Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2022.0042

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Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2022.0005

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s42003-022-03427-4

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1348-0421.12964

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/gkab1137

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Publishing type：Research paper (scientific journal)  

DOI： 10.1242/jcs.258850

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

INTRODUCTION: The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are smaller, their resorptive activity is high which helps in efficient destruction of nasal turbinate bones of pigs. METHODS: The proteome of bone marrow-derived macrophages differentiated into osteoclasts with either RANKL or PMT was analysed. The results were verified by characterizing the metabolic activity using Seahorse analysis, a protein translation assay, immunoblots, real-time PCR as well as flow cytometry-based monitoring of mitochondrial activity and ROS production. A Gαq overexpression system using ER-Hoxb8 cells was used to identify Gαq-mediated metabolic effects on osteoclast differentiation and function. RESULTS: PMT induces the upregulation of metabolic pathways, which included strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components were expressed more efficiently, cells also displayed increased mitochondrial respiration. The heterotrimeric G protein Gαq plays a central role in this hypermetabolic cell activation as it triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. This seems to be caused by a direct interaction between STAT3 and OPA1 resulting in enhanced mitochondrial respiration. Overexpression of Gαq mimicked the hypermetabolic phenotype observed for PMT-induced osteoclasts and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. In addition, rheumatoid arthritis (RA) patients showed an increase in GNAQ expression, especially in the synovial fluid. DISCUSSION: Our study suggests that Gαq plays a key role in PMT-induced osteoclastogenesis. Enhanced expression of GNAQ at the site of inflammation in RA patients indicates its pathophysiological relevance in the context of inflammatory bone disorders.

DOI： 10.3389/fcimb.2022.1016299

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.3390/cancers13143613

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.15335

Publishing type：Research paper (scientific journal)  

DOI： 10.1093/mmy/myaa001

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Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.15186

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbmt.2020.05.013

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.15401

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2020.0007

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1039/d0sc00317d

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

DOI： 10.1590/1678-4685-GMB-2019-0017

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1177/1010428320965279

Publishing type：Research paper (scientific journal)  

DOI： 10.1177/0963689720976567

Publishing type：Research paper (scientific journal)  

DOI： 10.1080/10428194.2019.1636986

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Epithelial cell polarity regulator Crumbs3 (Crb3), a mammalian homolog within the Drosophila Crb gene family, was initially identified as an essential embryonic development factor. It is recently implicated in tumor suppression, though its specific functions are controversial. We here demonstrate that Crb3 strongly promotes tumor invasion and metastasis of human colon adenocarcinoma cells. Crb3 centrality to tumor migration was supported by strong expression at invasive front and metastatic foci of colonic adenocarcinoma of the patient tissues. Accordingly, two different Crb3-knockout (KO) lines, Crb3-KO (Crb3 -/-) DLD-1 and Crb3-KO WiDr from human colonic adenocarcinomas, were generated by the CRISPR-Cas9 system. Crb3-KO DLD-1 cells exhibited loss of cellular mobility in vitro and dramatic suppression of liver metastases in vivo in contrast to the wild type of DLD-1. Unlike DLD-1, Crb3-KO WiDr mobility and metastasis were unaffected, which were similar to wild-type WiDr. Proteome analysis of Crb3-coimmunopreciptated proteins identified different respective fibroblast growth factor receptor (FGFR) isotypes specifically bound to Crb3 isoform a through their intracellular domain. In DLD-1, Crb3 showed membranous localization of FGFR1 leading to its functional activation, whereas Crb3 bound to cytoplasmic FGFR4 in WiDr without FGFR1 expression, leading to cellular growth. Correlative expression between Crb3 and FGFR1 was consistently detected in primary and metastatic colorectal cancer patient tissues. Taking these together, Crb3 critically accelerates cell migration, namely invasion and metastasis of human colon cancers, through specific interaction to FGFR1 on colon cancer cells.

DOI： 10.1002/ijc.32336

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

We previously demonstrated that CRM1, a major nuclear export factor, accumulates at Hox cluster regions to recruit nucleoporin-fusion protein Nup98HoxA9, resulting in robust activation of Hox genes (Oka et al., 2016). However, whether this phenomenon is general to other leukemogenic proteins remains unknown. Here, we show that two other leukemogenic proteins, nucleoporin-fusion SET-Nup214 and the NPM1 mutant, NPM1c, which contains a nuclear export signal (NES) at its C-terminus and is one of the most frequent mutations in acute myeloid leukemia, are recruited to the HOX cluster region via chromatin-bound CRM1, leading to HOX gene activation in human leukemia cells. Furthermore, we demonstrate that this mechanism is highly sensitive to a CRM1 inhibitor in leukemia cell line. Together, these findings indicate that CRM1 acts as a key molecule that connects leukemogenic proteins to aberrant HOX gene regulation either via nucleoporin-CRM1 interaction (for SET-Nup214) or NES-CRM1 interaction (for NPM1c).

DOI： 10.7554/eLife.46667

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Publishing type：Research paper (scientific journal)  

DOI： 10.1002/ajh.25612

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

PIWI-interacting RNAs (piRNAs), a subclass of germ cell-specific noncoding small RNAs, are essential for de novo DNA methylation of retrotransposon genes in embryonic testes. PIWIL2/MILI, one of three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably via piRNA, and normal spermatogenesis. In vitro analysis using germline stem cells (GS cells) revealed that glycerol-3-phosphate acyltransferase 2 (GPAT2), which is a mitochondrial outer membrane protein involved in generation of lysophosphatidic acid (LPA) and highly expressed in testes, plays important roles in spermatogenesis. Namely, GPAT2 binds to PIWIL2 and is closely involved in the biogenesis of piRNAs; this process is independent of its enzymatic activity on LPA. However, GS cells recapitulate only a limited phase of spermatogenesis and the biological functions of GPAT2 remain largely unknown. In this study, we generated GPAT2-deficient mice and conducted comprehensive analyses. The deficient mice showed defective piRNA production and subsequent de-silencing of IAP and Line-1 retrotransposons in fetal testes. In addition, apoptosis of pachytene spermatocytes was observed. These abnormalities were all common to the phenotype of PIWIL2-deficient mice, in which piRNA production was impaired. GPAT2-deficient mice exhibited apoptosis in spermatogonia at the neonatal stage, which was not observed in PIWIL2-deficient mice. These data show that GPAT2 plays a critical role in preventing apoptosis in spermatogonia.

DOI： 10.1093/biolre/ioz056

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Publishing type：Research paper (scientific journal)  

DOI： 10.1093/biolre/ioz056

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Publishing type：Research paper (scientific journal)  

DOI： 10.1002/ijc.32336

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Publishing type：Research paper (scientific journal)  

DOI： 10.1242/bio.032631

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Publishing type：Research paper (scientific journal)  

DOI： 10.1242/bio.032631

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1073/pnas.1705682114

Publishing type：Research paper (scientific journal)  

Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (gamma-TuRC) via Galectin-3-binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression.

DOI： 10.1073/pnas.1705682114

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-017-16386-2

Publishing type：Research paper (scientific journal)  

Nuclear pore complexes (NPCs) maintain cellular homeostasis by mediating nucleocytoplasmic transport. Although cyclin-dependent kinases (CDKs) regulate NPC assembly in interphase, the location of NPC assembly on the nuclear envelope is not clear. CDKs also regulate the disappearance of pore-free islands, which are nuclear envelope subdomains; this subdomain gradually disappears with increase in homogeneity of the NPC in response to CDK activity. However, a causal relationship between pore-free islands and NPC assembly remains unclear. Here, we elucidated mechanisms underlying NPC assembly from a new perspective by focusing on pore-free islands. We proposed a novel framework for image-based analysis to automatically determine the detailed 'landscape'of pore-free islands from a large quantity of images, leading to the identification of NPC intermediates that appear in pore-free islands with increased frequency in response to CDK activity. Comparison of the spatial distribution between simulated and the observed NPC intermediates within pore-free islands showed that their distribution was spatially biased. These results suggested that the disappearance of pore-free islands is highly related to de novo NPC assembly and indicated the existence of specific regulatory mechanisms for the spatial arrangement of NPC assembly on nuclear envelopes.

DOI： 10.1038/s41598-017-16386-2

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.biochem.6b01098

Publishing type：Research paper (scientific journal)  

Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the, H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

DOI： 10.1021/acs.biochem.6b01098

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/srep44976

Publishing type：Research paper (scientific journal)  

<p>To promote the use of sewage sludge on farmland, a flocculant compatible with agricultural application is required. In general, an inorganic flocculant such as aluminium sulfate is used at large sewage disposal plants. However, there is concern that aluminium accumulates when it is returned to farmland, and therefore a biodegradable rather than an inorganic flocculant is required. To obtain biodegradable flocculants produced by microorganisms, the latter were isolated from soils and sewage sludges, and the flocculation activity of the culture broths was evaluated. Here, real sewage sludge was used for the evaluation because practical use was regarded as an important consideration. As a result, two strains, V13 and RK14, were selected and identified as <i>Bacillus megaterium</i> and <i>Bacillus licheniformis</i>, respectively. It was also found that flocculation was caused by poly-γ-glutamic acid (γ-PGA). γ-PGA from RK14 cells had considerably higher molecular weight than commercial γ-PGA, and the enantiomeric composition differed from that of γ-PGA from other <i>B. licheniformis</i>. To obtain mutants with high γ-PGA productivity, RK14 cells were treated with ethyl methanesulfonate, and a mutant (RK14-46) was selected. The culture medium suitable for γ-PGA production by RK14-46 cells was totally different from that by RK-14 cells. The γ-PGA production reached 21.1 g/L when RK14-46 cells were cultured in the optimum semi-synthetic medium at 30°C for 2 d. Furthermore, the combination of γ-PGA obtained from RK14-46 cells and chitosan, a cationic flocculant, was very effective for flocculation of real sewage sludge.</p>

DOI： 10.1252/jcej.16we158

CiNii Article

Publishing type：Research paper (scientific journal)  

Functions of septin cytoskeletal polymers in tumorigenesis are still poorly defined. Their role in the regulation of cytokinesis and cell migration were proposed to contribute to cancer associated aneuploidy and metastasis. Overexpression of Septin 9 (Sept9) promotes migration of cancer cell lines. SEPT9 mRNA and protein expression is increased in breast tumors compared to normal and peritumoral tissues and amplification of SEPT9 gene was positively correlated with breast tumor progression. However, the existence of multiple isoforms of Sept9 is a confounding factor in the analysis of Sept9 functions. In the present study, we analyze the protein expression of Sept9_i2, an uncharacterized isoform, in breast cancer cell lines and tumors and describe its specific impact on cancer cell migration and Sept9 cytoskeletal distribution. Collectively, our results showed that, contrary to Sept9_i1, Sept9_i2 did not support cancer cell migration, and induced a loss of subnuclear actin filaments. These effects were dependent on Sept9_i2 specific N-terminal sequence. Sept9_i2 was strongly down-regulated in breast tumors compared to normal mammary tissues. Thus our data indicate that Sept9_i2 is a negative regulator of breast tumorigenesis. We propose that Sept9 tumorigenic properties depend on the balance between Sept9_i1 and Sept9_i2 expression levels.

