Katsumi Kito

Accumulation of NEDD8 in neuronal and glial inclusions of neurodegenerative disorders

NEDD8 (neural precursor cell expressed, developmentally down‐regulated 8) is a ubiquitin‐like protein that controls vital biological events through its conjugation to members of the cullin family, which are components of certain ubiquitin E3  ligases.  Recent  studies  have  shown  that  NEDD8 is incorporated into Lewy bodies (LBs) in Parkinsons disease, Mallory bodies in alcoholic liver disease and Rosenthal fibres in astrocytoma. In order to examine whether NEDD8 plays a role in the formation of ubiquitinated inclusions, we performed immunohistochemical staining of brain tissue from patients with various neurodegenerative disorders, using an affinity‐purified polyclonal antibody raised against NEDD8 that did not cross‐react with ubiquitin. In LB disease, NEDD8 immunoreactivity was present in almost all of the LBs and Lewy neurites. Moreover, NEDD8 immunoreactivity was found in a variety of ubiquitinated inclusions, including neuronal and oligodendroglial inclusions in multiple system atrophy, neurofibrillary tangles in Alzheimers disease, ubiquitinated inclusions in motor neurone disease, and intranuclear inclusions in triplet repeat diseases. These findings suggest that NEDD8 is involved in the formation of various ubiquitinated inclusions via the ubiquitin‐proteasome system.

NEDD8 protein is involved in ubiquitinated inclusion bodies.

Proteolysis by the ubiquitin–proteasome system is considered to play a pathological role in several degenerative diseases that involve ubiquitinated inclusion bodies. In recent years, several ubiquitin‐like proteins have been isolated, but it is uncertain whether their roles are associated with protein degradation through the ubiquitin–proteasome system. NEDD8 (neural precursor cell‐expressed and developmentally down‐regulated gene), which consists of 81 amino acid residues, possesses the highest sequence similarity to ubiquitin. Recent studies have indicated that NEDD8 is covalently ligated to cullin family proteins, which are components of certain ubiquitin E3 ligases, by a pathway analogous to that of ubiquitin. Thus, by focusing on the structural and functional association between NEDD8 and ubiquitin, it would be of interest to know whether the NEDD8 system is involved in pathological disorders of the ubiquitin–proteasome system. This study has examined the immunohistochemical distribution of NEDD8 protein by using a highly purified antibody in normal tissues and in tissues known to contain ubiquitinated inclusions. NEDD8 protein expression was widely observed in most types of tissues. Furthermore, accumulation of the NEDD8 protein was commonly observed in ubiquitinated inclusion bodies, including Lewy bodies in Parkinsons disease, Mallory bodies in alcoholic liver disease, and Rosenthal fibres in astrocytoma. Two of ten cases of neurofibrillary tangles and senile plaques from patients with Alzheimers disease showed intense staining for NEDD8 as well as for ubiquitin. These findings suggest the possibility that the NEDD8 system is involved in the metabolism of these inclusion bodies via the ubiquitin–proteasome system. Copyright

Incidence of p53 and Ha‐ras gene mutations in chemically induced rat mammary carcinomas

To determine whether p53 alterations, which are frequent in human breast cancers, are also common in rat mammary tumors, we examined 40 tumors from 24 rats treated with 7,12‐dimethylbenz[a]anthracene (DMBA) and 34 tumors from 14 rats treated with N‐nitroso‐N‐methylurea (NMU) (an N‐nitroso compound). DMBA and NMU are known genotoxic mutagens. The entire coding regions of the p53 and Ha‐ras genes were examined for mutations by polymerase chain reaction single‐strand conformational polymorphism analysis and by direct sequencing. One of the 40 DMBA‐induced mammary tumors had a p53 mutation, a single‐base substitution (???AGC→???GGC) at codon 307, resulting in an amino‐acid change from Ser to Gly. No mutations were found in NMU‐induced tumors. The incidence of Ha‐ras gene mutation was 79% (27 of 34) at codon 12 in the NMU group and 23% (nine of 40) at codon 61 in the DMBA group. Thus, p53 mutation, in contrast to Ha‐ras mutation, did not seem to be a prerequisite for carcinogenesis in chemically induced rat mammary tumors.

Novel orthotopic implantation model of human malignant pleural mesothelioma (EHMES-10 cells) highly expressing vascular endothelial growth factor and its receptor.

Malignant pleural mesothelioma (MPM) is closely related to exposure to asbestos, and a rapid increase in the number of MPM patients in Japan is estimated in the years 2010–2050. The purpose of the present study was to establish a clinically relevant animal model that shows human patient‐like progression of MPM. Here, we demonstrate that a human MPM cell line (EHMES‐10) inoculated orthotopically (thoracic cavity) into severe combined immunodeficiency (SCID) mice produces highly vascularized thoracic tumors with pleural dissemination and bloody pleural effusions by 5 weeks, suggesting a patient‐like progression of this cell line after orthotopic inoculation. EHMES‐10 cells overexpressed vascular endothelial growth factor (VEGF), a molecule responsible for malignant effusions, and its receptor. Treatment with cisplatin, but not gemcitabine, significantly inhibited the production of pleural effusions, but it was not effective for thoracic tumors, consistent with chemotherapy refractory characteristics of MPM in patients. Our patient‐like orthotopic model using EHMES‐10 cells overexpressing VEGF and its receptor may be useful for examining the molecular pathogenesis of MPM and may contribute to the development of novel treatment strategies for MPM. (Cancer Sci 2006; 97: 183 –191)

Expression and phosphorylation of TOPK during spermatogenesis

Among normal organs and tissues, the MAPKK‐like mitotic protein kinase TOPK is expressed exclusively in the testis. We analyzed the expression and phosphorylation of TOPK to address the functional role of this kinase during spermatogenesis. TOPK protein is expressed mainly in the cytosol of spermatocytes and spermatids, but not in spermatids and spermatogonia in situ. TOPK‐Thr‐9, a cdk1/cyclin B target residue, was specifically phosphorylated during mitotic and meiotic phases, while TOPK‐Thr‐198, a key amino acid for the ATP pocket, was constantly phosphorylated irrespective of the cell cycle. These data indicate that spermatogenic germ cells with vital proliferation activity express TOPK. As TOPK‐Thr‐9 was phosphorylated during both mitosis and meiosis, TOPK was indicted to play a role in cytokinesis and/or chromosomal segregation but not in DNA replication.

