RESUMO
Polyglutamine (polyQ) diseases are a group of hereditary neurodegenerative disorders caused by expansion of a glutamine repeat in responsible gene products. To date, the pathogenesis of polyQ diseases is still not very clear, but many researches suggest that phosphorylation of mutant proteins plays a critical role on the process of Huntington's disease, dentatorubral-pallidoluysian atrophy, spinal bulbar muscular atrophy, spinocerebellar ataxia1 and spinocerebellar ataxia 3/Machado-Joseph disease.
Assuntos
Humanos , Transtornos Heredodegenerativos do Sistema Nervoso , Genética , Metabolismo , Doença de Huntington , Genética , Metabolismo , Doença de Machado-Joseph , Genética , Metabolismo , Atrofia Muscular Espinal , Peptídeos , Genética , Metabolismo , Fosforilação , Fisiologia , Degenerações Espinocerebelares , Genética , Metabolismo , Expansão das Repetições de Trinucleotídeos , Genética , Fisiologia , Repetições de Trinucleotídeos , GenéticaRESUMO
OBJECTIVE@#To explore the relationship between the sequence variation of the promoter region (-1543 approximately -1160) of STK11 gene and the risk of developing Peutz-Jeghers syndrome (PJS).@*METHODS@#The sequences of the promoter region of 14 PJS patients (7 patients are inherited and the other 7 patients are sporadic) and 42 normal individuals were PCR amplified and then sequenced.@*RESULTS@#A new single nucleotide polymorphism (SNP) G/T (-1275) in STK11 promoter region was identified. The frequency of genotype GG, GT, and TT was 53.3%, 26.7%, and 20%, respectively among PJS patients and 33.3%, 64.3%, and 2.4%, respectively among the normal individuals. The frequency of genotype GG and TT among patients was significantly higher than that among the normal individuals, and the frequency of genotype GT among patients was significantly lower than that among the normal individuals (chi(2)=8.521, P<0.05).@*CONCLUSION@#G/T(-1275) in STK11 promoter region is a new SNP. The genotype of this new SNP may relate to the risk of developing Peutz-Jeghers syndrome (PJS) deserve further research.
Assuntos
Humanos , Sequência de Bases , Frequência do Gene , Genótipo , Dados de Sequência Molecular , Síndrome de Peutz-Jeghers , Genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Genética , Proteínas Serina-Treonina Quinases , GenéticaRESUMO
OBJECTIVE@#To screen for proteins interacting with ataxin-3 by yeast two-hybrid system 3, and to discuss the function of ataxin-3 and pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).@*METHODS@#First we sub-cloned the full reading frame of both wild-type and mutant ataxin-3 into carrier pGBKT7 (ataxin-3-bait), and then screened human brain cDNA library with ataxin-3-bait.@*RESULTS@#We found five positive clones in 6.5 x 10(6) transformers. After sequencing, we knew all of them were novel ataxin-3 interacting proteins. Three were corresponded to the known sequences coding the known proteins, which were human Rho GDP dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2. Another two of the five were unknown.@*CONCLUSION@#Small ubiquitin-like modifier 1 probably interacted with ataxin-3, suggesting that the sumoylation probably participated in post-translation modifying of ataxin-3 and pathogenesis of SCA3/MJD.
