasm Metastasis , Oncogene Proteins , Genetics , Metabolism , Protein Deglycase DJ-1 , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To investigate the frequencies of three polymorphisms in DJ-1 (g.168-185del; SNP405, refSNP ID:rs3766606 and 293 G/A) and their association with sporadic Parkinson's disease.</p><p><b>METHODS</b>An association study was performed to determine the genotype of each subject using polymerase chain reaction, restriction fragment length polymorphism and sequence analysis in 192 patients with sporadic Parkinson's disease and 198 healthy controls.</p><p><b>RESULTS</b>In the g.168-185del locus, the Ins/Ins genotype was common and the frequency of Del allele was very low (0.38%). The SNP of 293G/A was not detected in both groups. In the SNP405 G/T site, the GT genotype frequency was significantly higher in patients with age of onset before 40 years than in controls (18.75% vs 5.54%, P=0.004, OR=6.30 95%CI:1.96-20.18).</p><p><b>CONCLUSION</b>The results suggest that the frequencies of the g.168-185del and 293G/A polymorphisms might be different between Chinese and European. The SNP405 GT genotype might be a risk factor for sporadic Parkinson's disease with early age of onset in Sichuan Han population.</p>
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Age of Onset , Asian People , Genetics , Base Sequence , Case-Control Studies , China , Gene Frequency , Genotype , Intracellular Signaling Peptides and Proteins , Genetics , Oncogene Proteins , Genetics , Parkinson Disease , Genetics , Pathology , Polymorphism, Genetic , Protein Deglycase DJ-1ABSTRACT
<p><b>OBJECTIVE</b>To assess the effect of small interfering RNA (siRNA)-mediated gene silencing of DJ-1 on the proliferation of human laryngeal carcinoma cell line Hep-2.</p><p><b>METHODS</b>Three siRNA sequences specific to DJ-1 gene were synthesized according to GenBank. Human laryngeal carcinoma cell line Hep-2 was cultured and divided into 4 groups: non-specific group (siRNA control) and 3 RNAi groups, transfected with specific DJ-1 siRNA (siRNA1, siRNA2, siRNA3). The mRNA and protein levels of DJ-1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot respectively. Cell apoptosis were analyzed by flow cytometry. The proliferation of Hep-2 cells was assessed by MTT assay.</p><p><b>RESULTS</b>DJ-1 siRNA down-regulated the mRNA and protein levels of DJ-1 in Hep-2 cells. After transfection, the expression of DJ-1 mRNA and protein levels in Hep-2 cells of the DJ-1 siRNA1 group were significantly lower than those of non-specific siRNA control group. MTT assay showed that DJ-1 siRNA1 group inhibited proliferation of Hep-2 cells. Flow cytometry showed that apoptosis rate of the DJ-1 siRNA1 group (15.7%) was significantly higher than that of non-specific siRNA control group (4.5%) or untransfected group (3.5%), t = 4.736, P < 0.01.</p><p><b>CONCLUSIONS</b>Specific siRNA targeting DJ-1 can effectively inhibit DJ-1 expression, resulting in the reduced proliferation and the enhanced apoptosis in Hep-2 cells.</p>
Subject(s)
Humans , Cell Line, Tumor , Cell Proliferation , Intracellular Signaling Peptides and Proteins , Genetics , Laryngeal Neoplasms , Genetics , Pathology , Oncogene Proteins , Genetics , Protein Deglycase DJ-1 , RNA Interference , RNA, Messenger , Genetics , RNA, Small Interfering , GeneticsABSTRACT
Multifunctional sperm protein 22 (SP22) is expressed ubiquitously and related to quite a few diseases. Located on the sperm surface, SP22 has an enzymatic activity that may assist sperm in penetrating into the ovum. SP22 may be carcinogenic in conspiracy with the factor ras. Among all species SP22 is highly conservative, which demonstrates its importance to life. More and more studies indicate that SP22 is directly correlated with male infertility and Parkinsons disease. This article summarizes recent researches on SP22 in the gene structure, protein structure and functional characteristics.
Subject(s)
Animals , Humans , Male , Rats , Gene Components , Microtubule-Associated Proteins , Chemistry , Genetics , Physiology , Protein Conformation , Protein Deglycase DJ-1 , Spermatozoa , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the mutation characteristics of DJ1 gene in Chinese patients with autosomal recessive early-onset Parkinsonism (AR-EP).</p><p><b>METHODS</b>Mutations of DJ1 gene were screened by polymerase chain reaction combined with DNA direct sequencing in index patients with AR-EP from 11 unrelated families.</p><p><b>RESULTS</b>No pathogenetic mutations in the DJ1 gene were detected in this group. Six intronic DJ1 polymorphisms (IVS1-15T-->C, IVS4+30T-->G, IVS4+45G-->A, IVS4+46G-->A, IVS5+31G-->A, g.168-185del) were found. Three of them (IVS1-15T-->C, IVS4+45G-->A, IVS4+46G-->A) were not reported previously.</p><p><b>CONCLUSION</b>DJ1 mutations were rare in Chinese patients with autosomal recessive early-onset Parkinsonism.</p>