DOI： 10.1038/srep44976

PubMed

Publishing type：Research paper (scientific journal)  

Dermokine is one of the most highly expressed proteins in differentiating keratinocytes. Mouse dermokine has been reported to be encoded by 22 exons, and its expression leads to three transcripts, β, γ, and α, which are transcribed from two different transcriptional start sites. The α isoform represents the carboxyl-terminal domain of the β isoform, whereas the γ isoform lacks this domain. To reveal the distributions and expression levels of each isoform in mice, we generated rat monoclonal antibodies against dermokine-β/γ and dermokine-β/α. In immunofluorescence studies, the expression levels of dermokine in the cytosol of the cultured mouse keratinocytes were significantly elevated by high levels of extracellular calcium. In Western blot analyses, the expression levels of dermokine-β and dermokine-α were increased in the presence of high calcium. Finally, we developed a monoclonal antibody-based sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and showed that the secreted dermokine-β into the culture medium from mouse keratinocytes was significantly increased in a manner dependent on the extracellular calcium concentration. These dermokine-specific antibodies have allowed us to gain new insights into the role of each dermokine isoform in cutaneous homeostasis.

DOI： 10.1089/mab.2016.0051

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2016.0051

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.celrep.2016.12.065

Publishing type：Research paper (scientific journal)  

Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-typespecific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

DOI： 10.1016/j.celrep.2016.12.065

PubMed

Publishing type：Research paper (scientific journal)  

<p>To promote the use of sewage sludge on farmland, a flocculant compatible with agricultural application is required. In general, an inorganic flocculant such as aluminium sulfate is used at large sewage disposal plants. However, there is concern that aluminium accumulates when it is returned to farmland, and therefore a biodegradable rather than an inorganic flocculant is required. To obtain biodegradable flocculants produced by microorganisms, the latter were isolated from soils and sewage sludges, and the flocculation activity of the culture broths was evaluated. Here, real sewage sludge was used for the evaluation because practical use was regarded as an important consideration. As a result, two strains, V13 and RK14, were selected and identified as <i>Bacillus megaterium</i> and <i>Bacillus licheniformis</i>, respectively. It was also found that flocculation was caused by poly-γ-glutamic acid (γ-PGA). γ-PGA from RK14 cells had considerably higher molecular weight than commercial γ-PGA, and the enantiomeric composition differed from that of γ-PGA from other <i>B. licheniformis</i>. To obtain mutants with high γ-PGA productivity, RK14 cells were treated with ethyl methanesulfonate, and a mutant (RK14-46) was selected. The culture medium suitable for γ-PGA production by RK14-46 cells was totally different from that by RK-14 cells. The γ-PGA production reached 21.1 g/L when RK14-46 cells were cultured in the optimum semi-synthetic medium at 30°C for 2 d. Furthermore, the combination of γ-PGA obtained from RK14-46 cells and chitosan, a cationic flocculant, was very effective for flocculation of real sewage sludge.</p>

DOI： 10.1252/jcej.16we158

CiNii Article

Publishing type：Research paper (scientific journal)  

DOI： 10.1242/jcs.186817

Publishing type：Research paper (scientific journal)  

The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si) RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner.

DOI： 10.1242/jcs.186817

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Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.13288

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.13286

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.13275

PubMed

Publishing type：Research paper (scientific journal)  

Pancreatic ductal adenocarcinoma is an aggressive tumor with a poor prognosis. Biomarkers that can detect the tumor in its early stages when it may be amenable to curative resection might improve prognosis. To discover novel markers expressed in primary pancreatic cancer, we generated a panel of monoclonal antibodies against pancreatic ductal adenocarcinoma cell line BxPC3 using a rat medial iliac lymph node method. The antigen recognized by 1B5A5 was expressed on the cell surface and secreted into the conditioned medium of BxPC3 cells, and characterized as glycoproteins with molecular mass between 60 and 90 kDa. A wide range of molecular weights of 1B5A5 antigen in several pancreatic cancer cell lines were observed. Immunohistochemistry using a human multiple organ tumor tissue array showed an enhanced expression of 1B5A5 antigen in pancreas, lung, stomach, breast, urinary bladder, colon, and cervix uteri cancers. Immunoprecipitation followed by proteomic analyses identified CEACAM6 as a 1B5A5 antigen. In addition, western blot analysis results indicated that the 1B5A5 epitope is located within an amino-terminal domain of CEACAM6. These results raised the possibility that our approach could lead to discovery of novel biomarkers for the early stage of cancers in a relatively short period of time.

DOI： 10.1089/mab.2016.0012

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Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2016.0012

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.13285

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.13274

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.13276

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.7554/eLife.09540

Publishing type：Research paper (scientific journal)  

The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9-dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation.

DOI： 10.7554/eLife.09540

PubMed

Publishing type：Research paper (scientific journal)  

Overexpression of the epidermal growth factor receptor (EGFR) gene and dysregulation of EGFR signaling are observed in various cancer cells, and EGFR is a validated target for cancer therapy. In the present study, we report on the generation of two rat anti-EGFR antibodies (clones 2C2D3 and 4H7F4) by using the rat lymph node method. Flow cytometric analysis and immunofluorescence showed that both antibodies specifically bound to EGFR on the surface of cancer cells. Competitive analysis demonstrated that the epitope of each antibody had no overlap with that of the therapeutic anti-EGFR antibody cetuximab. These results suggest that 2C2D3 and 4H7F4 are potentially useful in EGFR-targeted cancer therapy.

DOI： 10.1089/mab.2015.0034

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Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2015.0034

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Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2015.03.005

Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.A115.008299

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.A115.008299

PubMed

Publishing type：Research paper (scientific journal)  

Zipper-interacting protein kinase (ZIPK) is known to regulate several functions such as apoptosis, smooth muscle contraction, and cell migration. While exogenously expressed GFP-ZIPK localizes to the cleavage furrow, role of ZIPK in cytokinesis is obscure. Here, we show that ZIPK is a major MRLC kinase during mitosis. Moreover, ZIPK siRNA-mediated knockdown causes delay of cytokinesis. The delay in cytokinesis of ZINC-knockdown cells was rescued by the exogenous diphosphorylation-mimicking MRLC mutant. Taken together, these findings suggest that ZIPK plays a role in the progression and completion of cytokinesis through MRLC phosphorylation. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2015.03.005

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Publishing type：Research paper (scientific journal)  

DOI： 10.1523/JNEUROSCI.5029-13.2015

Publishing type：Research paper (scientific journal)  

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs. The antigen recognized by MAb2 is expressed on the cell surface of undifferentiated hESCs
three diffused bands with molecular mass between 30 and 60kDa in the lysates of hESCs were diminished during hESC differentiation into neural cells. The expression of MAb2 antigen was also observed on the plasma membrane of lung cancer cells, and MAb2 detected 55, 50, and 35kDa protein bands in the cell lysates. Immunoprecipitation followed by proteomics analyses identified CD147/basigin as a MAb2 antigen. Finally, the positive expression of CD147/basigin protein in undifferentiated hESCs was confirmed. These results suggested that CD147/basigin could be another undifferentiated hESC marker.

DOI： 10.1089/mab.2014.0075

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2014.0075

PubMed

Publishing type：Research paper (scientific journal)  

Cell positioning and neuronal network formation are crucial for proper brain function. Disrupted-in-Schizophrenia 1 (DISC1) is anterogradely transported to the neurite tips, together with Lis1, and functions in neurite extension via suppression of GSK3 beta activity. Then, transported Lis1 is retrogradely transported and functions in cell migration. Here, we show that DISC1-binding zinc finger protein (DBZ), together with DISC1, regulates mouse cortical cell positioning and neurite development in vivo. DBZ hindered Ndel1 phosphorylation at threonine 219 and serine 251. DBZ depletion or expression of a double-phosphorylated mimetic form of Ndel1 impaired the transport of Lis1 and DISC1 to the neurite tips and hampered microtubule elongation. Moreover, application of DISC1 or a GSK3 beta inhibitor rescued the impairments caused by DBZ insufficiency or double-phosphorylated Ndel1 expression. We concluded that DBZ controls cell positioning and neurite development by interfering with Ndel1 from disproportionate phosphorylation, which is critical for appropriate anterograde transport of the DISC1-complex.

DOI： 10.1523/JNEUROSCI.5029-13.2015

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/gku1346

Publishing type：Research paper (scientific journal)  

Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated intomyotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications.

DOI： 10.1093/nar/gku1346

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M114.550848

Publishing type：Research paper (scientific journal)  

Human embryonic stem cells (hESCs) are pluripotent stem cells from early embryos, and their self-renewal capacity depends on the sustained expression of hESC-specific molecules and the suppressed expression of differentiation-associated genes. To discover novel molecules expressed on hESCs, we generated a panel of monoclonal antibodies against undifferentiated hESCs and evaluated their ability to mark cancer cells, as well as hESCs. MAb7 recognized undifferentiated hESCs and showed a diffuse band with molecular mass of &gt; 239 kDa in the lysates of hESCs. Although some amniotic epithelial cells expressed MAb7 antigen, its expression was barely detected in normal human keratinocytes, fibroblasts, or endothelial cells. The expression of MAb7 antigen was observed only in pancreatic and gastric cancer cells, and its levels were elevated in metastatic and poorly differentiated cancer cell lines. Analyses of MAb7 antigen suggested that the clustered NeuAc alpha 2-3Gal beta O-linked oligosaccharides on DMBT1 (deleted in malignant brain tumors 1) were critical for MAb7 binding in cancer cells. Although features of MAb7 epitope were similar with those of TRA-1-60, distribution of MAb7 antigen in cancer cells was different from that of TRA-1-60 antigen. Exposure of a histone deacetylase inhibitor to differentiated gastric cancer MKN74 cells evoked the expression of MAb7 antigen, whereas DMBT1 expression remained unchanged. Cell sorting followed by DNA microarray analyses identified the down-regulated genes responsible for the biosynthesis of MAb7 antigen in MKN74 cells. In addition, treatment of metastatic pancreatic cancer cells with MAb7 significantly abrogated the adhesion to endothelial cells. These results raised the possibility that MAb7 epitope is a novel marker for undifferentiated cells such as hESCs and cancer stem-like cells and plays a possible role in the undifferentiated cells.

