应用伴侣分子在大肠杆菌表达系统高效表达家蝇天蚕素

目的应用伴侣分子在大肠杆菌表达系统中高效表达家蝇天蚕素,研究伴侣分子对家蝇天蚕素基因在大肠杆菌中表达的影响。方法RT-PCR克隆家蝇的天蚕素成熟肽(mature cecropin,MC)与泛素(ubiquitin,UBI)基因,利用生物信息学方法分析硫氧还蛋白(thioredoxin,Trx)-MC和MC分子结构及Trx-MC分子和Trx-UBI-MC两种融合蛋白mRNA5'端的二级结构的异同。分别构建pET32a-MC和pET32a-UBI-MC两种重组质粒,转化入大肠杆菌(E.coli)BL21(DE3)中。检测Trx-MC融合蛋白的表达和pET32a-MC和pET32a-UBI-MC在相同诱导时间及诱导剂浓度的条件下,融合蛋白表达量的差异。结果生物信息学分析结果显示MC分子被Trx分子包裹在其内部,MC分子的细胞毒性被去除,使其可以在大肠杆菌中正常表达。分子生物学实验结果进一步验证了生物信息学分析结果的正确性。同时,也显示UBI分子的加入,未影响融合蛋白质Trx-UBI-MC与Trx-MC的mRNA5'端二级结构。并且Trx-UBI-MC融合蛋白在全菌蛋白中的含量明显高于Trx-MC。结论Trx作为伴侣...     

Expression of expanded polyglutamine targets profilin for degradation and alters actin dynamics

Huntington's disease is caused by polyglutamine expansion in the huntingtin protein. Huntingtin directly interacts with profilin, a major actin monomer sequestering protein and a key integrator of signals leading to actin polymerization. We observed a progressive loss of profilin in the cerebral cortex of Huntington's disease patients, and in cell culture and Drosophila models of polyglutamine disease. This loss of profilin is likely due to increased degradation through the ubiquitin proteasome system. Profilin loss reduces the F/G actin ratio, indicating a shift in actin polymerization. Overexpression of profilin abolishes mutant huntingtin toxicity in cells and partially ameliorates the morphological and functional eye phenotype and extends lifespan in a transgenic polyglutamine Drosophila model. These results indicate a link between huntingtin and profilin and implicate profilin in Huntington's disease pathogenesis.     

Ganglioglioma with neurofibrillary tangles (NFTs): neoplastic NFTs share antigenic determinants with NFTs of Alzheimer's disease

A cerebral ganglioglioma contained abundant neurofibrillary tangles (NFTs) of the paired helical filament (PHF) type. The NFTs in the tumor were argyrophilic and Congo red and thioflavin-S positive. Immunohistochemically, the NFTs were reactive with antibodies to phosphorylated neurofilament protein, PHF/tau and abiquitin. The demonstration in the neoplasm of abnormally phosphorylated and ubiquitinated cytoskeletal components, similar in morphology and in immunoreactivity to those seen in NFTs of Alzheimer's disease, suggest that similar pathogenetic mechanisms may operate in both conditions.     

A comprehensive genetic study of the proteasomal subunit S6 ATPase in German Parkinson’s disease patients

Dysfunction of proteasomal protein degradation is involved in neurodegeneration in Parkinson's disease (PD). Recently we identified the regulatory proteasomal subunit S6 ATPase as a novel interactor of synphilin-1, which is a substrate of the ubiquitin-ligase Parkin (PARK2) and an interacting protein of alpha-synuclein (PARK1). To further investigate a potential role in the pathogenesis of PD, we performed a detailed mutation analysis of the S6 ATPase gene in a large sample of 486 German sporadic and familial PD patients. Direct sequencing revealed two novel intronic variants. An insertion/deletion variant in intron 5 of the S6 ATPase gene was more frequent in patients compared to controls. Moreover, this variant was significantly more frequent in early-onset compared to late-onset PD patients. The identification of a genetic link between a regulatory proteasomal subunit and PD further underscores the relevance of disturbed protein degradation in PD.     

