Jun. 2013, German Research and Innovation Forum Tokyo/German Chamber of Commerce and Industry in Japan, German Innovation Award Gottfried Wagner Prize 2013　　　　　　　　　　　　　　　
FRET-Based Biosensors for Accurate Prediction of Responses to Molecular Target Drugs in Chronic Myeloid Leukemia
OHBA Yusuke

SARS-CoV-2 uptake and inflammatory response in senescent endothelial cells are regulated by the BSG/VEGFR2 pathway.
Yuya Sakurai, Yoichiro Fujioka, Nako Maishi, Ryo Takeda, Yusuke Ohba, Michihito Sasaki, Takahito Teshirogi, Wataru Ito, Yasuhiro Hida, Aya Matsuda, Kanta Kido, Yasuko Orba, Hirofumi Sawa, Kyoko Hida
Proceedings of the National Academy of Sciences of the United States of America, 122, 31, e2502724122, 05 Aug. 2025, [International Magazine]
English, Scientific journal, Aging is a risk factor for severe COVID-19, characterized by vascular endothelial dysfunction. Although possible susceptibility of vascular endothelial cells (ECs) to SARS-CoV-2 infection has been suggested, the details of entry into cells have not been clarified. Previously, we reported that in an aged mouse model of severe COVID-19, ECs show a massive viral uptake and inflammatory response. Here, we focused on the endocytic capacity of senescent ECs. We found that the senescent ECs showed high endocytic capacity and SARS-CoV-2 virus uptake. This triggers an nuclear factor-kappa B (NF-κB) pathway-mediated inflammatory response. Further, Basigin enhanced endocytosis in the senescent ECs by activating the intracellular vascular endothelial growth factor signaling. Thus, EC senescence is associated with enhanced SARS-CoV-2 endocytosis and subsequent vascular endothelial dysfunction. This could prove a potential target for treating severe COVID-19 in older adults.

The crucial role of intercellular calcium wave propagation triggered by influenza A virus in promoting infection.
Fumiya Kozawa, Tomokazu Tamura, Naoki Takahashi, Taishi Kakizuka, Taro Ichimura, Rumi Shimada, Yasuyuki Hashimoto, Hironoshin Onizuska, Sayaka Kashiwagi, Tomoko Kamasaki, Maho Amano, Takeharu Nagai, Takasuke Fukuhara, Yoichiro Fujioka, Yusuke Ohba
Cell communication and signaling : CCS, 23, 1, 361, 361, Cold Spring Harbor Laboratory, 02 Aug. 2025, [International Magazine]
English, Scientific journal, Abstract

Influenza A viruses (IAVs) initially infect a few host cells, and the infection subsequently spreads to neighboring cells. However, the molecular mechanisms underlying this expansion remain unclear. Here, we show that IAV infection upregulates the frequency of intercellular calcium wave propagations (iCWPs) that mediate the spread of IAVs. ADP released from initially infected cells mediated iCWPs via the P2Y₁receptor. The generation of iCWPs and spread of viral infection were inhibited by a P2Y₁antagonist. Enhanced endocytosis in the surrounding cells that received ADP signaling upregulated viral entry. Expression of IAV matrix protein 2 (M2) in initially infected cells triggered iCWPs through ADP diffusion, thereby increasing infection, whereas an ion permeability-deficient mutation of M2 or inhibition of its ion channel activity suppressed iCWPs. Therefore, intercellular calcium signaling is essential for early expansion and potential establishment of IAV infection, which offers a promising target for IAV prophylaxis.

Intranasal administration of stem cell-derived exosome alleviates cognitive impairment against subarachnoid hemorrhage.
Shuho Gotoh, Masahito Kawabori, Sho Yamaguchi, Yo Nakahara, Erika Yoshie, Kohtarou Konno, Yuki Mizuno, Yoichiro Fujioka, Yusuke Ohba, Yuji Kuge, Masahiko Watanabe, Miki Fujimura
Experimental neurology, 386, 115143, 115143, Apr. 2025, [International Magazine]
English, Scientific journal, INTRODUCTION: Brain damage caused by subarachnoid hemorrhage (SAH) currently lacks effective treatment, leading to stagnation in the improvement of functional outcomes for decades. Recent studies have demonstrated the therapeutic potential of exosomes released from mesenchymal stem cells (MSC), which effectively attenuate neuronal apoptosis and inflammation in neurological diseases. Due to the challenge of systemic dilution associated with intravenous administration, intranasal delivery has emerged as a novel approach for targeting the brain. In this study, we investigate the effects of intranasally administered MSC-derived exosomes in a SAH animal model and elucidate their mode of action. METHODS: Exosomes were isolated from the cell supernatants of amnion-derived MSC. SAH was induced in 8-week-old Sprague-Dawley rats using an autologous blood prechiasmatic cistern injection model. A total of 1.2 × 1010 particles of exosomes in 200 μL of PBS or PBS alone were intranasally administered immediately and 24 h post-injury. Neurological function was assessed up to 7 days after injury, and histological analysis was performed to evaluate their anti-apoptotic and anti-inflammatory effects. The biodistribution of exosomes was assessed using PET/CT imaging of 64Cu labeled exosome. In vitro analyses were performed using primary glial cells and cell lines to evaluate the anti-inflammatory effects of the exosomes. RESULTS: Animals treated with exosomes exhibited significant improvement in cognitive function compared with PBS treated animal. Apoptotic cells and inflammation were reduced for the exosome group in the hippocampal CA1 area and in cortex, resulting in better neuronal cell survival. Blood brain barrier permeability was also preserved in the exosome group. Nuclear imaging revealed that exosomes were primarily transferred to the olfactory nerve and cerebrum; furthermore, exosomes were also observed in the trigeminal nerve and brainstem, where exosomes were co-localized with microglia and with endothelial cells. In vitro assessment showed that exosome administration ameliorated inflammation and prevented the death of glial cells. CONCLUSIONS: MSC-derived exosomes were successfully transferred into the brain through intranasal administration and alleviated brain damage following SAH.