DOI： 10.1074/jbc.M114.550848

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1186/2040-2392-5-33

Publishing type：Research paper (scientific journal)  

DOI： 10.1002/ijc.28550

Publishing type：Research paper (scientific journal)  

DOI： 10.1002/glia.22636

Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09168451.2014.917259

Publishing type：Research paper (scientific journal)  

Recent studies have shown changes in myelin genes and alterations in white matter structure in a wide range of psychiatric disorders. Here we report that DBZ, a central nervous system (CNS)-specific member of the DISC1 interactome, positively regulates the oligodendrocyte (OL) differentiation in vivo and in vitro. In mouse corpus callosum (CC), DBZ mRNA is expressed in OL lineage cells and expression of DBZ protein peaked before MBP expression. In the CC of DBZ-KO mice, we observed delayed myelination during the early postnatal period. Although the myelination delay was mostly recovered by adulthood, OLs with immature structural features were more abundant in adult DBZ-KO mice than in control mice. DBZ was also transiently upregulated during rat OL differentiation in vitro before myelin marker expression. DBZ knockdown by RNA interference resulted in a decreased expression of myelin-related markers and a low number of cells with mature characteristics, but with no effect on the proliferation of oligodendrocyte precursor cells. We also show that the expression levels of transcription factors having a negative-regulatory role in OL differentiation were upregulated when endogenous DBZ was knocked down. These results strongly indicate that OL differentiation in rodents is regulated by DBZ.

DOI： 10.1002/glia.22636

Publishing type：Research paper (scientific journal)  

ハプロイド突然変異体AQ-37に基づいて、マウスマクロファージを強力に活性化できる2倍体パン用イーストを構築し、その活性化に対する要因を検討した。得られた3種の2倍体株(BQ-10、BQ-48、BQ-55)のうち、BQ-55が最高のマクロファージ活性化能を示したため、2倍体パン用イーストとして詳細な評価を行った。BQ-55の増殖能はAQ-37より約5倍高かった。2倍体株BQ-55はヒト免疫細胞も活性化した。マウスRAW264.7細胞およびヒト末梢血単核球細胞において、BQ-55はTNF-α分泌を有意に促進した。この活性化に対する要因を解明するため、その細胞壁構造、特にβグルカン構造を解析した結果、分岐の長さ、β-1,6-グルカンがその要因の一つであることが示唆された。

Publishing type：Research paper (scientific journal)  

Background: Changes in serotonin transporter (SERT) function have been implicated in autism. SERT function is influenced by the number of transporter molecules present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. Thus, we searched for novel SERT-binding proteins and investigated whether the expression of one such protein was affected in subjects with autism.
Methods: Novel SERT-binding proteins were examined by a pull-down system. Alterations of SERT function and membrane expression upon knockdown of the novel SERT-binding protein were studied in HEK293-hSERT cells. Endogenous interaction of SERT with the protein was evaluated in mouse brains. Alterations in the mRNA expression of SERT (SLC6A4) and the SERT-binding protein in the post-mortem brains and the lymphocytes of autism patients were compared to nonclinical controls.
Results: N-ethylmaleimide-sensitive factor (NSF) was identified as a novel SERT-binding protein. NSF was co-localized with SERT at the plasma membrane, and NSF knockdown resulted in decreased SERT expression at the cell membranes and decreased SERT uptake function. NSF was endogenously co-localized with SERT and interacted with SERT. While SLC6A4 expression was not significantly changed, NSF expression tended to be reduced in post-mortem brains, and was significantly reduced in lymphocytes of autistic subjects, which correlated with the severity of the clinical symptoms.
Conclusions: These data clearly show that NSF interacts with SERT under physiological conditions and is required for SERT membrane trafficking and uptake function. A possible role for NSF in the pathophysiology of autism through modulation of SERT trafficking, is suggested.

DOI： 10.1186/2040-2392-5-33

PubMed

Publishing type：Research paper (scientific journal)  

Cetuximab is a chimeric IgG1 monoclonal antibody (mAb) that targets the extracellular domain of epidermal growth factor receptor (EGFR). Oncogenic KRAS mutations in tumors have been shown to be a negative predictor of the response of colorectal cancer (CRC) to cetuximab treatment. Cetuximab exerts its therapeutic effects through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC). However, the influence of KRAS mutations on cetuximab-mediated ADCC is not fully understood. Here, we investigated cetuximab-mediated ADCC in two pairs of isogenic CRC cells with or without a KRAS mutation. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and NK92, a natural killer (NK) cell line that exogenously expresses Fc?RIIIa (CD16a), were used as effector cells. In an ADCC assay, perforin-dependent target cell lysis was not affected by the KRAS mutation status. On the other hand, perforin-independent ADCC was observed only in CRC cells with wild-type KRAS, but not in cells with mutant KRAS. Neutralizing experiments revealed that the Fas-Fas ligand (FasL) interaction was responsible for the induction of apoptosis and perforin-independent ADCC. Furthermore, the presence of effector cells clearly enhanced the growth-inhibitory effect of cetuximab only in CRC cells with wild-type KRAS, but not in those with mutant KRAS. These findings suggest that ADCC is an important mode of action of cetuximab and that KRAS mutation impairs the therapeutic effect exerted by cetuximab-mediated ADCC.

DOI： 10.1002/ijc.28550

Publishing type：Research paper (scientific journal)  

Diploid baker's yeast capable of strongly activating a mouse macrophage was constructed based on haploid mutant AQ-37 obtained previously. The obtained strain BQ-55 activated also human immune cells. To clarify a factor for the activation, the cell wall structure, especially the beta-glucan structure, was analyzed, suggesting that the length of branching, beta-1,6-glucan, may be one of the factors.

DOI： 10.1080/09168451.2014.917259

PubMed

Publishing type：Research paper (scientific journal)  

Type I insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that is involved in the transformation of cells, cancer proliferation, and metastatic events in various types of cancer. The present study reports on the generation of a mouse monoclonal antibody (MAb) to IGF-1R using the mouse lymph node method. MAb 1B1, which reacts specifically with the extracellular domain of IGF-1R, was obtained using flow cytometry (FCM) screening using MCF-7 cells. Using immunostaining, MAb 1B1 detected IGF-1R mainly on the plasma membrane of MCF-7 cells. MAb 1B1 would be useful in FCM and immunofluorescence assays to detect IGF-1R-expressing cells for basic and clinical research. © Copyright 2014, Mary Ann Liebert, Inc. 2014.

DOI： 10.1089/mab.2013.0083

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2013.0083

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1567-1364.12098

Publishing type：Research paper (scientific journal)  

A physiological function of the -glucans which constitute the cell wall of Saccharomyces cerevisiae is to activate immune cells. Here, we focused on the immunostimulation ability of S.cerevisiae itself to give this ability to fermented foods including yeast. Previously, we found that in S.cerevisiae the deletion of MCD4 gene causes exposure of -glucans on the cell surface and that the mcd4 deletion mutant strongly enhances immunity in vitro and in vivo. However, this is not a practical strain but a genetically modified strain with an antibiotic resistance gene, and growth was very slow. The aim of this study was to acquire a practical strain capable of strongly activating a macrophage. The parental strain y-21 was mutated with ethyl methanesulfonate, and the resulting strain was screened. Two mutants (AP-57 and AQ-37) were obtained. AQ-37 had the same fermentation capacity as y-21. In addition, a mutation point of AQ-37 was identified, suggesting that the mutation of NDD1 gene affects the cell wall structure and confers a high ability for macrophage stimulation. The obtained yeast may activate immune cells in materials to which the yeast is added.

DOI： 10.1111/1567-1364.12098

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bej.2013.12.011

Publishing type：Research paper (scientific journal)  

INI1/hSNF5/BAF47, which has an SNF5 domain, belongs to the SWI/SNF family. This family is known as ATP-dependent regulators of gene expression by remodeling chromatin structure during cell differentiation. However, the detailed function of INI1/hSNF5/BAF47 is unclear. Here we report the generation of a specific monoclonal antibody for INI1/hSNF5/BAF47 by the mouse iliac lymph node method. The obtained antibody recognized two isoforms of INI1/hSNF5/BAF47 in immunoblotting and precisely recognized the nuclear localization of INI1/hSNF5/BAF47 in immunostaining. This antibody can contribute to further elucidation of the mechanisms of gene expression regulation by INI1/hSNF5/BAF47 during cell differentiation. © 2014, Mary Ann Liebert, Inc.

DOI： 10.1089/mab.2013.0065

PubMed

Publishing type：Research paper (scientific journal)  

The CCAAT/enhancer-binding protein (C/EBP)β belongs to the C/EBP family of proteins that possesses a basic leucine zipper DNA-binding domain. These proteins bind DNA by dimerization and play a role in the transcriptional regulation of various cells. There are six different types of C/EBPs, and some form isoforms through the use of alternative translation initiation sites. The functional analysis of the C/EBP family is therefore difficult to achieve. Here we report on the production of specific monoclonal antibodies against mouse C/EBPβ using a rat medial iliac lymph node method. Immunoblotting using C/EBPβ monoclonal antibodies identified two types of isoforms, while immunostaining revealed a subnuclear localization for C/EBPβ. Use of this antibody should contribute to the further elucidation of the transcriptional regulatory function of C/EBPβ. © 2014, Mary Ann Liebert, Inc.

DOI： 10.1089/mab.2013.0069

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1002/yea.2995

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2013.0069

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2013.0065

PubMed

Publishing type：Research paper (scientific journal)  

Microbial fuel cells are attracting attention as one of the systems for producing electrical energy from organic compounds. We used commercial baker's yeast (Saccharomyces cerevisiae) for a glucose fuel cell because the yeast is a safe organism and relatively high power can be generated in the system. In the present study, a milliliter (mL)-scale dual-chamber fuel cell was constructed for evaluating the power generated by a variety of yeasts and their mutants, and the optimum conditions for high performance were investigated. When carbon fiber bundles were used as an electrode in the fuel cell, high volumetric power density was obtained. The maximum power produced per volume of anode solution was 850 W/m(3) under optimum conditions. Furthermore, the power was examined using seven kinds of yeast. In Kluyveromyces marxianus, not only the power but also the power per consumed glucose was high. Moreover, it was suggested that xylose is available as fuel for the fuel cell. The fuel cell powered by K. marxianus may prove to be helpful for the effective utilization of woody biomass. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2013.12.011

Publishing type：Research paper (scientific journal)  

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. -Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of -glucosidase activity using p-nitrophenyl--glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright (c) 2014 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.2995

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2013.10.134

Publishing type：Research paper (scientific journal)  

Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy. © 2013 Elsevier Inc.