White matter lesions in the brain with frontotemporal lobar degeneration with motor neuron disease: TDP-43-immunopositive inclusions co-localize with p62, but not ubiquitin

Recently, TDP-43 was established as a major component of the ubiquitinated inclusions found in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with motor neuron disease (FTLD-MND). However, differences in the underlying pathogenesis between ALS and FTLD-MND remain yet to be elucidated. Originally, TDP-43-immunopositive inclusions were found in neuronal cells and reported to be ubiquitinated. This study shows that TDP-43-positive inclusions were distributed throughout the subcortical white matter except for the occipital lobe in the FTLD-MND brain, but not in the ALS brain. TDP-43-positive inclusions were also prominent features of pathologically proven FTLD-MND cases (p-FTLD-MND) without history of apparent clinical cognitive decline. A substantial fraction of these inclusions was also p62-immunoreactive, and another noteworthy feature was that those inclusions did not stain positively for ubiquitin. Significant correlations between immunoreactivity for TDP-43 and p62 were observed, particularly in p-FTLD-MND (Pearson correlation coefficient, 0.976). Furthermore, TDP-43 extracted from white matter appeared to be uncleaved. These results indicate that pathological changes might take place within the white matter also in the brain with FTLD-MND, but in a different manner than within the gray matter.     

Frontotemporal lobar degeneration with ubiquitin-positive, but TDP-43-negative inclusions

Frontotemporal lobar degeneration (FTLD) can be pathologically subdivided into tau-positive and tau-negative types. The most common tau-negative variant is FTLD with ubiquitin-immunoreactive lesions (FTLD-U). Recently, the TAR DNA binding protein 43 (TDP-43) was identified in neuronal inclusions in FTLD-U. After applying TDP-43 immunohistochemistry to a series of 44 cases of FTLD-U with no secondary pathology, three cases (7%) were identified with ubiquitin- and p62-positive neuronal cytoplasmic inclusions (NCI) that were negative for TDP-43. All the three cases had marked brain atrophy with striking atrophy of the striatum. Cases 1 and 2 presented at ages 43 and 38, respectively, as behavioral variant frontotemporal dementia (1 with positive family history) and had ubiquitin- and p62-positive NCI in frontotemporal neocortex and dentate granule cells of the hippocampus. Case 3 presented with the corticobasal syndrome. Unlike the other two cases, ubiquitin- and p62-positive NCI were also visible on hematoxylin and eosin stain. There were no neuronal intranuclear inclusions. Electron microscopic examination of the NCI in cases 2 and 3 revealed granulofilamentous inclusions. These cases confirm the existence of TDP-43-negative FTLD-U and extend the clinical and pathological spectrum of this disorder. The findings raise the possibly of an as yet identified protein that may play a pathogenic role in tau-negative FTLD.     

Sporadic amyotrophic lateral sclerosis: two pathological patterns shown by analysis of distribution of TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions

A nuclear protein, 43-kDa TAR DNA-binding protein (TDP-43), was recently identified as a component of the ubiquitinated inclusions (UIs) in frontotemporal lobar degeneration (FTLD-U) and sporadic amyotrophic lateral sclerosis (SALS). In the present study using immunohistochemistry, we examined various regions of the nervous system in a series of 35 SALS cases using a polyclonal antibody against TDP-43. Seven of the 35 cases had disease durations of more than 10 years with artificial respiratory support (ARS; duration: 69-156 months). In all cases, TDP-43-immunoreactive (ir) neuronal and glial cytoplasmic inclusions (NCIs and GCIs) were found together in many regions, including the histologically affected lower motor neuron nuclei. Cluster analysis of the distribution pattern of TDP-43-ir NCIs for cases without ARS (n = 28) identified two types (type 1, n = 16; type 2, n = 12). Type 2 was distinguished from type 1 by the presence of TDP-43-ir NCIs in the frontotemporal cortex, hippocampal formation, neostriatum and substantia nigra, and was significantly associated with dementia. Eleven of the 28 cases showed UIs in the hippocampal dentate granule cells, all of which had type-2 distribution pattern. Cases with ARS (n = 7) were also classified into the same types (type 1, n = 5; type 2, n = 2). Cases having type-1 distribution pattern (n = 21) showed no evident neuronal loss in most of the non-motor neuron nuclei where TDP-43-ir NCIs were present, whereas cases having type-2 distribution pattern (n = 14) often showed evident neuronal loss in the frontotemporal cortices, amygdaloid nuclei and substantia nigra. These findings indicate that SALS is a multisystem degenerative disease widely affecting both neurons and glial cells with a heterogeneous pattern of TDP-43-ir NCI distribution (SALS showing type-2 distribution pattern being closely linked to FTLD-U), and that long-term survival supported by a respirator has no apparent influence on the TDP-43 neuronal distribution pattern.     