BT-DNBS: a novel cyanine-based turn-on fluorescent probe with large Stokes shift for sensitive and selective detection of biothiols in live-cell imaging.
Shuai Zhang, Yoichiro Fujioka, Yusuke Ohba, Koji Yamada
RSC advances, 15, 1, 135, 141, 02 Jan. 2025, [International Magazine]
English, Scientific journal, Detecting biothiols like glutathione (GSH), homocysteine (Hcy), and cysteine (Cys) is key to understanding their roles in health and disease. We developed BT-DNBS, a cyanine-based turn-on fluorescent probe with a dinitrobenzenesulfonyl (DNBS) quencher group. Upon biothiol interaction, the quencher is cleaved, restoring fluorescence. The resulting probe BT-NH shows a maximum emission wavelength at 630 nm and a large Stokes shift (≈200 nm), enhancing detection accuracy. Low cytotoxicity and high time resolution make BT-DNBS suitable for live-cell imaging. Imaging of A431 cells confirmed intracellular biothiol detection, with NEM pre-treatment reducing fluorescence, verifying specificity. BT-DNBS holds promise for biomedical research, particularly in disease diagnostics.

Intravenous Administration of Mesenchymal Stem Cell-Derived Exosome Alleviates Spinal Cord Injury by Regulating Neutrophil Extracellular Trap Formation through Exosomal miR-125a-3p.
Yutaka Morishima, Masahito Kawabori, Kazuyoshi Yamazaki, Soichiro Takamiya, Sho Yamaguchi, Yo Nakahara, Hajime Senjo, Daigo Hashimoto, Sakiko Masuda, Yoichiro Fujioka, Yusuke Ohba, Yuki Mizuno, Yuji Kuge, Miki Fujimura
International journal of molecular sciences, 25, 4, 18 Feb. 2024, [International Magazine]
English, Scientific journal, Spinal cord injury (SCI) leads to devastating sequelae, demanding effective treatments. Recent advancements have unveiled the role of neutrophil extracellular traps (NETs) produced by infiltrated neutrophils in exacerbating secondary inflammation after SCI, making it a potential target for treatment intervention. Previous research has established that intravenous administration of stem cell-derived exosomes can mitigate injuries. While stem cell-derived exosomes have demonstrated the ability to modulate microglial reactions and enhance blood-brain barrier integrity, their impact on neutrophil deactivation, especially in the context of NETs, remains poorly understood. This study aims to investigate the effects of intravenous administration of MSC-derived exosomes, with a specific focus on NET formation, and to elucidate the associated molecular mechanisms. Exosomes were isolated from the cell supernatants of amnion-derived mesenchymal stem cells using the ultracentrifugation method. Spinal cord injuries were induced in Sprague-Dawley rats (9 weeks old) using a clip injury model, and 100 μg of exosomes in 1 mL of PBS or PBS alone were intravenously administered 24 h post-injury. Motor function was assessed serially for up to 28 days following the injury. On Day 3 and Day 28, spinal cord specimens were analyzed to evaluate the extent of injury and the formation of NETs. Flow cytometry was employed to examine the formation of circulating neutrophil NETs. Exogenous miRNA was electroporated into neutrophil to evaluate the effect of inflammatory NET formation. Finally, the biodistribution of exosomes was assessed using 64Cu-labeled exosomes in animal positron emission tomography (PET). Rats treated with exosomes exhibited a substantial improvement in motor function recovery and a reduction in injury size. Notably, there was a significant decrease in neutrophil infiltration and NET formation within the spinal cord, as well as a reduction in neutrophils forming NETs in the circulation. In vitro investigations indicated that exosomes accumulated in the vicinity of the nuclei of activated neutrophils, and neutrophils electroporated with the miR-125a-3p mimic exhibited a significantly diminished NET formation, while miR-125a-3p inhibitor reversed the effect. PET studies revealed that, although the majority of the transplanted exosomes were sequestered in the liver and spleen, a notably high quantity of exosomes was detected in the damaged spinal cord when compared to normal rats. MSC-derived exosomes play a pivotal role in alleviating spinal cord injury, in part through the deactivation of NET formation via miR-125a-3p.