DOI： 10.1016/j.bbrc.2013.10.134

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1091/mbc.E13-05-0239

PubMed

Publishing type：Research paper (scientific journal)  

The X-linked gene Rnf12 encodes the ubiquitin ligase really interesting new gene (RING) finger LIM domain-interacting protein (RLIM)/RING finger protein 12 (Rnf12), which serves as a major sex-specific epigenetic regulator of female mouse nurturing tissues. Early during embryogenesis, RLIM/Rnf12 expressed from the maternal allele is crucial for the development of extraembryonic trophoblast cells. In contrast, in mammary glands of pregnant and lactating adult females RLIM/Rnf12 expressed from the paternal allele functions as a critical survival factor for milk-producing alveolar cells. Although RLIM/Rnf12 is detected mostly in the nucleus, little is known about how and in which cellular compartment(s) RLIM/Rnf12 mediates its biological functions. Here we demonstrate that RLIM/Rnf12 protein shuttles between nucleus and cytoplasm and this is regulated by phosphorylation of serine S214 located within its nuclear localization sequence. We show that shuttling is important for RLIM to exert its biological functions, as alveolar cell survival activity is inhibited in cells expressing shuttling-deficient nuclear or cytoplasmic RLIM/Rnf12. Thus regulated nucleocytoplasmic shuttling of RLIM/Rnf12 coordinates cellular compartments during mammary alveolar cell survival.

DOI： 10.1091/mbc.E13-05-0239

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1468-3083.2012.04598.x

Publishing type：Research paper (scientific journal)  

Background Although dermokine-β, a glycoprotein expressed in epithelial cells, does not have significant homology to other proteins, its carboxyl-terminal domain shares a high pI value with many cytokines, suggesting similar functions. Objective To better understand the biology of dermokine, we here determined its localization under pathological conditions and examined factors that regulate its expression. Methods We generated an anti-human dermokine-β/γ monoclonal antibody cross-reacting with the mouse protein. Using this antibody, immunohistological staining and Western blotting of dermokine-β/γ were performed with various tissue samples. Results Although human dermokine-β/γ was expressed in almost all granular layers, upper spinous layers of the skin were also stained with anti-dermokine-β/γ antibody in inflammatory skin disorders. Dermokine-β/γ was expressed in keratoacanthoma and a part of well-differentiated squamous cell carcinoma (SCC). However, dermokine-β/ γ was not detected in poorly differentiated SCC or tumours derived from non-keratinocytes. In mice, dermokine-β/γ-expressed keratinocytes were increased in models of contact hypersensitivity, ultraviolet-irradiated skin injury and wound healing. Consistent with expanded distribution in inflammatory skin diseases, proinflammatory cytokines such as interleukin-1β, interleukin-12, and tumour necrosis factor-α augmented dermokine-β/γ expression in cultured human keratinocytes. In contrast, growth factors including epidermal growth factor, insulin-like growth factor-I, keratinocyte growth factor and transforming growth factor-α significantly reduced dermokine expression. Conclusion These results provide novel insights into the physiological and pathological significance of dermokine in the epidermis. © 2012 European Academy of Dermatology and Venereology.

DOI： 10.1111/j.1468-3083.2012.04598.x

PubMed

Publishing type：Research paper (scientific journal)  

Pou5f1/Oct4, a member of the POU transcription factor family, is exclusively expressed in embryonic stem cells, which are involved in self-renewal and maintaining pluripotency. In the present study, we report on the establishment of a monoclonal antibody that is specific for Oct4 using the rat medial iliac lymph node method. In an immunoblotting analysis, our antibody detected endogenous Oct4. In addition, immunocytochemical staining using the antibody revealed the nuclear localization of Oct4. This monoclonal antibody has the potential for use in the further analysis of Oct4 function in stem cells. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

DOI： 10.1089/mab.2013.0004

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2013.0004

PubMed

Publishing type：Research paper (scientific journal)  

Modification of histone plays a critical role in the epigenetic regulation of gene expression. However, unlike the widely studied roles of histone methylation or acetylation of histone H3, relatively little is known about the molecular mechanisms involved in translating histone phosphorylation into a specific outcome. The present study reports on the development of antibodies (MAbs) directed against phosphorylated histone H3 (S10, T11, S28, S31, and T32), produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat or mouse. The MAbs produced specifically recognize different sites of phosphorylation on histone H3. All of these MAbs are suitable for immunoblotting and immunofluorescence analysis. We believe that these antibodies should significantly facilitate our efforts to investigate epigenetic regulation. © Mary Ann Liebert, Inc.

DOI： 10.1089/mab.2012.0105

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2012.0105

PubMed

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Publishing type：Research paper (scientific journal)  

DOI： 10.1096/fj.12-213900

Publishing type：Research paper (scientific journal)  

Pasteurella multocida is the causative agent of a number of epizootic and zoonotic diseases. Its major virulence factor associated with atrophic rhinitis in animals and dermonecrosis in bite wounds is P. multocida toxin (PMT). PMT stimulates signal transduction pathways downstream of heterotrimeric G proteins, leading to effects such as mitogenicity, blockade of apoptosis, or inhibition of osteoblast differentiation. On the basis of Gα , it was demonstrated that the toxin deamidates an essential glutamine residue of the Gαi2 subunit, leading to constitutive activation of the G protein. Here, we studied the specificity of PMT for its G-protein targets by mass spectrometric analyses and by utilizing a monoclonal antibody, which recognizes specifically G proteins deamidated by PMT. The studies revealed deamidation of 3 of 4 families of heterotrimeric G proteins (Gα , Gαi and Gα of mouse or human origin) by PMT but not by a catalytic inactive toxin mutant. With the use of G-protein fragments and chimeras of responsive or unresponsive G proteins, the structural basis for the discrimination of heterotrimeric G proteins was studied. Our results elucidate substrate specificity of PMT on the molecular level and provide evidence for the underlying structural reasons of substrate discrimination. © FASEB. i2 q/11 1,2,3, 12/13

DOI： 10.1096/fj.12-213900

PubMed

Publishing type：Research paper (scientific journal)  

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Publishing type：Research paper (scientific journal)  

DOI： 10.1210/me.2012-1256

Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/gks1010

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Deep sequencing approaches, such as chromatin immunoprecipitation by sequencing (ChIP-seq), have been successful in detecting transcription factor-binding sites and histone modification in the whole genome. An approach for comparing two different ChIP-seq data would be beneficial for predicting unknown functions of a factor. We propose a model to represent co-localization of two different ChIP-seq data. We showed that a meaningful overlapping signal and a meaningless background signal can be separated by this model. We applied this model to compare ChIP-seq data of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation with a large amount of peak-called data, including ChIP-seq and other deep sequencing data in the Encyclopedia of DNA Elements (ENCODE) project, and then extracted factors that were related to RNA polymerase II CTD serine 2 in HeLa cells. We further analyzed RNA polymerase II CTD serine 7 phosphorylation, of which their function is still unclear in HeLa cells. Our results were characterized by the similarity of localization for transcription factor/histone modification in the ENCODE data set, and this suggests that our model is appropriate for understanding ChIP-seq data for factors where their function is unknown.

DOI： 10.1093/nar/gks1010

Publishing type：Research paper (scientific journal)  

Testosterone is a final product of androgenic hormone biosynthesis, and Leydig cells are known to be the primary source of androgens. In the mammalian testis, two distinct populations of Leydig cells, the fetal and the adult Leydig cells, develop sequentially, and these two cell types differ both morphologically and functionally. It is well known that the adult Leydig cells maintain male reproductive function by producing testosterone. However, it has been controversial whether fetal Leydig cells can produce testosterone, and the synthetic pathway of testosterone in the fetal testis is not fully understood. In the present study, we generated transgenic mice in which enhanced green fluorescence protein was expressed under the control of a fetal Leydig cell-specific enhancer of the Ad4BP/SF-1 (Nr5a1) gene. The transgene construct was prepared by mutating the LIM homeodomain transcription factor (LHX9)-binding sequence in the promoter, which abolished promoter activity in the undifferentiated testicular cells. These transgenic mice were used to collect highly pure fetal Leydig cells. Gene expression and steroidogenic enzyme activities in the fetal Leydig cells as well as in the fetal Sertoli cells and adult Leydig cells were analyzed. Our results revealed that the fetal Leydig cells synthesize only androstenedione because they lack expression of Hsd17b3, and fetal Sertoli cells convert androstenedione to testosterone, whereas adult Leydig cells synthesize testosterone by themselves. The current study demonstrated that both Leydig and Sertoli cells are required for testosterone synthesis in the mouse fetal testis. (Molecular Endocrinology 27: 63-73, 2013)

DOI： 10.1210/me.2012-1256

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

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DOI： 10.1016/j.febslet.2012.06.022

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Dermokine-beta is abundant in stratified epithelia and in differentiating cultured keratinocytes. In this study, we investigated the role of dermokine-beta in differentiation of keratinocytes. Treatment of keratinocytes or skin tumor cells with dermokine-beta attenuated phosphorylation of extracellular-signal-regulated kinase (ERK). Exposure of cells to dermokine-beta, as well as its carboxyl-terminus domain peptide, interrupted phosphorylation of ERK and stimulated dermokine gene expression. Inhibition of ERK signaling by its specific inhibitor also increased dermokine expression level. A combination of chemical cross-linking and immunoprecipitation, followed by proteomics analyses, identified glucose-regulated protein 78 (GRP78) as a dermokine-beta-associated protein. Blockage of GRP78 expression by a specific siRNA abrogated actions of dermokine-beta. These findings provide novel insights into the physiological significance of dermokine-beta in the epidermis.

