Ubiquitin immunoreactivity of paired helical filaments differs in Alzheimer’s disease and corticobasal degeneration

To establish whether there is a relationship between ubiquitination and ultrastructural appearance of filaments, we compared the ubiquitin immunoreactivity of paired helical filaments (PHFs) in Alzheimer’s disease (AD) and corticobasal degeneration (CBD). PHFs in these disorders share a limited similarity since filaments in CBD are wider and twisted at longer intervals than those in AD, and also display less ultrastructural stability. Preparations enriched in SDS-soluble filaments were isolated from AD and CBD brains and subjected to tau and ubiquitin immunogold labeling. Both preparations contained mostly dispersed individual PHFs, which labeled for the amino and carboxyl termini of tau. Immunolabeling of ubiquitin was variable, however, being more intense in AD than CBD samples. SDS-insoluble filaments were prepared from PHFs by boiling in the presence of SDS and 2-mercaptoethanol and collected by sedimentation. In both disorders, the pellets contained highly aggregated and bundled filaments, which were devoid of the amino but not the carboxyl terminal region of tau. Again, ubiquitin labeling was more intense in AD than CBD filaments. The present results suggest that ubiquitination has limited influence on SDS solubility, aggregation and bundling of PHFs; however, it may be one of the factors responsible for the ultrastructural variability and/or stability of filaments. Received: 6 March 1998 / Revised, accepted: 14 May 1998     

Using simultaneous, tandem gene replacements to study expression of the multicopy ubiquitin-fusion (FUS) gene family of Trypanosoma cruzi

Many genes in trypanosomes exist as members of multicopy gene families. Due to this fact it is frequently difficult to determine if specific members of a gene family are expressed. We describe here a strategy for simultaneous tandem gene replacement in T. cruzi which leads to the replacement of the gene of interest by a silent reporter gene, the expression of which can be assayed in stable transformants. To determine if the FUSI gene (one of 5 copies of the ubiquitin-fusion, FUS, gene family) was expressed, stable G418-resistant transformants were isolated in which the tandemly arrayed CUB2.65 and FUSI genes were precisely replaced by the neomycin phosphotransferase (neo^r) and chloramphenicol acetyltransferase (CAT) genes, respectively. All stable clones carrying the tandem gene replacements were shown to express the CAT activity indicating that FUSI is expressed in mid-log epimastigotes. Northern blot analysis of parasites carrying the tandem gene replacements indicated that at least one other member of the FUS gene family is expressed and that there were no apparent polar effects on the expression of genes downstream of the replacement events. These experiments have demonstrated the utility of tandem gene replacements as a means of inserting a nonselected reporter gene into the chromosome, facilitating the molecular genetic analysis of the expression of multicopy gene families.     

大鼠膀胱泛素样分子的分离纯化及其N-端氨基酸序列分析

作者用醋酸冲洗膀胱粘膜,所得冲洗液经反相高压液相层析纯化出泛素样分子,N-端部分氨基酸序列测定显示其N-端25个氨基酸序列与人泛素一致。结合粘膜上皮抗菌多肽分子的发现,作者由此提出粘膜上皮也可能是一类免疫细胞的新见解。     

Lewy bodies are ubiquitinated

Summary The nature of Lewy bodies (LBs) in the brain stem and cerebral cortex in five cases of diffuse Lewy body disease and one case of Parkinson's disease with dementia were investigated immunocytochemically with various antibodies to cytoskeletal proteins, paired helical filaments (PHF) and ubiquitin. Antibodies to 200-kDa component of neurofilament, tau and PHF showed no significant reactions with most of LBs. Antibodies to high-molecular weight microtubule-associated proteins (HMWMAPs) moderately stained the periphery of a few of LBs. A monoclonal antibody to PHF (DF2) which recognizes ubiquitin, and polyclonal antibodies to ubiquitin immunostained virtually all of the typical and cortical LBs as intensely as Alzheimer's neurofibrillary tangles and senile plaque neurites: the periphery of LBs was darkly stained, whereas the central core of typical LBs and central zone of cortical LBs were less intensely stained or remained unstained. Immunoelectron microscopy of the LBs with DF2 revealed that immune reaction products were located on the filaments exclusively in the periphery of LBs, but not on those in the center. These findings suggest that both types of LBs are immunocytochemically indistinguishable despite some structural differences, and that peripherally located filaments in LBs are tagged with ubiquitin, an element required for the ATP-dependent proteolysis system in the cell. Antibodies to ubiquitin are the most useful marker of LBs ever known.Supported in part by grants-in-aid from the Ministry of Education, Science and Culture, Japan     