血管内皮細胞におけるSARS-CoV-2侵入機構の解析(Analysis of SARS-CoV-2 uptake mechanism in vascular endothelial cells)　　　　　　　　　　　　　　　
桜井 優弥, 間石 奈湖, 藤岡 容一郎, 大場 雄介, 武田 遼, 佐々木 道仁, 大場 靖子, 樋田 泰浩, 澤 洋文, 樋田 京子
日本病理学会会誌, 113, 1, 369, 369, (一社)日本病理学会, Feb. 2024
English

Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.
Masatoshi Maeki, Shuya Uno, Kaisei Sugiura, Yusuke Sato, Yoichiro Fujioka, Akihiko Ishida, Yusuke Ohba, Hideyoshi Harashima, Manabu Tokeshi
ACS applied materials & interfaces, 16, 2, 2110, 2119, 17 Jan. 2024, [International Magazine]
English, Scientific journal, RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.

Strength in numbers: Unleashing the potential of trans-scale scope AMATERAS for massive cell quantification.
Taro Ichimura, Taishi Kakizuka, Yuki Sato, Yoichiro Fujioka, Yusuke Ohba, Kazuki Horikawa, Takeharu Nagai
Biophysics and physicobiology, 21, Supplemental, e211017, Biophysical Society of Japan, 2024, [Domestic magazines]
English, Scientific journal, Singularity biology is a scientific field that targets drastic state changes in multicellular systems, aiming to discover the key cells that induce the state change and investigate the mechanisms behind them. To achieve this goal, we developed a trans-scale optical imaging system (trans-scale scope), that is capable of capturing both macroscale changes across the entire system and the micro-scale behavior of individual cells, surpassing the cell observation capabilities of traditional microscopes. We developed two units of the trans-scale scope, named AMATERAS-1 and -2, which demonstrated the ability to observe multicellular systems consisting of over one million cells in a single field of view with sub-cellular resolution. This flagship instrument has been used to observe the dynamics of various cell species, with the advantage of being able to observe a large number of cells, allowing the detection and analysis of rare events and cells such as leader cells in multicellular pattern formation and cells that spontaneously initiate calcium waves. In this paper, we present the design concept of AMATERAS, the optical configuration, and several examples of observations, and demonstrate how the strength-in-numbers works in life sciences.

Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles.
Shunyi Li, Hiroki Toriumi, Daisuke Takahashi, Tomoko Kamasaki, Yoichiro Fujioka, Satoru Nagatoishi, Jinting Li, Yiwei Liu, Takanatsu Hosokawa, Kouhei Tsumoto, Yusuke Ohba, Yoshiki Katayama, Daisuke Murakami, Koji Hase, Takeshi Mori
Biomaterials, 303, 122381, 122381, Dec. 2023, [International Magazine]
English, Scientific journal, Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has many disadvantages related to efficiency, safety, long-term duration, and patient compliance. Dendritic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde crosslinked antigen to mannoprotein (MAN) have been established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency of DCs. To overcome this, we developed a MAN-coated ovalbumin (OVA) nanoparticle (MDO), which uses intermolecular disulfide bond to crosslink OVA and MAN. MDO effectively targeted DCs resulting in tolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or glutaraldehyde crosslinked nanoparticles. In vitro and in vivo experiments showed that DCs exposed to MDO induced Treg cells. Moreover, MDO had low reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new approach for the efficient and safe treatment of allergies.

Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5.
Tomokazu Tamura, Daichi Yamasoba, Yoshitaka Oda, Jumpei Ito, Tomoko Kamasaki, Naganori Nao, Rina Hashimoto, Yoichiro Fujioka, Rigel Suzuki, Lei Wang, Hayato Ito, Yukie Kashima, Izumi Kimura, Mai Kishimoto, Masumi Tsuda, Hirofumi Sawa, Kumiko Yoshimatsu, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Yutaka Suzuki, Yusuke Ohba, Isao Yokota, Keita Matsuno, Kazuo Takayama, Shinya Tanaka, Kei Sato, Takasuke Fukuhara
Communications biology, 6, 1, 772, 772, 24 Jul. 2023, [International Magazine]
English, Scientific journal, The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.

Interaction between PI3K and the VDAC2 channel tethers Ras-PI3K-positive endosomes to mitochondria and promotes endosome maturation.
Aya O Satoh, Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Asuka Nanbo, Maho Amano, Yusuke Ohba
Cell reports, 42, 3, 112229, 112229, 28 Mar. 2023, [International Magazine]
English, Scientific journal, Intracellular organelles of mammalian cells communicate with one another during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identify voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 tethers endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and promotes clathrin-independent endocytosis, as well as endosome maturation at membrane association sites. With an optogenetics system to induce mitochondrion-endosome association, we find that, in addition to its structural role in such association, VDAC2 is functionally implicated in the promotion of endosome maturation. The mitochondrion-endosome association thus plays a role in the regulation of clathrin-independent endocytosis and endosome maturation.