DOI： 10.1016/j.febslet.2012.06.022

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DOI： 10.1038/emboj.2012.136

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Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation. The EMBO Journal (2012) 31, 2994-3007. doi: 10.1038/emboj.2012.136; Published online 8 May 2012

DOI： 10.1038/emboj.2012.136

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DOI： 10.1016/j.fob.2012.09.002

Publishing type：Research paper (scientific journal)  

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is synthesized in the ER, transported along the exocytic pathway, and expressed on the plasma membrane as a type I transmembrane protein. Upon extracellular stimulation, HB-EGF, either proHB-EGF or the shed form HB-EGF-CTF, undergoes endocytosis and is then transported retrogradely to the ER. In this study, we showed the essential contribution of the short cytoplasmic tail of HB-EGF (HB-EGF-cyto) to the bidirectional intracellular trafficking between the ER and plasma membrane and revealed several critical amino acids residues that are responsible for internalization from the plasma membrane and ER targeting. We suggest that these anterograde and retrograde sorting signals within HB-EGF-cyto are strictly regulated by protein modification and conformation. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.fob.2012.09.002

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DOI： 10.1038/cdd.2011.54

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Publishing type：Research paper (scientific journal)  

SF-1 (Steroidogenic Factor 1, NR5A1) is a tissue-specific transcription factor critical for the growth, development and differentiation of steroidogenic and a few other endocrine tissues. But how SF-1 regulates cell growth is not entirely clear. Here we found that SF-1 was localized to the centrosome in addition to the nucleus, and SF-1 depletion by shRNA caused centrosome over-duplication, aberrant mitosis and genomic instability, leading to a reduction of cell number. Centrosome amplification defect was rescued by both wild-type SF-1 and transcription-defective SF-1-G35E, suggesting a non-genomic activity of SF-1 involved in centrosome homeostasis. In addition, we identified in SF-1 a centrosome localization signal, whose overexpression led to reduced localization of both SF-1 and gamma-tubulin to the centrosome. Our results uncover a novel role of SF-1 in the control of centrosome homeostasis and genomic stability. Cell Death and Differentiation (2011) 18, 1836-1844; doi:10.1038/cdd.2011.54; published online 13 May 2011

DOI： 10.1038/cdd.2011.54

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DOI： 10.1186/1471-2164-12-516

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DOI： 10.1091/mbc.E10-10-0809

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Nuclear import of karyophilic proteins is carried out by a variety of mechanisms. We previously showed that two basic helix-loop-helix proteins, NeuroD1 and E47, synergistically affect each other&apos;s nuclear import. In this study, we dissected the molecular pathways underlying nuclear import of the NeuroD1/E47 heterodimer. In vitro nuclear import assays indicated that importin alpha family members are the major nuclear import receptors for E47. However, inhibition of importin alpha resulted in cytoplasmic retention of E47 that could be rescued by its binding partner, NeuroD1, through heterodimerization. In addition, nuclear import of NeuroD1 was importin alpha independent but importin beta 1 dependent. In primary neurons, localization of endogenous E47 was not affected by importin alpha inhibition, suggesting that neuronal E47 could be imported into the nucleus as a heterodimer with NeuroD1 by using importin beta 1 alone. We also found that E47 had similar nuclear import characteristics in C2C12 cells, where E47 heterodimerized with MyoD, another helix-loop-helix protein, suggesting functional conservation within the same family of transcription factors. Collectively, our data reveal that E47 is imported into the nucleus via multiple pathways, depending on the molecular binding mode, establishing a previously uncharacterized cross-talk between two distinct nuclear import pathways.

DOI： 10.1091/mbc.E10-10-0809

Publishing type：Research paper (scientific journal)  

Background: Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII), which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq.
Results: We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII.
Conclusions: We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

DOI： 10.1186/1471-2164-12-516

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1742-4658.2011.08197.x

Publishing type：Research paper (scientific journal)  

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase (G(q), G(12/13) and G(i))-dependent pathways, by deamidating a glutamine residue in the alpha subunit of these GTPases. However, the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cell-based assays. In the present study, we developed rat monoclonal antibodies, specifically recognizing the deamidated G alpha(q), to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated G alpha(q) only under reducing conditions. The C-terminal region of PMT, C-PMT, was more active than the full-length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other domains. These results not only support previous observations on toxicity, but also provide insights into the enzymatic nature of PMT. In addition, we present several lines of evidence that G alpha(11), as well as G alpha(q), could be a substrate for PMT.

DOI： 10.1111/j.1742-4658.2011.08197.x

Publishing type：Research paper (scientific journal)  

DOI： 10.1093/toxsci/kfq307

Publishing type：Research paper (scientific journal)  

In the present study, protein lysates from microdissected glutathione S-transferase placental-form-positive (GST-P(+)) foci and hepatocellular carcinomas from livers of rats treated with N-diethylnitrosamine followed by phenobarbital at doses of 0 and 500 ppm in the diet for 10 and 33 weeks were analyzed using QSTAR Elite liquid chromatography with tandem mass spectrometry and iTRAQ technology. Among 75 proteins, a total of 27 and 50 proteins displaying significant quantitative changes comparing with adjacent normal-appearing liver tissue were identified in GST-P(+) foci of initiation control and promotion groups, respectively, which are related to transcription, protein folding, cytoskeleton filaments reorganization, cell cycle control, nuclear factor (erythroid-derived 2)-like 2 (NRF2)-mediated oxidative stress responses, lipid metabolism, glutathione metabolism, oxidative phosphorylation, and signal transduction. Furthermore, Ingenuity Pathway and bioinformatic analyses revealed that expression changes of genes encoding proteins with altered expression detected in GST-P(+) foci are likely to be controlled by c-myc, NRF2, aryl hydrocarbon receptor, nuclear factor kappa B, and hepatocyte nuclear factor 4 transcriptional factors. Coordinated overexpression of mitochondrial chaperons prohibitin (PHB) and prohibitin 2 (PHB2), septin 9 (SEPT9), neurabin 1, and other cytoskeletal and functional proteins in areas of GST-P(+) foci during initiation and/or promotion stages of rat hepatocarcinogenesis was associated with induction of cell proliferation and might be responsible for the neoplastic transformation of rat liver preneoplastic lesions. Newly discovered elevation of PHB, PHB2, and SEPT9 in GST-P(+) foci and tumors, imply that they might play important role in the onset of liver cancer and be of potential values in the studies of hepatocarcinogenesis.

DOI： 10.1093/toxsci/kfq307

PubMed

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DOI： 10.1089/hyb.2010.0065

Publishing type：Research paper (scientific journal)  

Nup96 is a component of the Nup107-160 complex, the largest subunit of the nuclear pore complex. Nup96 is generated as a precursor protein with Nup98. However, the mechanism by which Nup96 contributes to cell function is not clear. We report here on the preparation of a monoclonal antibody (MAb) directed against mouse Nup96. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The antibody, MAb 4H5, specifically recognized Nup96, as evidenced by immunoblotting using the whole cell lysates. In immunostaining using MAb 4H5, a nuclear rim staining pattern was observed. This antibody will be useful in immunoblotting and immunolocalization experiments, as well as further analyses of the biological function and cellular dynamics of this protein.

DOI： 10.1089/hyb.2010.0065

Publishing type：Research paper (scientific journal)  

DOI： 10.1091/mbc.E09-12-1041

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DOI： 10.1091/mbc.e09-12-1041

PubMed

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DOI： 10.1089/hyb.2009.0107

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DOI： 10.1089/hyb.2009.0106

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DOI： 10.1089/hyb.2009.0117

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DOI： 10.1111/j.1365-2443.2010.01406.x

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Nuclear pore complexes (NPCs) are &apos;supramolecular complexes&apos; on the nuclear envelope assembled from multiple copies of approximately 30 different proteins called nucleoporins (Nups) that provide aqueous channels for nucleocytoplasmic transport during interphase. Although the structural aspects of NPCs have been characterized in detail, NPC formation and its regulation, especially during interphase, are poorly understood. In this study, using the temperature-sensitive RCC1 mutant tsBN2, a baby hamster kidney 21 cell line, we found that a lack of RCC1 activity inhibited NPC formation during interphase, suggesting that RanGTP is required for NPC formation during interphase in mammalian cells. Utilizing the reversible RCC1 activity in tsBN2 cells, we established a live-cell system that allows for the inhibition or initiation of NPC formation by changes in temperature. Our system enables the examination of NPC formation during interphase in living cells. As a lack of RCC1 decreased some Nups containing unstructured phenylalanine-glycine repeats in the NPC structure, we propose that RCC1 is also involved in maintaining NPC integrity during interphase in mammalian cells.

DOI： 10.1111/j.1365-2443.2010.01406.x

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CHD1 is a subfamily member of the CHD family, which possesses a chromodomain, a helicase domain, and a DNA-binding domain. The CHD family regulates gene expression by contributing to ATP-dependent chromatin remodeling. CHD1 exists in the transcriptionally active region and alters the chromatin structure. Little is known about the function of endogenous CHD1, however, and studies have been hindered by the lack of an antibody specific for CHD1 in mammals. In the present study, we established a monoclonal antibody specifically against CHD1 using the rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody showed specific binding to CHD1, allowing us to identify the deduced full-length CHD1. In addition, cell immunostaining clearly revealed the nuclear localization of CHD1. This monoclonal antibody will be useful for further analysis of CHD1 function in mammals.

DOI： 10.1089/hyb.2009.0106

PubMed

Publishing type：Research paper (scientific journal)  

Myogenic determination 1 (MyoD) is a myogenic regulatory factor (MRF) possessing a basic domain and a helix-loop-helix domain. MRFs play a critical role in myoblast fate and terminal differentiation. MyoD is a transcriptional factor that induces transcription by binding with gene regulatory factors expressed in skeletal muscle. As a master gene, MyoD also determines skeletal muscle differentiation. In this study, we established a monoclonal antibody specific for MyoD using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against MyoD could identify full-length MyoD. Moreover, immunocytochemical staining revealed a change in the expression of MyoD at the skeletal muscle differentiation stage. This monoclonal antibody against MyoD allows for further studies to elucidate the mechanism by which MyoD influences skeletal muscle differentiation.

DOI： 10.1089/hyb.2009.0117

Publishing type：Research paper (scientific journal)  

Dhx9/NDHII/RHA is a member of the DEAH family of proteins, which possess a double-stranded RNA-binding domain (dsRBD) and a helicase domain. The DEAH protein family plays a critical role in RNA metabolism. DEAH family members function as ATP-dependent RNA helicases and regulation of transcription. In the present study, we report the establishment of a monoclonal antibody specific for Dhx9 using the rat medial iliac lymph node method. Immunoblot analysis using our antibody against Dhx9 detected full-length Dhx9. In addition, immunocytochemical staining using our antibody against Dhx9 revealed the nuclear localization of Dhx9. This monoclonal antibody against Dhx9 will allow for further detailed studies of Dhx9 expression.

DOI： 10.1089/hyb.2009.0107

PubMed

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DOI： 10.1089/hyb.2009.0092

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DOI： 10.1089/hyb.2009.0090

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CHD2 is a member of the CHD family that contains chromodomain, helicase domain as well as DNA-binding domain. The CHD family is involved in gene expression and transcription by ATP-dependent chromatin remodeling. Analysis of mutant mouse revealed that CHD2 is involved in development as well as hematopoiesis, which suggests the involvement of CHD2 in gene expression. However, CHD2 has not yet been analyzed biochemically as there is no specific antibody against it. Here, we report on the establishment of specific monoclonal antibody (MAb) against CHD2 utilizing a rat medial iliac lymph node method. Through cell immunostaining utilizing established MAb to CHD2, we confirmed that CHD2 was localized in euchromatin. Additionally, IP-Western revealed that the expression level of full-length CHD2 did not change during the differentiation stage. Additionally, a specific signal was confirmed around 95 kDa at the undifferentiated stage. This clearly indicated that CHD2 was involved in specific gene expression at this stage. Thus, this antibody can contribute to elucidating the function of CHD2 in cell expression.