Assuntos
Humanos , Ataxina-3 , Encéfalo , Metabolismo , Biblioteca Gênica , Proteínas do Tecido Nervoso , Genética , Metabolismo , Proteínas Nucleares , Genética , Metabolismo , Mapeamento de Interação de Proteínas , Proteínas Repressoras , Genética , Metabolismo , Degenerações Espinocerebelares , Genética , Metabolismo , Técnicas do Sistema de Duplo-Híbrido , Leveduras , GenéticaRESUMO
<p><b>OBJECTIVE</b>To investigate over-expression of wild-type alpha-synuclein inducing the aberrant aggregation of alpha-synuclein in HEK293 cell in vitro.</p><p><b>METHODS</b>The cDNA encoding the human alpha-synuclein without the stop code was cloned into PGEM T-easy vector. Using enzyme map and DNA sequencing analyzed and determined the recombinant plasmid, and then sub-clone the alpha-synuclein cDNA fragment into pEGFP-N1 vector. The recombinant plasmids alpha-synuclein-pEGFP were transfected into HEK293 cells by lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence, anti-alpha-synuclein immunocytochemistry. The inclusions in the cultured cells were identified with HE staining.</p><p><b>RESULTS</b>The restriction enzyme map suggested that eukaryotic expression vector for human wild-type alpha-synuclein gene was constructed successfully. By EGFP fluorescence, anti-alpha-synuclein immunocytochemistry, it could be observed that the alpha-synuclein protein could aggregate in cytoplasm and the Lewy body-like inclusions found in cytoplasm of cultured cells.</p><p><b>CONCLUSION</b>The over-expression of wild-type alpha-synuclein can induce protein aberrant aggregation and Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro.</p>
Assuntos
Humanos , Células Cultivadas , Expressão Gênica , Imuno-Histoquímica , Corpos de Inclusão , Metabolismo , Corpos de Lewy , Metabolismo , Doença de Parkinson , Genética , Metabolismo , alfa-Sinucleína , Genética , MetabolismoRESUMO
OBJECTIVE@#To determine the frequency of different subtypes of spinocerebellar ataxias (SCAs) in the Han nationality of Hunan province in China.@*METHODS@#The mutations of SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, and dentatorulral-pallidoluysian (DRPLA) were detected with the polymerase chain reaction (PCR), denaturing polyacrylamide gel and DNA sequencing techniques in 139 autosomal dominant SCA families and 61 sporadic SCA patients.@*RESULTS@#Of the 139 families, 11 (7.9%) were positive for SCA1, 9(6.5%) were positive for SCA2, 71 (51.1%) were positive for SCA3, 4 (2.9%) were positive for SCA6, 2 (1.4%) were positive for SCA7, and none was positive for SCA17 and DRPLA. There was 1 SCA2 patient, 3 SCA3 patients, 1 SCA6 patient in the 61 sporadic SCA patients.@*CONCLUSION@#The frequency of SCA3 is substantially higher than that of SCA1 and SCA2 in the autosomal dominant SCA patients in the Han nationality of Hunan province. SCA6 and SCA7 are rare subtypes.
Assuntos
Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ataxina-1 , Ataxina-3 , Ataxina-7 , Ataxinas , China , Etnologia , Análise Mutacional de DNA , Proteínas do Tecido Nervoso , Genética , Proteínas Nucleares , Genética , Proteínas Repressoras , Genética , Ataxias Espinocerebelares , Classificação , Diagnóstico , Genética , Repetições de Trinucleotídeos , GenéticaRESUMO
<p><b>OBJECTIVE</b>This study sought to isolate and identify the proteins that interact with ataxin-3, to confirm the interacted domain, and to provide new clues for exploring the function of ataxin-3 and the pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).</p><p><b>METHODS</b>Yeast two-hybrid screen (MATCHMAKER GAL4 Two-Hybrid System 3) and regular molecular biologic techniques were undertaken to screen human brain cDNA library with mutant ataxin-3 bait. Two baits from both normal and mutant C-terminus of ataxin-3 were created by subcloned methods to determine which domain of ataxin-3 interacts with the putative associated proteins and to find out optimal candidate proteins that interact with C-terminus of ataxin-3. Confocal microscope was used to observe whether ataxin-3 co-localized with the obtained interacting proteins in mammalian cells.</p><p><b>RESULTS</b>Five novel ataxin-3 interacting proteins were obtained, among which were three known proteins, namely human rhodopsin guanosine diphosphate dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2; the other two were unknown. Interacting domain analysis revealed that an unknown protein interacted with the C-terminus near the polyglutamine tract of ataxin-3, the other four all interacted with the N-terminus. In the nucleus of SH-SY5Y cell, small ubiquitin-like modifier 1 co-localized with the wild-type ataxin-3 and with the intranuclear aggregates formed by the mutant ataxin-3.</p><p><b>CONCLUSION</b>An unknown protein probably interacting with C-terminus of ataxin-3 is firstly discovered, and the initiative findings suggest first that the interaction of small ubiquitin-like modifier 1 with N-terminus of ataxin-3 and the relevant sumoylation probably participate in the post-translation modifying of ataxin-3 and in the pathogenesis of SCA3/MJD.</p>