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses
Tomokazu Tamura, Shiho Torii, Kentaro Kajiwara, Itsuki Anzai, Yoichiro Fujioka, Kisho Noda, Shuhei Taguwa, Yuhei Morioka, Rigel Suzuki, Yuzy Fauzyah, Chikako Ono, Yusuke Ohba, Masato Okada, Takasuke Fukuhara, Yoshiharu Matsuura
PLOS Pathogens, 18, 6, e1010593, e1010593, Public Library of Science (PLoS), 03 Jun. 2022
Scientific journal, Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of E^rns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

Stimulation of the mitochondrial calcium uniporter mitigates chronic heart failure-associated ventricular arrhythmia in mice.
Hikaru Hagiwara, Masaya Watanabe, Yoichiro Fujioka, Takahide Kadosaka, Takuya Koizumi, Taro Koya, Motoki Nakao, Rui Kamada, Taro Temma, Kazufumi Okada, Jose Antonio Moreno, Ohyun Kwon, Hisakata Sabe, Yusuke Ohba, Toshihisa Anzai
Heart rhythm, 19, 10, 1725, 1735, 31 May 2022, [International Magazine]
English, Scientific journal, BACKGROUND: An aberrant increase in the diastolic calcium concentration ([Ca2+]i) level is a hallmark of heart failure (HF) and the cause of delayed afterdepolarization and ventricular arrhythmia (VA). Although mitochondria play a role in regulating [Ca2+]i, whether they can compensate for the [Ca2+]i abnormality in ventricular myocytes is unknown. OBJECTIVE: We investigated whether enhanced Ca2+ uptake of mitochondria may compensate for an abnormal increase in the [Ca2+]i of ventricular myocytes in HF to effectively mitigate VA. METHODS: We used a HF mouse model, in which myocardial infarction was induced by permanent left anterior descending coronary artery ligation. The mitochondrial Ca2+ uniporter was stimulated by kaempferol. Ca2+ dynamics and membrane potential were measured using an epifluorescence microscope, a confocal microscope, and the perforated patch-clamp technique. VA was induced in the Langendorff-perfused hearts, and the hemodynamic parameters were measured using a microtip transducer catheter. RESULTS: Protein expression of the mitochondrial Ca2+ uniporter, as assessed by its subunit expression, did not change between HF and sham mice. Treatment of cardiomyocytes with kaempferol, isolated from HF mice at 28 days after coronary ligation, reduced the appearance of aberrant diastolic [Ca2+]i waves and sparks and spontaneous action potentials. Kaempferol effectively reduced the VA occurring in Langendorff-perfused hearts. Intravenous administration of kaempferol did not markedly affect the left ventricular hemodynamic parameters. CONCLUSION: The effects of kaempferol in HF of mice implied that mitochondria may have the potential to compensate for abnormal [Ca2+]i. Mechanisms involved in mitochondrial Ca2+ uptake may provide novel targets to treat HF-associated VA.

A method for the generation of pseudovirus particles bearing SARS coronavirus spike protein in high yields.
Yoichiro Fujioka, Sayaka Kashiwagi, Aiko Yoshida, Aya O Satoh, Mari Fujioka, Maho Amano, Yohei Yamauchi, Yusuke Ohba
Cell structure and function, 47, 1, 43, 53, 28 Apr. 2022, [Domestic magazines]
English, Scientific journal, The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 spike protein in high yield. We found that a large proportion of pseudovirions produced with the conventional transient expression system lacked coronavirus spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus spike protein allowed the efficient production of progeny pseudoviruses decorated with spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.Keywords: severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, pseudovirus, vesicular stomatitis virus (VSV), spike protein.

Direct visualization of glucagon-like peptide-1 secretion by fluorescent fusion proteins.
Atsushi Tsuzuki, Yoichiro Fujioka, Aiko Yoshida, Sayaka Kashiwagi, Maho Amano, Tohru Hira, Akinobu Nakamura, Hideaki Miyoshi, Tatsuya Atsumi, Yusuke Ohba
Journal of diabetes investigation, 13, 7, 1134, 1139, 04 Apr. 2022, [Domestic magazines]
English, Scientific journal, Live-cell imaging with fluorescent proteins (FPs) is a powerful tool for investigating the exocytosis processes of hormones. However, the secretion process of glucagon-like peptide-1 (GLP-1) has not been visualized by FPs, which might be because tagging FPs inhibits GLP-1 synthesis through the post-translational processing from proglucagon. Here, we have developed FP-tagged GLP-1 by inserting FPs into the middle of GLP-1 and adding the proglucagon signal peptide. Confocal imaging confirmed that GLP-1 fused to FPs with high folding efficiency showed granular structure, in which secretory vesicle markers colocalized. The fluorescence intensity of FP in the culture supernatant from cells treated with KCl or forskolin was significantly increased compared with those from untreated cells. Furthermore, FP-tagged GLP-1 enables direct visualization of stimulation-dependent exocytosis of GLP-1 at a single granule resolution with total internal reflection fluorescence microscopy. FP-tagged GLP-1 might facilitate the screening of GLP-1 secretagogues and the discovery of new antidiabetic drugs.