DOI： 10.1089/hyb.2009.0090

Publishing type：Research paper (scientific journal)  

The septin family of GTPase proteins has been shown to be important for cell division, cytoskeletal organization, and membrane-remodeling events. Septin 9 (SEPT9) is a member of the septin family (also designated MSF/eseptin/Sint1) and has been implicated in tumorigenesis. The present study reports on the preparation and properties of a monoclonal antibody (MAb) directed against SEPT9. The antibody was produced by hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 7B5 specifically recognized SEPT9, as evidenced by immunoblotting using a variety of extracts from cultured cells. In immunostaining using MAb 7B5, a filamentous pattern near the plasma membrane was observed. The MAb 7B5 promises to be useful in immunoblotting and immunostaining experiments in various cells and tissues to determine the expression levels of SEPT9, as well as to further the analysis of the biological function of this protein.

DOI： 10.1089/hyb.2009.0092

PubMed

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DOI： 10.1089/hyb.2009.0069

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DOI： 10.1089/hyb.2009.0101

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DOI： 10.1089/hyb.2009.0078.MAb

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CHD5 (chromodomain/helicase/DNA-binding protein 5) is a member of the CHD subfamily of chromatin remodeling Swi/Snf proteins, and has been recently identified as a tumor suppressor in a diverse range of human cancers. We report here on the establishment of a hybridoma cell line for producing a monoclonal antibody against CHD5 by the rat medial iliac lymph node method. Immunoblotting analyses indicated that this antibody, MAb 5A10, specifically recognizes endogenous CHD5. In immunostaining using the antibody, a nuclear staining pattern was observed. The monoclonal antibody will be useful in immunoblotting and immunolocalization experiments in a variety of cells and tissues, as well as in further studies of the biological function and cellular dynamics of this protein.

DOI： 10.1089/hyb.2009.0069

PubMed

Publishing type：Research paper (scientific journal)  

In mammals, primordial germ cells (PGCs) are generated in the extra-embryonic epiblast, and thereafter migrate into the developing gonads. Following the development of the gonads to the testes or ovaries, germ cells mature into sperms or eggs. In the present study, we report production and characterization of monoclonal antibodies (MAb) that recognize PGCs. Extracts from E12.5 mouse embryonic gonads were immunized as an antigen, and hybridomas were generated using the rat medial iliac lymph node method. The hybridoma supernatants were screened by immunohistochemical analyses of E12.5 mouse embryonic sections. The antibody, referred to herein as MAb 5B5, provided strong signals on PGCs. Moreover, immunofluorescence analyses using a variety of the tissue sections of mouse embryos revealed that MAb 5B5 also recognizes the ventricular zone of the cerebral cortex and the neural canal in the spinal cord in which neural specific stem cells are present in abundance. Based on these findings, MAb 5B5 might recognize stem cell-associated antigens.

DOI： 10.1089/hyb.2009.0101

PubMed

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DOI： 10.1089/hyb.2009.0078.MAb

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DOI： 10.1016/j.taap.2009.09.013

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DOI： 10.1038/nn.2451

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To search for a reliable biomarker of preneoplastic lesions arising early in mouse hepatocarcinogenesis the proteomes of microdissected basophilic foci, hepatocellular adenomas (HCAs), carcinomas (HCCs) and normal-appearing liver of B6C3F1 mice initiated with diethylnitrosamine (DEN) were analysed on anionic (Q10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays. Significant overexpression of cytokeratin 8 (CK8; m/z 54, 565), cytokeratin 18 (CK18; m/z 47,538) proteins was found in basophilic foci as well as in HCAs and HCCs. Furthermore, immunohistochemistry demonstrated profound overexpression of CK8 and CK18 proteins (CK8/18) in all basophilic foci, mixed cell type foci, HCAs and HCCs in B6CF1 and C57BL/6J mice initiated with DEN. A strong correlation between CK8/18-positive foci development and multiplicity of liver tumors in 1360171 and C57Bl/6J mice was further observed. Moreover, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with neoplastic transformation of mice liver basophilic foci. Elevation of CK8/18 was strongly correlated with induction of cell proliferation in basophilic foci and tumors. In conclusion, our data imply that CK8/18 is a novel reliable marker of preneoplastic lesions arising during mouse hepatocarcinogenesis which might be used for prediction of tumor development and evaluation of environmental agents as well as drugs and food additives using mouse liver tests. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.taap.2009.09.013

PubMed

Publishing type：Research paper (scientific journal)  

We report the characterization of a new member of the low-density lipoprotein receptor (LDLR) gene family designated LRP10. Human LRP10 cDNA encodes a 1905 amino acid type I membrane protein consisting of five functional domains characteristic of the LDLR gene family. CHO-ldlA7 cells transfected with human LRP10 cDNA bound LDLR-associated protein, but not beta-VLDL and HDL. Human LRP10 transcripts were primarily found in the brain, muscle and heart. in situ hybridization of the rat brain showed that the transcripts were intensely present in the cerebral cortex, hippocampus, choroid plexus, ependyma and granular layer. In the developing rat brain, transcript levels gradually increased from postnatal day 1 to 20. Immunofluorescence analysis indicated that LRP10 was observed in the ventricular zone of the embryonic day 14.5 mouse cerebral cortex. The present studies suggest that LRP10 may play a significant role in the brain physiology other than lipoprotein metabolism. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.12.033

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Behavioral circadian rhythms are controlled by a neuronal circuit consisting of diverse neuronal subgroups. To understand the molecular mechanisms underlying the roles of neuronal subgroups within the Drosophila circadian circuit, we used cell-type specific gene-expression profiling and identified a large number of genes specifically expressed in all clock neurons or in two important subgroups. Moreover, we identified and characterized two circadian genes, which are expressed specifically in subsets of clock cells and affect different aspects of rhythms. The transcription factor Fer2 is expressed in ventral lateral neurons; it is required for the specification of lateral neurons and therefore their ability to drive locomotor rhythms. The Drosophila melanogaster homolog of the vertebrate circadian gene nocturnin is expressed in a subset of dorsal neurons and mediates the circadian light response. The approach should also enable the molecular dissection of many different Drosophila neuronal circuits.

DOI： 10.1038/nn.2451

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DOI： 10.11491/scej.2010f.0.677.0

CiNii Article

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DOI： 10.1089/hyb.2009.0041

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DOI： 10.1089/hyb.2009.0048

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DOI： 10.1089/hyb.2009.0063.MAb

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Glyoxalase I (GLO1) is a key enzyme that plays a role in the detoxification of methylglyoxal (MG), a toxic cellular metabolite produced during glycolysis. The present study reports on the preparation and properties of a monoclonal antibody (MAb) directed against mouse GLO1. The antibody was produced by hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 6F10 specifically recognized GLO1, as evidenced by immunoblotting using a variety of extracts from cultured cells. In immunostaining using MAb 6F10, a diffuse cytoplasmic and nuclear staining pattern was observed. The MAb 6F10 promises to be useful in immunoblotting and immunostaining experiments in various cells and tissues to determine the expression levels of GLO1, as well as to further analyze the biological function of this protein.

DOI： 10.1089/hyb.2009.0048

PubMed

Publishing type：Research paper (scientific journal)  

Brm-related gene-1 (Brg1) is a catalytic subunit of the SWI/SNF chromatin remodeling enzyme complex that has ATPase activity. This complex facilitates chromatin remodeling for gene expression by utilizing energy for ATP hydrolysis. It is well known that the SWI/SNF chromatin remodeling enzyme complex is essential for cell differentiation, cell cycle regulation, and embryogenesis. Here we report the establishment of a hybridoma cell line for producing an antibody against Brg1 subunit by the rat medial iliac lymph node method. Immunoblot analysis showed that our antibody can specifically recognize Brg1. It was revealed by immunocytochemistry that Brg1 is located in euchromatin of C2C12 myoblast nuclei. These data suggested this antibody is useful for analyzing molecular function of Brg1 protein in cells.

DOI： 10.1089/hyb.2009.0041

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DOI： 10.1089/hyb.2009.0063.MAb

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CiNii Article

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DOI： 10.1016/j.jbiosc.2009.08.378

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CiNii Article

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DOI： 10.1016/j.jbiosc.2009.08.378

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DOI： 10.1089/hyb.2008.0084

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DOI： 10.1089/hyb.2008.0096.MAb

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Ad4BP/SF-1 (adrenal4 binding protein/steroidogenic factor-1[NR5A1]) is an essential nuclear receptor required for animal reproduction and endocrine regulation. The present study reports on monoclonal antibodies (MAbs) directed against mouse Ad4BP/SF-1, which were produced by the hybridization of mouse myeloma cells with lymph node cells of an immunized rat. The produced MAbs reacted with both recombinant and endogenous Ad4BP/SF-1. These MAbs will be useful in immunolocalization and immunoblotting experiments conducted on different tissue types to determine the levels of expression of Ad4BP/SF-1 throughout development, as well as further analyses of the biological function and cellular dynamics of this protein.

DOI： 10.1089/hyb.2008.0084

PubMed

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DOI： 10.1089/hyb.2008.0096.MAb

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DOI： 10.1016/j.bbrc.2008.11.028

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Methylglyoxal (MG) is an endogenous metabolite in glycolysis and forms stable adducts primarily with arginine residues of intracellular proteins. The biological role of this modification in cell function is not known. In the present study, we found that a MG-detoxification enzyme glyoxalase I (GLO1) is mainly expressed in the ventricular zone (VZ) at embryonic clay 16 which neural stein and progenitor cells localize. Moreover, immunohistochemical analysis revealed that argpyrimidine, a major MG-arginine adduct, is predominantly produced in cortical plate neurons not VZ during cerebral Cortex development and is exclusively located in the nucleus. Immunoblotting experiment showed that the formation of argpyrimidine occurs oil some nuclear proteins of cortical neurons. To our knowledge, this is first report of the argpyrimidine formation in the nucleus of neuron. These findings Suggest that GLO1, which is dominantly expressed in the embryonic VZ, reduces the intracellular level of MG and Suppresses the formation of argpyrimidine in neural stern and progenitor cells. Argpyrimidine May contribute to the neural differentiation and/or the maintenance of the differentiated state via the modification of nuclear proteins. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.11.028

PubMed

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DOI： 10.1089/hyb.2008.0010

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DOI： 10.1089/hyb.2008.0023.MAb

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DOI： 10.1089/hyb.2008.0023.MAb

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DOI： 10.1210/me.2007-0444

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Ad4BP/SF-1 [adrenal4 binding protein/steroidogenic factor-1 (NR5A1)] is a factor important for animal reproduction and endocrine regulation, and its expression is tightly regulated in the gonad, adrenal gland, ventromedial hypothalamic nucleus, and pituitary gonadotrope. Despite its functional significance in the pituitary, the mechanisms underlying pituitary-specific expression of the gene remain to be uncovered. In this study, we demonstrate by transgenic mouse assays that the pituitary gonadotrope-specific enhancer is localized within the sixth intron of the gene. Functionally, the enhancer recapitulates endogenous Ad4BP/SF-1 expression in the fetal Rathke&apos;s pouch to the adult pituitary gonadotrope. Structurally, the enhancer consists of several elements conserved among animal species. Mutational analyses confirmed the significance of these elements for the enhancer function. One of these elements was able to interact both in vitro and in vivo with Pitx2 (pituitary homeobox 2), demonstrating that pituitary homeobox 2 regulates Ad4BP/SF-1 gene transcription in the pituitary gonadotrope via interaction with the gonadotrope-specific enhancer.