Modulation of S‐100 genes response to growth conditions in human epithelial tumor cells

Many new members of the S‐100 genes are known to be associated with cell differentiation, malignant transformation, and cell cycle. Of the S‐100 genes examined In the present study, calcyclin, calpactin I light chain and calvasculln were expressed In most human epithellal tumor cells, and their expression levels differed according to various growth conditions. Their transcribed levels differed depending on each cell line, but their expression patterns were similarly changed under growth‐modulatory conditions. Their messenger RNA levels increased parallel to the S phase population of cells, and decreased at G1/G2 phases. In contrast, this expression diminished in tumor cells under growth‐Inhibitory conditions, such as treatment with topolsomerase II inhibitor VP‐16 or phorbol 12‐myristate 13‐acetate.

Immunohistochemical localization of NUB1, a synphilin-1-binding protein, in neurodegenerative disorders

Recently, we showed that NUB1 is a synphilin-1-interacting protein and that NUB1, as well as synphilin-1, accumulates in Lewy bodies in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and glial cytoplasmic inclusions in multiple system atrophy (MSA). In this study, an investigation was further conducted to elucidate the immunohistochemical localization of NUB1 in various neurodegenerative disorders. In controls, anti-NUB1 antibody weakly immunolabeled neuronal perikarya. In PD and DLB, cortical and brainstem-type Lewy bodies, pale bodies and Lewy neurites were strongly immunolabeled with anti-NUB1. In MSA, NUB1 immunoreactivity was found in the intracytoplasmic inclusions of both neuronal and oligodendroglial cells, neuronal nuclear inclusions, and swollen neurites. No NUB1 immunoreactivity was found in a variety of other neuronal or glial inclusions in other disorders, including Alzheimer’s disease, Pick’s disease, progressive supranuclear palsy, corticobasal degeneration, motor neuron disease and triplet-repeat diseases. These findings indicate that the abnormal accumulation of NUB1 is specific for α-synucleinopathy lesions. However, yeast two-hybrid assay demonstrated that NUB1 did not directly interact with α-synuclein.

Identification of anti-α-enolase autoantibody as a novel serum marker for endometriosis

Diagnosis of endometriosis needs invasive maneuvers. New serum marker that possesses both high sensitivity and high specificity has long been desired. To establish novel serum marker for endometriosis, serum autoantibodies (autoAbs) were investigated using proteomic approach. AutoAbs in sera of endometriotic patients and healthy controls were analyzed using a mesothelial cell line, 2‐DE and Western blotting. Proteins in reacted spots were identified using MALDI TOF‐MS with MASCOT analysis. ELISAs were established using recombinant proteins and autoAb‐titers were estimated in sera of endometriotic patients, disease and healthy controls. Several autoAbs were identified. Anti‐α‐enolase (Eno1)‐autoAb levels in endometriotic patients were significantly elevated compared with both healthy and disease controls. Sensitivity and specificity of serum anti‐Eno1‐autoAb was nearly comparable to serum CA125. When anti‐Eno1‐autoAb and CA125 assays were combined, diagnostic sensitivity and accuracy improved. Serum anti‐Eno1‐autoAb can be a new serum endometriotic marker and it is useful as a supplement assay for CA125. This study validates further clinical evaluation of this novel marker.

Neither MafA/L‐Maf nor MafB is essential for lens development in mice

The importance of the large Maf transcription factor family has been investigated in lens development in the chick, Xenopus and mammals. Previously we reported that c‐maf‐deficient mice exhibit severe defects in lens fibre cells. Here, we report the roles of other large Mafs, MafA/L‐Maf and MafB, during mouse lens development. MafA/L‐Maf and MafB were expressed in lens epithelial cells and fibre cells at E12.5 but had largely disappeared from the lens at E18.5. The lens of mafA‐, mafB‐deficient and mafA::mafB double‐deficient mice developed normally. In c‐maf‐deficient mice, the pattern of expression of MafA and MafB differed from their expression in wild‐type mice. Moreover, the expression of crystallin genes was unchanged in mafA‐, mafB‐ and mafA::mafB double‐deficient lens. These results indicate that c‐Maf alone is essential for lens development, and that MafA/L‐Maf and MafB are dispensable in mice.

Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein ☆ ☆☆

PRPK (p53-related protein kinase) has been reported as a novel protein kinase which binds to the tumor suppressor protein p53 and induces phosphorylation of p53 at Ser 15. To identify novel binding partners of PRPK, we performed a yeast two-hybrid screening and isolated an expressed sequence tag CGI-121 by which a 20-kDa protein was encoded. We demonstrated the protein-protein interaction of CGI-121 with PRPK in vivo and in vitro. The protein expression of CGI-121 was observed in many cell lines and was immunocytochemically identified in both the nucleus and cytosol. Although PRPK interacted with both CGI-121 and p53, several attempts to demonstrate an association between CGI-121 and p53 were unsuccessful. In addition, coprecipitation of p53 using recombinant PRPK was inhibited by adding recombinant CGI-121 in vitro, suggesting that CGI-121 could act as a potent inhibitor of the binding of PRPK to p53.