Imaging technology that enables simultaneous visualization of weak interaction interfaces and cell responses
Aiko Yoshida, Yoichiro Fujioka, Maho Amano, Yusuke Ohba
Drug Delivery System, 37, 2, 102, 111, Japan Society of Drug Delivery System, 25 Mar. 2022
Scientific journal

Schlafen family member 11 indicates favorable prognosis of patients with head and neck cancer following platinum-based chemoradiotherapy.
Seijiro Hamada, Satoshi Kano, Junko Murai, Takayoshi Suzuki, Nayuta Tsushima, Takatsugu Mizumachi, Masanobu Suzuki, Tsuyoshi Takashima, Daiki Taniyama, Naoya Sakamoto, Yoichiro Fujioka, Yusuke Ohba, Akihiro Homma
Frontiers in oncology, 12, 978875, 978875, 2022, [International Magazine]
English, Scientific journal, Recently, Schlafen family member 11 (SLFN11) has been reported to increase the sensitivity of cancer cells to DNA-damaging agents, including platinum derivatives; thus, SLFN11 may be a predictive biomarker for platinum-based chemoradiotherapy (CRT). In this study, we examined whether SLFN11 expression was associated with the therapeutic outcome of platinum-based CRT in head and neck squamous cell carcinoma (HNSCC). We performed immunohistochemical analyses for SLFN11 expression in 161 HNSCC tissues from patients who had been administered cisplatin-based CRT and examined the correlation between SLFN11 expression and progression-free survival (PFS). Additionally, SLFN11 expression was examined in 10 paired samples obtained before and after CRT in patients with local failure. Furthermore, in vitro experiments were performed using several HNSCC cell lines and isogenic SLFN11-knockout cells to assess the association between SLFN11 expression and drug sensitivity. PFS was found to be significantly better in the SLFN11-positive group than in the SLFN11-negative group among the 161 patients (5-year PFS: 78.8% vs. 52.8%, respectively, p < 0.001). Similar results were observed for the PFS at each primary site. The percentage of SLFN11 positivity was lower in tumor samples from patients with local failure after CRT than that in the corresponding primary tumors before CRT in 8 of 10 cases. Results of the in vitro assay demonstrated that SLFN11-knockout cells exhibited reduced sensitivity to DNA-damaging agents but not to the non-DNA-damaging agent docetaxel. Our findings suggest that SLFN11 may serve as a potential biomarker for predicting the response of HNSCC patients to platinum-based CRT.

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis
Tadasuke Tsukiyama, Juqi Zou, Jihoon Kim, Shohei Ogamino, Yuki Shino, Takamasa Masuda, Alessandra Merenda, Masaki Matsumoto, Yoichiro Fujioka, Tomonori Hirose, Sayuri Terai, Hidehisa Takahashi, Tohru Ishitani, Keiichi I. Nakayama, Yusuke Ohba, Bon-Kyoung Koo, Shigetsugu Hatakeyama
Nature Communications, 11, 1, Springer Science and Business Media LLC, Dec. 2020
Scientific journal,
Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.

Calcium Wave Promotes Cell Extrusion.
Yasuto Takeuchi, Rika Narumi, Ryutaro Akiyama, Elisa Vitiello, Takanobu Shirai, Nobuyuki Tanimura, Keisuke Kuromiya, Susumu Ishikawa, Mihoko Kajita, Masazumi Tada, Yukinari Haraoka, Yuki Akieda, Tohru Ishitani, Yoichiro Fujioka, Yusuke Ohba, Sohei Yamada, Yoichiroh Hosokawa, Yusuke Toyama, Takaaki Matsui, Yasuyuki Fujita
Current biology : CB, 30, 4, 670, 681, 24 Feb. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.

An antiviral drug screening platform with a FRET biosensor for measurement of arenavirus Z assembly
Tatsuaki Mizutani, Yusuke Ohba, Satoshi Mizuta, Jiro Yasuda, Shuzo Urata
Cell Structure and Function, Japan Society for Cell Biology, 2020
Scientific journal

Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins.
Sayaka Kashiwagi, Yoichiro Fujioka, Aya O Satoh, Aiko Yoshida, Mari Fujioka, Prabha Nepal, Atsushi Tsuzuki, Ozora Aoki, Sarad Paudel, Hitoshi Sasajima, Yusuke Ohba
Cell structure and function, 44, 2, 183, 194, 26 Dec. 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (avGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type avGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pH-sensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.Key words: fluorescent protein, organelle, fusion protein, mitochondria.

Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function.
Sayaka Kashiwagi, Yoichiro Fujioka, Takeshi Kondo, Aya O Satoh, Aiko Yoshida, Mari Fujioka, Hitoshi Sasajima, Maho Amano, Takanori Teshima, Yusuke Ohba
Cell structure and function, 44, 2, 195, 204, 26 Dec. 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.