DOI： 10.1210/me.2007-0444

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The detailed properties of the enzyme from Pseudomonas aeruginosa, which catalyzes the N-acyl linkage between myristic acid and the N-terminal glycine residue of the octapeptide GNAAAARR-NH_2(PKA) in aqueous solution without ATP and CoA, were studied. The substrate specificity for the acyl peptide in the synthetic reaction was examined, and it was found that at least eight amino acid residues are required for the reaction and that the N-terminal glycine residue is not absolutely essential for the reaction because the activity was detected using the octapeptide that has an N-terminal alanine. The activity was also strongly affected by the amino acid sequence because the activity was very weak in the reaction using GARASVLS-NH_2(HIV-1p17^<gag>). The substrate specificity for fatty acids was also examined. In the reactions using lauric acid and decanoic acid, only slight activities were detected; however, those activities were very small compared with the activity in the reaction using myristic acid. In addition, the degradation of myristoyl PKA by the enzyme was detected, although there are only a few reports on demyristoylation. The optimum pH and temperature of the degradation reaction were consistent with those of the synthetic reaction. The degradation reaction was inhibited by divalent cations.

CiNii Article

Publishing type：Research paper (scientific journal)  

The detailed properties of the enzyme from Pseudomonas aeruginosa, which catalyzes the N-acyl linkage between myristic acid and the N-terminal glycine residue of the octapeptide GNAAAARR-NH_2(PKA) in aqueous solution without ATP and CoA, were studied. The substrate specificity for the acyl peptide in the synthetic reaction was examined, and it was found that at least eight amino acid residues are required for the reaction and that the N-terminal glycine residue is not absolutely essential for the reaction because the activity was detected using the octapeptide that has an N-terminal alanine. The activity was also strongly affected by the amino acid sequence because the activity was very weak in the reaction using GARASVLS-NH_2(HIV-1p17^<gag>). The substrate specificity for fatty acids was also examined. In the reactions using lauric acid and decanoic acid, only slight activities were detected; however, those activities were very small compared with the activity in the reaction using myristic acid. In addition, the degradation of myristoyl PKA by the enzyme was detected, although there are only a few reports on demyristoylation. The optimum pH and temperature of the degradation reaction were consistent with those of the synthetic reaction. The degradation reaction was inhibited by divalent cations.

CiNii Article

Publishing type：Research paper (scientific journal)  

The detailed properties of the enzyme from <i>Pseudomonas aeruginosa</i>, which catalyzes the <i>N</i>-acyl linkage between myristic acid and the <i>N</i>-terminal glycine residue of the octapeptide GNAAAARR-NH₂ (PKA) in aqueous solution without ATP and CoA, were studied. The substrate specificity for the acyl peptide in the synthetic reaction was examined, and it was found that at least eight amino acid residues are required for the reaction and that the <i>N</i>-terminal glycine residue is not absolutely essential for the reaction because the activity was detected using the octapeptide that has an <i>N</i>-terminal alanine. The activity was also strongly affected by the amino acid sequence because the activity was very weak in the reaction using GARASVLS-NH₂ (HIV-1p17^gag). The substrate specificity for fatty acids was also examined. In the reactions using lauric acid and decanoic acid, only slight activities were detected; however, those activities were very small compared with the activity in the reaction using myristic acid. In addition, the degradation of myristoyl PKA by the enzyme was detected, although there are only a few reports on demyristoylation. The optimum pH and temperature of the degradation reaction were consistent with those of the synthetic reaction. The degradation reaction was inhibited by divalent cations.<br>

DOI： 10.1263/jbb.105.282

CiNii Article

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DOI： 10.1083/jcb.200710022

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DOI： 10.1093/nar/gkm556

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Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

DOI： 10.1083/jcb.200710022

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The nuclear RNA export factor (NXF) family proteins have been implicated in various aspects of post-transcriptional gene expression. This study shows that mouse NXF7 exhibits heterologous localization, i.e. NXF7 associates with translating ribosomes, stress granules (SGs) and processing bodies (P-bodies), the latter two of which are believed to be cytoplasmic sites of storage, degradation and/or sorting of mRNAs. By yeast two-hybrid screening, a series of heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as possible binding partners for NXF7. Among them, hnRNP A3, which is believed to be involved in translational control and/or cytoplasmic localization of certain mRNAs, formed a stable complex with NXF7 in vitro. Although hnRNP A3 was not associated with translating ribosomes, it was co-localized with NXF7 in P-bodies. After exposing to oxidative stress, NXF7 trans-localized to SGs, whereas hnRNP A3 did not. In differentiated neuroblastoma Neuro2a cells, NXF7 was co-localized with hnRNP A3 in cell body and neurites. The amino terminal half of NXF7, which was required for stable complex formation with hnRNP A3, coincided with the region required for localization in both P-bodies and neuronal RNA granules. These findings suggest that NXF7 plays a role in sorting, transport and/or storage of mRNAs through interactions with hnRNP A3.

DOI： 10.1093/nar/gkm556

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DOI： 10.1074/jbc.M611703200

Publishing type：Research paper (scientific journal)  

Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.

DOI： 10.1074/jbc.M611703200

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bmc.2007.02.008

Publishing type：Research paper (scientific journal)  

Ceramides act as a second messenger of the apoptotic signaling process. The allylic alcohol portion comprising the C-3, C-4, and C-5 carbons is essential for this function. The suggestion has been made that this alcohol moiety is oxidized in mitochondria to a carbonyl moiety, with the generation of reactive oxygen species. However, there is no established precedent for the apoptotic performance of 3-ketoceramides thus presumed. In this work, we have synthesized three different types of short-chain 3-ketoceramides, that is, (2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A), (2S,4E,6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-ol (B), and (2S,4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene (C), and demonstrated that these 3-ketoceramides are capable of inducing effective apoptosis in human leukemia HL-60 cells. In particular, the two monoenoic compounds, A and C, are far more powerful than the corresponding alcoholic analogue, N-acetyl-D-erythro-sphingosine. Observations of DNA fragmentation, caspase-3 activation, and cytochrome c release from mitochondria provide substantiated evidence for mitochondrial apoptosis and the effects of exogenous glutathione on these phenomena are also discussed. (c) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmc.2007.02.008

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M608898200

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Many studies have examined transcriptional regulation during the initiation of skeletal muscle differentiation; however, there is less information regarding transcriptional control during adult myogenesis and during the maintenance of the differentiated state. MyoD and the mammalian SWI/SNF chromatinremodeling enzymes containing the Brg1 ATPase are necessary to induce myogenesis in cell culture models and in developing embryonic tissue, whereas myogenin and Brg1 are critical for the expression of the late genes that induce terminal muscle differentiation. Here, we demonstrate that myogenin also binds to its own promoter during the late stages of embryonic muscle development. As is the case during embryonic myogenesis, MyoD and Brg1 co-localize to the myogenin promoter in primary adult muscle satellite cells. However, in mature myofibers, myogenin and Brg1 are preferentially co-localized to the myogenin promoter. Thus, the myogenin promoter is occupied by different myogenic factors at different times of myogenesis. The relevance of myogenin in the continued expression from its own promoter is demonstrated in culture, where we show that myogenin, in the absence of MyoD, is capable of maintaining its own expression by recruiting the Brg1 ATPase to modify promoter chromatin structure and facilitate myogenin expression. Finally, we utilized in vivo electroporation to demonstrate that Brg1 is required for the continued production of the myogenin protein in newborn skeletal muscle tissue. These findings strongly suggest that the skeletal muscle phenotype is maintained by myogenin and the continuous activity of Brg1-based SWI/SNF chromatin-remodeling enzymes.

DOI： 10.1074/jbc.M608898200

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DOI： 10.1242/jcs.03207

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Nuclear pores are sophisticated gateways on the nuclear envelope that control macromolecular transport between the cytoplasm and nucleoplasm. So far the structural and functional aspects of nuclear pores have been extensively studied, but their distribution and density, which might reflect nuclear organization and function, remain unknown. Here, we report the cell-cycle-dependent dynamics of nuclear pores. Large distinct subdomains lacking nuclear pores are present on the nuclear surface of HeLaS3 cells in early cell-cycle stages. Such 'pore-free islands' gradually become dispersed in G1-S phase. Surprisingly, the islands are enriched with inner nuclear membrane proteins lamin A/C and emerin, but exclude lamin B. Lamin-A/C-enriched pore-free islands were also observed in human normal diploid fibroblasts and several cell lines, showing the generality of this phenomenon. Knockdown and ectopic expression analyses demonstrated that lamin A/C, but not emerin, plays an essential structural and regulatory role in the nuclear pore distribution and the formation of pore-free islands. These data thus provide strong evidence that the dynamics of nuclear pores are regulated by the reorganization of inner nuclear structures.

DOI： 10.1242/jcs.03207

Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M602280200

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Small ubiquitin-related modifiers, SUMO-2/3 and SUMO-1, are involved in gene regulation and nuclear structures. However, little is known about the roles of SUMO, in heterochromatin formation of mammalian cells. Here we demonstrate that SUMOs directly interact with human MCAF1, which forms complexes with either the methyl-CpG-binding protein MBD1 or SETDB1, which trimethylates histone H3 at lysine 9 (H3-K9) in the presence of MCAF1. Modification of MBD1 with either SUMO-2/3 or SUMO-1 facilitated the interaction between MBD1 and MCAF1, suggesting that SUMOylation links the methylation of DNA and histones. In a cultured human cell line, SUMOs were localized in MBD1- and MCAF1-containing heterochromatin regions that were enriched in trimethyl-H3-K9 and the heterochromatin proteins HP1beta and HP1gamma. Specific knockdown of either SUMO-2/3 or SUMO-1 induced dissociation of MCAF1, trimethyl-H3-K9, and the HP1 proteins from the MBD1-containing heterochromatin foci, suggesting a requirement for SUMOs for heterochromatin assembly. These findings provide insights into the roles of SUMOylation in the regulation of heterochromatin formation and gene silencing.