Assuntos
Humanos , Canais Iônicos Sensíveis a Ácido , Ataxina-3 , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde , Genética , Metabolismo , Microscopia Confocal , Mutação , Proteínas do Tecido Nervoso , Genética , Metabolismo , Proteínas Nucleares , Genética , Metabolismo , Plasmídeos , Genética , Ligação Proteica , Proteínas Recombinantes de Fusão , Genética , Metabolismo , Proteínas Repressoras , Genética , Metabolismo , Proteína SUMO-1 , Genética , Metabolismo , Canais de Sódio , Genética , Metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
<p><b>OBJECTIVE</b>To assess the frequency of spinocerebellar ataxia (SCA), including the subtypes of SCA1, SCA2, SCA3/Machado-Joseph disease(MJD), SCA6, SCA7, SCA8, SCA10, SCA12, SCA14, SCA17 and dentatorubro-pallidoluysian atrophy (DRPLA) in Han population in the Chinese mainland, and to specifically characterize the mainland Chinese patients with SCA6 in terms of clinical and molecular features.</p><p><b>METHODS</b>Using a molecular approach, the authors investigated SCA in 120 families with dominantly inherited ataxias and in 60 patients with sporadic ataxias. Clinical and molecular features of SCA6 were further characterized in 13 patients from 4 families.</p><p><b>RESULTS</b>SCA3/MJD was the most common type of autosomal dominant SCA in the Han population, accounting for 83 patients from 59 families(49.2%), followed by SCA2(8, 6.7%), SCA1(7, 5.8%), SCA6(4, 3.3%), SCA7(1,0.8%), SCA8 (0), SCA10 (0), SCA12(0), SCA14 (0), SCA17(0) and DRPLA(0). The genes responsible for 41(34.2%) of dominantly inherited SCA families remained undetermined. Among the 60 patients with sporadic ataxias in the present series, 3(5.0%) were found to harbor SCA3 mutations while none were found to harbor SCA6 mutations. In the 4 families with SCA6, significant anticipation was found with no genetic instability on transmission.</p><p><b>CONCLUSION</b>The present authors firstly found and reported a geographic cluster of families with SCA6 subtype in the Chinese mainland, which were initially identified in Hans reported of the Chinese mainland.</p>
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Povo Asiático , Genética , China , Saúde da Família , Frequência do Gene , Genes Dominantes , Reação em Cadeia da Polimerase , Ataxias Espinocerebelares , Diagnóstico , Etnologia , Genética , Repetições de Trinucleotídeos , GenéticaRESUMO
OBJECTIVE@#To construct the eukaryotic expression vector of MJD1 with normal copies of CAG trinucleotide repetition and MJD1 with CAG trinucleotide repetition expansion mutation respectively, and to determine whether the polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.@*METHODS@#The coding sequence of wild-type MJD1 and mutant MJD1 was amplified by PCR from pAS2-1-MJD20Q and pAS2-1-MJD68Q respectively. After being digested with BamH I and Hind III, the PCR products were inserted into pcDNA3. 1-Myc-His(-) B. The recombinant plasmids pcDNA3.1-Myc-His(-) B-MJD20Q and pcDNA3.1-Myc-His(-) B-MJD68Q were identified by enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into SH-SYSY cells and the expression of MJD1 in the transfected cells was analyzed by Western blot. The immunofluorescence of the transfected cells was examined using a confocal microscope to observe the formation of intranuclear aggregation.@*RESULTS@#Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into pcDNA3. 1-Myc-His(-) B. The expression of MJD1 in the transfected cells was confirmed by Western blot; The SH-SY5Y cells transfected with pcDNA3. 1-Myc-His(-) B-MJD68Q showed the formation of intranuclear aggregation, but the cells transfected with pcDNA3.1-Myc-His(-) B-MJD20Q did not show such phenomenon.@*CONCLUSION@#The eukaryotic expression vectors of MJD1 has been successfully constructed; The polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.