Argentophilic intracytoplasmic inclusions in multiple system atrophy

Summary Argentophilic intracytoplasmic glial inclusions were recently reported in olivo-ponto-cerebellar atrophy (OPCA). We examined the brains of 3 cases of OPCA [2 with striato-nigral degeneration (SND) and 1 without SND], 1 case of pure autonomic failure (PAF) without pathology of OPCA or SND, as well as 36 controls including 2 cases of Holmes' type cerebellar cortical atrophy and 2 cases of Joseph's disease. Although the inclusions were tubulin-positive, the immunoreactivity was different from that of the dendrites. Electron microscopically, the microtubular structures composing the inclusion were fuzzy with granular material. These findings may indicate that the microtubules composing the inclusions are modified. Inclusion-bearing cells appeared to be oligodendrocytes while many of them had larger and lighter nuclei than those of normal-looking oligodendrocytes without the inclusions. The inclusions were widely distributed in a characteristic fashion beyond the typical lesions of OPCA, SND and PAF. The distribution pattern was essentially the same in the case of PAF and 3 cases of OPCA irrespective of the presence or absence of OPCA or SND lesions. In contrast, argentophilic inclusions were not observed in other types of spinocerebellar degeneration, in Holmes' type cerebellar cortical atrophy or in Joseph's disease. It is suggested, in line with other studies, that the inclusion may be specific to OPCA and related disorders which include PAF and a useful marker to distinguish OPCA from other neurodegenerative diseases.     

Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines

Ubiquitin specific proteases (USPs) regulate the production and recycling of ubiquitin and are thereby critically involved in the control of cell growth, differentiation, and apoptosis. Increasing evidence implicates deregulation of USPs in malignant transformation but there is very little information on the overall and specific activity of USPs in normal and tumor tissues. We have used a chemistry-based functional proteomics approach to profile the activities of individual USPs in biopsies of human papillomavirus (HPV) carrying cervical carcinoma and adjacent normal tissue. To assess the contribution of HPV proteins, USP activity was also compared in HPV positive and negative cervical carcinoma cell lines and HPV E6/E7 immortalized human keratinocytes. The activity of the C-terminal hydrolases UCH-L3 and UCH37 was upregulated in the majority of tumor tissues compared to the adjacent normal tissues. UCH-L1 activity was lower in a significant proportion of the tumors but to a less extent in advanced tumors. In accordance with the relatively low UCH-L1 activity in tumor biopsies, UCH-L1 was detected only in one out of eight cervical carcinoma lines. UCH-L1, UCH-L3, USP7, and USP9X activity was upregulated following HPV E6/E7 immortalization of keratinocytes, suggesting a role of these enzymes in growth transformation.     

Neonatal Fc receptor stimulation induces ubiquitin c-terminal hydrolase-1 overexpression in podocytes through activation of p38 mitogen-activated protein kinase

Ubiquitin c-terminal hydrolase-1 is overexpressed in renal podocytes in some immune complex-mediated glomerulonephritides, an effect closely related to extensive podocyte injury. Neonatal Fc receptor is newly recognized to be present on human renal podocytes. It is presumed that neonatal Fc receptor serves as a sensor for immune stimulation transduction and is involved in the pathogenesis of podocyte injury. In our current study, we found that neonatal Fc receptor was constitutively expressed in normal podocytes and up-regulated by immune stimulation induced by antithymocyte serum. An increase in neonatal Fc receptor expression was observed in human podocytes within diseased glomeruli in 97 cases of various glomerulonephritides. The expression percentage was significantly higher in immune-mediated disease, including membranous nephropathy (46.7%), immunoglobin A nephropathy (66.7%), lupus nephritis (87.5%), and acute proliferative glomerulonephritis (100%), than in normal kidney samples (16.7%) (P < .05), whereas there was no significant difference between minimal-change disease and normal kidney. Further study showed that neonatal Fc receptor up-regulated the expression of ubiquitin c-terminal hydrolase-1 via activation of p38 in podocytes subjected to immune stimulation in vitro. These data suggest that neonatal Fc receptor acts as an immune sensor that evokes an inflammatory response, which may lead to functional and morphological changes in podocytes in glomerulonephritides.     

胆道肿瘤组织中ubiquitin的表达及临床意义

目的研究人胆道系统肿瘤中泛素（ubiquitin）蛋白的表达与胆道系统肿瘤不同病理分型的关系及其临床意义。方法采用免疫组织化学法结合蛋白印迹技术检测人胆道肿瘤组织、正常胆道组织及炎性组织中泛素蛋白的表达,分析人胆道肿瘤组织中泛素蛋白的表达情况与不同临床病理情况之间的相关性。结果人胆道肿瘤组织中泛素蛋白的表达显著高于正常组织及炎性组织中泛素蛋白的表达（P〈0．05）。泛素蛋白的表达与病理组织学分级有明显相关性（P〈0．05）。结论泛素蛋白在人胆道肿瘤组织中表达水平升高,可能与胆道系统肿瘤的恶性程度、疾病的进程有密切的关系。     

Role of cytochrome P450 2E1 in protein nitration and ubiquitin-mediated degradation during acetaminophen toxicity

It is well established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400 mg/kg) for 4 and 24 h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.