NSAIDs enhance proteasomic degradation of survivin, a mechanism of gastric epithelial cell injury and apoptosis

NSAIDs cause severe gastrointestinal injury, in part by suppressing survivin, an inhibitor of apoptosis protein, both in cultured gastric epithelial cells and in human and rat gastric mucosa. The mechanism(s) of survivin down-regulation by NSAIDs is unclear. In this study, we examined whether NSAID treatment decreases survivin mRNA expression and/or enhances degradation of survivin protein via ubiquitin proteasome system in rat gastric mucosal, RGM-1 cells, and whether survivin overexpression prevents indomethacin-induced cell injury and apoptosis. Effects of indomethacin on survivin mRNA expression, survivin protein half-life and ubiquitination were examined in RGM-1 cells. Proteasome inhibitors were utilized to prevent indomethacin-induced survivin protein degradation in RGM-1 cells. The effects of stable overexpression of survivin on indomethacin-induced RGM-1 cell injury and apoptosis were examined. Results showed: (1) Indomethacin treatment did not alter survivin mRNA expression, but significantly reduced survivin protein half-life from 1.5h to approximately 1h and increased survivin ubiquitination. (2) Inhibition of ubiquitin proteasome prolonged survivin protein half-life to over 2h and inhibited indomethacin-induced survivin degradation. (3) Overexpression of survivin significantly reduced indomethacin-induced cell injury and apoptosis. In conclusion, indomethacin treatment enhances degradation of survivin via the ubiquitin proteasome machinery in RGM-1 cells, and maintenance of survivin levels is important for prevention of gastric epithelial cell injury and apoptosis.     

Ubiquitin-immunoreactive filamentous inclusions in anterior horn cells of Guamanian and non-Guamanian amyotrophic lateral sclerosis

Summary Immunohistochemical studies with an antibody to ubiquitin revealed the presence of filamentous inclusions in spinal anterior horn cells in all of six patients with Guamanian amyotrophic lateral sclerosis (ALS) and one of six cases of parkinsonism-dementia complex (PD) on Guam. Similar ubiquitin-reactive filamentous inclusions were found in all of seven non-Guamanian sporadic ALS patients examined. No similar inclusions were seen in six normal controls or in non-ALS patients who had chromatolytic neurons. The filamentous inclusions differed from spinal neurofibrillary tangles, a characteristic feature of Guamanian ALS and PD, since they were restricted to anterior horn cells and did not react with anti-tau antibody. The chromatolytic neurons of non-ALS patients occasionally had weak diffuse immunoreactivity, but no focal inclusions were detected. These results suggest that ubiquitin-reactive focal filamentous inclusions may reflect a characteristic degenerative process of anterior horn cells of motor neuron disease.     

Ubiquitin-positive achromatic neurons in corticobasal degeneration

A 66-year-old woman presented with an alien limb syndrome without dementia. The course of her illness was unremitting and at autopsy 6 years later her diagnosis was confirmed as corticobasal degeneration without Alzheimer-type pathology. Although the presence of ballooned achromatic cortical neurons and cell loss from the substantia nigra distinguishes such patients, the site and density of achromatic neurons has not previously been quantified. We show that immunohistochemistry for the cell stress protein ubiquitin selectively stains these achromatic neurons, whereas they do not stain for abnormally phosphorylated tau protein. Phosphorylated neurofilament antibodies recognise both ballooned and non-ballooned neurons. In this case, high densities of ubiquitin-positive ballooned neurons were found in frontal cortical regions with the highest densities in layers V and VI of the anterior cingulate cortex. In addition, high densities of ubiquitin-positive ballooned neurons were found in the insular cortex, claustrum and amygdala. These results confirm past reports of frontal pathology, but show that there is also considerable pathology in insular and parahippocampal cortical regions and some subcortical regions. Our findings suggest that the distribution and staining characteristics of ballooned neurons in corticobasal degeneration may help to differentiate these cases pathologically, while the absence of dementia appears to be an important clinical criterion.