Assuntos
Humanos , Ataxina-1 , Ataxina-3 , Ataxinas , Sequência de Bases , Células Eucarióticas , Metabolismo , Vetores Genéticos , Complexo Mediador , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Genética , Neuroblastoma , Metabolismo , Patologia , Proteínas Nucleares , Genética , Plasmídeos , Genética , Receptores dos Hormônios Tireóideos , Genética , Proteínas Recombinantes , Genética , Proteínas Repressoras , Genética , Transfecção , Células Tumorais CultivadasRESUMO
<p><b>BACKGROUND</b>Dominantly inherited spinocerebellar ataxia (SCA) is a clinically and genetically heterogeneous group of neurodegenerative disorders. This study was to further assess the frequency of SCA1 (spinocerebellar ataxia type 1), SCA2, SCA3/MJD (spinocerebellar ataxia type 3/Machado-Joseph disease), SCA6, SCA7, SCA8, SCA10, SCA12, SCA14, SCA17 and DRPLA (dentatorubro-pallidoluysian atrophy) in mainland Chinese, and to specifically characterize mainland Chinese patients with SCA6 in terms of clinical and molecular features.</p><p><b>METHODS</b>Using a molecular approach, we investigated SCA in 120 mainland Chinese families with dominantly inherited ataxias and in 60 mainland Chinese patients with sporadic ataxias. Clinical and molecular features of SCA6 were further characterized in 13 patients from 4 families.</p><p><b>RESULTS</b>SCA3/MJD was the most common type of autosomal dominant SCA in mainland Chinese, accounting for 83 patients from 59 families (49.2%), followed by SCA2 [8 (6.7%)], SCA1 [7 (5.8%)], SCA6 [4 (3.3%)], SCA7 [1 (0.8%)], SCA8 (0%), SCA10 (0%), SCA12 (0%), SCA14 (0%), SCA17 (0%) and DRPLA (0%). The genes responsible for 41 (34.2%) of dominantly inherited SCA families remain to be determined. Among the 60 patients with sporadic ataxias in the present series, 3 (5.0%) was found to harbor SCA3 mutations while none was found to harbor SCA6 mutations. In the 4 families with SCA6, significant anticipation was found in the absence of genetic instability on transmission.</p><p><b>CONCLUSION</b>A geographic cluster of families with SCA6 subtype was initially identified in a mainland Chinese population.</p>
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Povo Asiático , Canais de Cálcio , Genética , Frequência do Gene , Genes Dominantes , Ataxias Espinocerebelares , Genética , Repetições de TrinucleotídeosRESUMO
<p><b>OBJECTIVE</b>To report a Chinese Charcot-Marie-Tooth disease type 2 (CMT2) family.</p><p><b>METHODS</b>All the members in the family were studied clinically,and 6 patients were studied electrophysiologically. Sural nerve biopsy was performed in the proband. PMP22 gene duplications were detected by highly polymorphic short tandem repeat. Point mutation analysis of PMP22, MPZ and NEFL gene was screened by PCR-SSCP combined with DNA direct sequencing. A genome-wide screening was carried out to the family.</p><p><b>RESULT</b>Except 2 who had weakness and atrophy in both proximal and distal muscles of the lower limbs, all patients presented muscle wasting and a predominating weakness of distal parts of the lower limbs, and mild to moderate sensory impairments. In 6 patients who were subjected to elctrophysiological examinations, median-nerve conduction velocity (NCV) of the median nerve was normal. Electromyograms (EMGs) revealed signs of denervation with large motor unit potentials, fibrillation potentials and positive sharp waves. Sural nerve biopsy of the proband confirmed the presence of axonal neuropathy with an important loss of large myelinating fibers and a large number of clusters with mostly thinly myelinated axons. PMP22, MPZ and NEFL gene mutations were not found. The results of genome-wide screening revealed a linkage of CMT2 to a locus at chromosome 12q24.</p><p><b>CONCLUSION</b>The results are consistent with the diagnosis of CMT2. This family represents a rare genetic type of CMT2 which can be designated as CMT2L.</p>
Assuntos
Adolescente , Adulto , Feminino , Humanos , Masculino , Povo Asiático , Doença de Charcot-Marie-Tooth , Genética , Patologia , Cromossomos Humanos Par 12 , Genética , Eletromiografia , LinhagemRESUMO
Objective To study the PINK1 aggresome formation and it's features in response to proteasomal inhibition.Methods Full-length PINK1 cDNA were amplified by polymerase chain reaction (PCR)from fetus brain cDNA library and subcloned into the EcoR I and BamH I sites of the vector pEGFP- N1.The integrity of the constructs was confirmed by sequencing.COS-7 cells were transiently transfected with PINK1-pEGFP-N1 using Lipofectamine 2000.Cells were treated by MG-132 in order to test the effect of proteasome inhibition on aggregation formation.The protein level of wild-type PINK1 with or without MG-132 treatment was confirmed by Western blot analysis.The formation of PINK1 aggregates was tested by fluorescence and the presence of ubiquitin,and ?-synuclein in PINK1 aggregates was examined by immunofluorescence and confocal microscopy.Results The expression level of PINK1 was significant increased into the form of aggregate in cells treated with MG-132;immunostaining for endogenous ubiquitin and ?-synuclein revealed a co-localization of both proteins in PINK1-positive aggregates.Conclusions In the presence of MG-132,overexpressed PINK1 forms into aggregates,whose components are ubiquitin and ?-synuclein.