Clinical efficacy and safety of first-line nilotinib therapy and evaluation of the clinical utility of the FRET-based drug sensitivity test.
Takeshi Kondo, Mari Fujioka, Shinichi Fujisawa, Kaori Sato, Masumi Tsuda, Takuto Miyagishima, Akio Mori, Hiroshi Iwasaki, Yasutaka Kakinoki, Satoshi Yamamoto, Yoshihito Haseyama, Seisho Ando, Motohiro Shindo, Shuichi Ota, Mitsutoshi Kurosawa, Yusuke Ohba, Takanori Teshima
International journal of hematology, 110, 4, 482, 489, Oct. 2019, [Peer-reviewed], [Domestic magazines]
English, Nilotinib is widely used for primary treatment of patients with chronic myelogenous leukemia (CML). We previously reported that use of an FRET-based drug sensitivity test at diagnosis efficiently predicts the response to treatment with imatinib or dasatinib. Here, we conducted a phase-II study to evaluate the efficacy and safety of nilotinib treatment and identify useful biomarkers, including results of the FRET-based drug sensitivity test, for predicting treatment response. Data from 42 patients were used in the analysis. Major molecular response (MMR), MR4, and MR4.5 rates at 12 months were 64.3, 42.9, and 28.6%, respectively. Grade 3/4 non-hematologic adverse events occurred in 11 patients (26.2%). The dose intensity of nilotinib (> 76.44%) and halving time (HT, < 13.312 days) were identified as significant factors for MMR at 12 months. However, when we focused on patients whose dose intensity of nilotinib was > 76.44%, the FRET-based drug sensitivity test became a predictive factor of MR4 achievement at 12 months. Our study reconfirmed the efficacy and safety of nilotinib treatment in CML patients. Moreover, our results suggest that the FRET-based drug sensitivity test is an independent predictor for achievement of MR4 in patients treated with a sufficient dose intensity of nilotinib.

Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer.
Nako Maishi, Hiroshi Kikuchi, Masumi Sato, Hiroko Nagao-Kitamoto, Dorcas A Annan, Shogo Baba, Takayuki Hojo, Misa Yanagiya, Yusuke Ohba, Genichiro Ishii, Kenkichi Masutomi, Nobuo Shinohara, Yasuhiro Hida, Kyoko Hida
International journal of molecular sciences, 20, 18, 17 Sep. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Tumor angiogenesis research and antiangiogenic drug development make use of cultured endothelial cells (ECs) including the human microvascular ECs among others. However, it has been reported that tumor ECs (TECs) are different from normal ECs (NECs). To functionally validate antiangiogenic drugs, cultured TECs are indispensable tools, but are not commercially available. Primary human TECs are available only in small quantities from surgical specimens and have a short life span in vitro due to their cellular senescence. We established immortalized human TECs (h-imTECs) and their normal counterparts (h-imNECs) by infection with lentivirus producing simian virus 40 large T antigen and human telomerase reverse transcriptase to overcome the replication barriers. These ECs exhibited an extended life span and retained their characteristic endothelial morphology, expression of endothelial marker, and ability of tube formation. Furthermore, h-imTECs showed their specific characteristics as TECs, such as increased proliferation and upregulation of TEC markers. Treatment with bevacizumab, an antiangiogenic drug, dramatically decreased h-imTEC survival, whereas the same treatment failed to alter immortalized NEC survival. Hence, these h-imTECs could be a valuable tool for drug screening to develop novel therapeutic agents specific to TECs or functional biological assays in tumor angiogenesis research.

A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection.
Yoichiro Fujioka, Aya O Satoh, Kosui Horiuchi, Mari Fujioka, Kaori Tsutsumi, Junko Sasaki, Prabha Nepal, Sayaka Kashiwagi, Sarad Paudel, Shinya Nishide, Asuka Nanbo, Takehiko Sasaki, Yusuke Ohba
Cell structure and function, 44, 1, 61, 74, 25 Apr. 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.

Antibody-free digital influenza virus counting based on neuraminidase activity.
Kazuhito V Tabata, Yoshihiro Minagawa, Yuko Kawaguchi, Mana Ono, Yoshiki Moriizumi, Seiya Yamayoshi, Yoichiro Fujioka, Yusuke Ohba, Yoshihiro Kawaoka, Hiroyuki Noji
Scientific reports, 9, 1, 1067, 1067, 31 Jan. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes. By analyzing 600,000 reactors, the practical limit of detection reached the order of 103 (PFU)/mL, only 10-times less sensitive than RT-PCR and more than 1000-times sensitive than commercial rapid test kits (RIDTs). Interestingly, neuraminidase activity differed among virions. The coefficient of variance was 30-40%, evidently broader than that of alkaline phosphatase measured as a model enzyme for comparison, suggesting the heterogeneity in size and integrity among influenza virus particles. Sensitivity to oseltamivir also differed between virions. We also tested DIViC using clinical gargle samples that imposes less burden for sampling while with less virus titre. The comparison with RIDTs showed that DIViC was largely superior to RIDTs in the sensitivity with the clinical samples although a few false-positive signals were observed in some clinical samples that remains as a technical challenge.