PubMed

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DOI： 10.1038/sj.emboj.7601225

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DOI： 10.1074/jbc.M512052200

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The microphthalmia transcription factor (Mitf) activates melanocyte-specific gene expression, is critical for survival and proliferation of melanocytes during development, and has been described as an oncogene in malignant melanoma. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that play a role in many developmental processes. To determine the requirement for SWI/SNF enzymes in melanocyte differentiation, we introduced Mitf into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, Brahma or Brahma-related gene 1 (BRG1). These dominant negative SWI/SNF components have been shown to inhibit gene activation events that normally require SWI/SNF enzymes. We found that Mitf-mediated activation of a subset of endogenous melanocyte-specific genes required SWI/SNF enzymes but that cell-cycle regulation occurred independently of SWI/SNF function. Activation of tyrosinase-related protein 1, a melanocyte-specific gene, correlated with SWI/SNF-dependent changes in chromatin accessibility at the endogenous locus. Both BRG1 and Mitf could be localized to the tyrosinase-related protein 1 and tyrosinase promoters by chromatin immunoprecipitation, whereas immunofluorescence and immunoprecipitation experiments indicate that Mitf and BRG1 co-localized in the nucleus and physically interacted. Together these results suggest that Mitf can recruit SWI/SNF enzymes to melanocyte- specific promoters for the activation of gene expression via induced changes in chromatin structure at endogenous loci.

DOI： 10.1074/jbc.M512052200

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

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PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.yexcr.2006.01.013

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SUMO modification plays a critical role in a number of cellular functions including nucleocytoplasmic transport, gene expression, cell cycle and formation of subnuclear structures such as promyelocytic leukemia (PML) bodies. In order to identify the sites where SUMOylation takes place in the cell, we developed an in situ SUMOylation assay using a semi-intact cell system and subsequently combined it with siRNA-based knockdown of nucleoporin RanBP2, also known as Nup358, which is one of the known SUMO E3 proteins. With the in situ SUMOylation assay, we found that both nuclear rim and PML bodies, besides mitotic apparatuses, are major targets for active SUMOylation. The ability to analyze possible SUMO conjugation sites would be a valuable tool to investigate where SUMO E3-like activities and/or SUMO substrates exist in the cell. Specific knockdown of RanBP2 completely abolished SUMOylation along the nuclear rim and dislocated RanGAP1 from the nuclear pore complexes. interestingly, the loss of RanBP2 markedly reduced the number of PML bodies, in contrast to other, normal-appearing nuclear compartments including the nuclear lamina, nucleolus and chromatin, suggesting a novel link between RanBP2 and PML bodies. SUMOylation facilitated by RanBP2 at the nuclear rim may be a key step for the formation of a particular subnuclear organization. Our data imply that SUMO E3 proteins like RanBP2 facilitate spatio-temporal SUMOylation for certain nuclear structure and function. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.yexcr.2006.01.013

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DOI： 10.1089/hyb.2006.25.51

PubMed

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The nuclear pore complex (NPC) is an enormous structure embedded in the double membrane of the nuclear envelope that acts as a passageway for nucleocytoplasmic transport. The vertebrate NPC is comprised of about 30 unique proteins. Nup62/p62, a major component of the NPC, has been reported to interact directly with several nuclear transport factors, including importin-beta and NTF2. However, it has not been shown how the interaction of Nup62/p62 with transport factors is involved in nucleocytoplasmic transport. The present study reports on the preparation of monoclonal antibodies (MAbs) directed against human Nup62/p62 and a functional analysis of Nup62/p62 using antibodies in living cells. Hybridomas producing the antibodies were produced by the hybridization of mouse myeloma cells with medial iliac lymph node cells from an immunized rat. These MAbs specifically recognized Nup62/p62 as evidenced by immunoblotting analysis using a nuclear membrane fraction. In the immunostaining using MAbs, a punctuate nuclear rim staining pattern was observed. Moreover, cytoplasmic injected-anti-Nup62/p62 MAbs were rapidly targeted to the nuclear pore of cultured cells and some of them inhibited normal cell division, causing the formation of abnormal nuclei. The antibodies described in this study provide the means for immunochemical analyses of the NPC protein Nup62/p62 in mammalian cells, and represent useful molecular tools that should permit a better understanding of the biological roles and cellular dynamics of this protein in nucleocytoplasmic transport, cell division, and nuclear organization.

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DOI： 10.1016/j.bmc.2005.10.029

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DOI： 10.1089/hyb.2005.24.244

PubMed

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Nup98 is a component of nuclear pore complexes, which are large protein assemblies embedded in the nuclear envelope. Previous studies have shown that Nup98 interacts with several transport factors and plays a critical part in nuclear trafficking. However, the mechanism by which Nup98 contributes to nuclear trafficking is not clear. The present study reports on the preparation of a monoclonal antibody (MAb) directed against human Nup98. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. This antibody, MAb 21110, specifically recognized Nup98, as evidenced by immunoblotting using a nuclear membrane fraction. In immunostaining using MAb 21110, a punctuate nuclear rim staining pattern was observed. This MAb will be useful in immunoblotting and immunolocalization experiments in various cells and tissues, as well as further analyses of the biological function and cellular dynamics of this protein.

DOI： 10.1089/hyb.2005.24.244

PubMed

Publishing type：Research paper (scientific journal)  

Nup98 is a component of nuclear pore complexes, which are large protein assemblies embedded in the nuclear envelope. Previous studies have shown that Nup98 interacts with several transport factors and plays a critical part in nuclear trafficking. However, the mechanism by which Nup98 contributes to nuclear trafficking is not clear. The present study reports on the preparation of a monoclonal antibody (MAb) directed against human Nup98. The antibody was produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat. This antibody, MAb 21110, specifically recognized Nup98, as evidenced by immunoblotting using a nuclear membrane fraction. In immunostaining using MAb 21110, a punctuate nuclear rim staining pattern was observed. This MAb will be useful in immunoblotting and immunolocalization experiments in various cells and tissues, as well as further analyses of the biological function and cellular dynamics of this protein.

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DOI： 10.1074/jbc.M413476200

PubMed

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The intracellular localization of a RING-IBR protein, RBCK1, possessing DNA binding and transcriptional activities, has been investigated. The endogenous RBCK1 was found in both the cytoplasm and nucleus. Particularly in the nucleus, it was localized in the granular structures, most likely nuclear bodies. In contrast, the over-expressed RBCK1 was detected exclusively in the cytoplasm. When the cells were treated with leptomycin B, the over-expressed RBCK1 accumulated in the nuclear bodies. These results suggest that RBCK1 possesses the signal sequences responsible for the nuclear-cytoplasmic translocation. Mutational analysis of RBCK1 has indicated that an N-terminal region containing Leu-142 and Leu-145 and a C-terminal one containing the RING-IBR domain serve as the nuclear export and localization signals, respectively. Thus, RBCK1 is a transcription factor dynamically shuttling between cytoplasm and nucleus. Furthermore, RBCK1 was found to interact with nuclear body proteins, CREB-binding protein (CBP), and promyelocytic leukemia protein (PML). Coexpression of RBCK1 with CBP significantly enhanced the transcriptional activity of RBCK1. Although PML per se showed no effect on the transcriptional activity of RBCK1, the CBP-enhanced activity was repressed by coexpression with PML, presumably through the interaction of PML and CBP. Taken together, our data demonstrate that RBCK1 is involved in transcriptional machinery in the nuclear bodies, and its transcriptional activity is regulated by nucleocytoplasmic shuttling. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M413476200

PubMed

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DOI： 10.1016/j.bbrc.2005.03.063

PubMed

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The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is involved in a variety of biological functions. STAT3 is activated by cytokines and growth factors via the phosphorylation of a tyrosine residue, dimerization, and subsequent nuclear translocation. However, the mechanism of its nuclear translocation is unclear. A study of the cytokine-stimulated import of STAT3 into the nucleus is reported herein. An oncostatin M (OSM)-dependent nuclear import assay system was first established in living cells. Using this system, we demonstrated that the microinjection of the importin alpha 5/NPI-1 mutant, an anti-importin P antibody, and the RanQ69L mutant inhibited the nuclear import of STAT3. Second, we showed that tyrosine-phosphorylated STAT3 associates, not only with importin alpha 5/NPI-1 but also with other importin alpha s, as a result of OSM stimulation, as evidenced by a solution binding assay. These findings suggest that the extracellular signal-dependent nuclear transport of STAT3 is mediated by various importin alpha s, importin beta, and Ran. (c) 2005 Elsevier Inc. All rights reserved.

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DOI： 10.1016/j.cub.2005.01.058

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DOI： 10.1016/j.jconrel.2004.11.024

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in order to enhance the nuclear import of exogenous genes, novel plasmid DNA/importin-beta conjugates, which consist of a biotinylated plasmid. DNA and a recombinant streptavidin-fused importin-beta, were prepared. The spacer length between plasmid DNA and biotin and the number of introduced biotin were adjusted. The microinjection of plasmid DNA/importin-beta conjugates into the cytoplasm of NIH3T3 cells resulted in the nuclear localization of conjugates and the higher expression efficiency, compared to intact plasmid DNA alone. These results indicate that plasmid DNA/importin-beta conjugates would be an important tool to enhance the nuclear localization of exogenous DNA in non-viral gene delivery system. (c) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jconrel.2004.11.024

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Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure [1]. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity [2, 3]. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation [4, 5]. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGIuR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGIuR5 activation and regulates spine morphology to stabilize the synaptic structure.

DOI： 10.1016/j.cub.2005.01.058

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DOI： 10.1089/hyb.2004.23.301

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DOI： 10.1089/hyb.2004.23.301

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DOI： 10.1016/j.legalmed.2004.05.004

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DOI： 10.1016/j.bmc.2003.10.040

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DOI： 10.1089/153685903771797110

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DOI： 10.1089/153685903771797110

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DOI： 10.1021/jm030125p

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DOI： 10.1021/jm030125p

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DOI： 10.1016/s0968-0896(03)00228-1

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DOI： 10.1021/jo0206824

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DOI： 10.1021/jo0206824

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DOI： 10.1021/bc025602h

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DOI： 10.1021/bc025602h

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DOI： 10.1016/s0960-894x(02)01026-0

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Other URL： http://search.jamas.or.jp/link/ui/2016232199

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