Budding of Ebola Virus Particles Requires the Rab11-Dependent Endocytic Recycling Pathway.
Asuka Nanbo, Yusuke Ohba
The Journal of infectious diseases, 218, suppl_5, S388-S396, 22 Nov. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, The Ebola virus-encoded major matrix protein VP40 traffics to the plasma membrane, which leads to the formation of filamentous viral particles and subsequent viral egress. However, the cellular machineries underlying this process are not fully understood. In the present study, we have assessed the role of host endocytic recycling in Ebola virus particle formation. We found that a small GTPase Rab11, which regulates recycling of molecules among the trans-Golgi network, recycling endosomes, and the plasma membrane, was incorporated in Ebola virus-like particles. Although Rab11 predominantly localized in the perinuclear region, it distributed diffusely in the cytoplasm and partly localized in the periphery of the cells transiently expressing VP40. In contrast, Rab11 exhibited a perinuclear distribution when 2 VP40 derivatives that lack ability to traffic to the plasma membrane were expressed. Finally, expression of a dominant-negative form of Rab11 or knockdown of Rab11 inhibited both VP40-induced clusters at the plasma membrane and release of viral-like particles. Taken together, our findings demonstrate that Ebola virus exploits host endocytic recycling machinery to facilitate the trafficking of VP40 to the cell surface and the subsequent release of viral-like particles for its establishment of efficient viral egress.

ダーモスコープを用いた術後植皮片の観察　　　　　　　　　　　　　　　
北村 真也, 柳 輝希, 高島 有香, 今福 恵輔, 秦 洋郎, 清水 宏, 藤岡 容一朗, 大場 雄介
日本皮膚科学会雑誌, 128, 12, 2665, 2665, (公社)日本皮膚科学会, Nov. 2018
Japanese

Infection of Epstein⁻Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines.
Asuka Nanbo, Harutaka Katano, Michiyo Kataoka, Shiho Hoshina, Tsuyoshi Sekizuka, Makoto Kuroda, Yusuke Ohba
Cancers, 10, 7, 19 Jul. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Infection of Epstein⁻Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.

Pretreatment evaluation of fluorescence resonance energy transfer-based drug sensitivity test for patients with chronic myelogenous leukemia treated with dasatinib.
Takeshi Kondo, Mari Fujioka, Masumi Tsuda, Kazunori Murai, Kohei Yamaguchi, Takuto Miyagishima, Motohiro Shindo, Takahiro Nagashima, Kentaro Wakasa, Nozomu Fujimoto, Satoshi Yamamoto, Masakatsu Yonezumi, Souichi Saito, Shinji Sato, Kazuei Ogawa, Takaaki Chou, Reiko Watanabe, Yuichi Kato, Shuichiro Takahashi, Yoshiaki Okano, Joji Yamamoto, Masatsugu Ohta, Hiroaki Iijima, Koji Oba, Satoshi Kishino, Junichi Sakamoto, Yoji Ishida, Yusuke Ohba, Takanori Teshima
Cancer science, 109, 7, 2256, 2265, Jul. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Tyrosine kinase inhibitors (TKI) are used for primary therapy in patients with newly diagnosed CML. However, a reliable method for optimal selection of a TKI from the viewpoint of drug sensitivity of CML cells has not been established. We have developed a FRET-based drug sensitivity test in which a CrkL-derived fluorescent biosensor efficiently quantifies the kinase activity of BCR-ABL of living cells and sensitively evaluates the inhibitory activity of a TKI against BCR-ABL. Here, we validated the utility of the FRET-based drug sensitivity test carried out at diagnosis for predicting the molecular efficacy. Sixty-two patients with newly diagnosed chronic phase CML were enrolled in this study and treated with dasatinib. Bone marrow cells at diagnosis were subjected to FRET analysis. The ΔFRET value was calculated by subtraction of FRET efficiency in the presence of dasatinib from that in the absence of dasatinib. Treatment response was evaluated every 3 months by the BCR-ABL1 International Scale. Based on the ΔFRET value and molecular response, a threshold of the ΔFRET value in the top 10% of FRET efficiency was set to 0.31. Patients with ΔFRET value ≥0.31 had significantly superior molecular responses (MMR at 6 and 9 months and both MR4 and MR4.5 at 6, 9, and 12 months) compared with the responses in patients with ΔFRET value <0.31. These results suggest that the FRET-based drug sensitivity test at diagnosis can predict early and deep molecular responses. This study is registered with UMIN Clinical Trials Registry (UMIN000006358).

A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells.
Yoichiro Fujioka, Shinya Nishide, Toyoyuki Ose, Tadaki Suzuki, Izumi Kato, Hideo Fukuhara, Mari Fujioka, Kosui Horiuchi, Aya O Satoh, Prabha Nepal, Sayaka Kashiwagi, Jing Wang, Mika Horiguchi, Yuko Sato, Sarad Paudel, Asuka Nanbo, Tadaaki Miyazaki, Hideki Hasegawa, Katsumi Maenaka, Yusuke Ohba
Cell host & microbe, 23, 6, 809, 818, 13 Jun. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.

体外診断薬開発のための慢性骨髄性白血病におけるBCR-ABL活性測定用改良型FRETバイオセンサー　　　　　　　　　　　　　　　
大場 雄介, 近藤 健, 豊嶋 崇徳
臨床薬理の進歩, 39, 18, 26, (公財)臨床薬理研究振興財団, Jun. 2018
Japanese, アミノ酸置換および核外移行シグナル(NES)の導入により、切断抵抗性が向上した改良型Picklesバイオセンサーを開発したので報告した。フェルスター共鳴エネルギー移動(FRET)に基づくバイオセンサーPickles 2.31のために開発されたオリジナルのプロトコールでは、評価対象細胞をCFPとYFPの蛍光強度比に従って選択する。この基準に従ってPickles 2.31とPickles 2.34 NESの特性を比較したところ、薬剤処理の有無にかかわらず基準を満たす細胞の数が5%から25%増加した。さらに、実際の薬効評価指標であるFRET効率(FRET/CFP蛍光強度比)についても単一細胞レベルで評価した。以前の報告において使用した定義では、FRET効率が2.04より高い細胞は有意に高いBCR-ABL活性を示す。この基準によれば、改良型バイオセンサーはコントロールサンプル中に四つのFRET-high細胞を検出できるが、プロトタイプでは2.04以上の細胞は一つしか検出されなかった。この増加は分析対象細胞数の増加が検出能の向上に寄与した結果と考えられた。

Dermoscopic evaluation for skin grafts after surgery; neo-vascularization correlates with survival of skin grafts: A prospective study
Shinya Kitamura, Teruki Yanagi, Yuka Inamura-Takashima, Keisuke Imafuku, Hiroo Hata, Yoichiro Fujioka, Yusuke Ohba, Hiroshi Shimizu
Journal of Dermatological Science, 90, 2, 213, 216, Elsevier Ireland Ltd, 01 May 2018, [Peer-reviewed]
English

miR-23a promotes invasion of glioblastoma via HOXD10-regulated glial-mesenchymal transition.
Kazuhiro Yachi, Masumi Tsuda, Shinji Kohsaka, Lei Wang, Yoshitaka Oda, Satoshi Tanikawa, Yusuke Ohba, Shinya Tanaka
Signal transduction and targeted therapy, 3, 33, 33, 2018, [Peer-reviewed], [International Magazine]
English, Glioblastoma is the most aggressive and invasive brain tumor and has a poor prognosis; elucidating the underlying molecular mechanisms is essential to select molecular targeted therapies. Here, we investigated the effect of microRNAs on the marked invasiveness of glioblastoma. U373 glioblastoma cells were infected with 140 different microRNAs from an OncomiR library, and the effects of the invasion-related microRNAs and targeted molecules were investigated after repeated Matrigel invasion assays. Screening of the OncomiR library identified miR-23a as a key regulator of glioblastoma invasion. In six glioblastoma cell lines, a positive correlation was detected between the expression levels of miR-23a and invasiveness. A luciferase reporter assay demonstrated that homeobox D10 (HOXD10) was a miR-23a-target molecule, which was verified by high scores from both the PicTar and miRanda algorithms. Forced expression of miR-23a induced expression of invasion-related molecules, including uPAR, RhoA, and RhoC, and altered expression of glial-mesenchymal transition markers such as Snail, Slug, MMP2, MMP9, MMP14, and E-cadherin; however, these changes in expression levels were reversed by HOXD10 overexpression. Thus, miR-23a significantly promoted invasion of glioblastoma cells with polarized formation of focal adhesions, while exogenous HOXD10 overexpression reversed these phenomena. Here, we identify miR-23a-regulated HOXD10 as a pivotal regulator of invasion in glioblastoma, providing a novel mechanism for the aggressive invasiveness of this tumor and providing insight into potential therapeutic targets.

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.
Asuka Nanbo, Junki Maruyama, Masaki Imai, Michiko Ujie, Yoichiro Fujioka, Shinya Nishide, Ayato Takada, Yusuke Ohba, Yoshihiro Kawaoka
PLoS pathogens, 14, 1, e1006848, Jan. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.

Epstein-Barr Virus Acquires Its Final Envelope on Intracellular Compartments With Golgi Markers.
Asuka Nanbo, Takeshi Noda, Yusuke Ohba
Frontiers in microbiology, 9, 454, 454, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Herpesvirus subfamilies typically acquire their final envelope in various cytoplasmic compartments such as the trans-Golgi network (TGN), and endosomes prior to their secretion into the extracellular space. However, the sites for the final envelopment of Epstein-Barr virus (EBV), a ubiquitous human gamma herpesvirus, are poorly understood. Here, we characterized the sites for the final envelopment of EBV in Burkitt's lymphoma cell lines induced into the lytic cycle by crosslinking cell surface IgG. Electron microscopy revealed the various stages of maturation and egress of progeny virions including mature EBV in irregular cytoplasmic vesicles. Immunofluorescence staining showed that gp350/220, the major EBV glycoprotein, and the viral capsid antigen, p18, efficiently colocalized with a cis-Golgi marker, GM130. gp350/220 partly colocalized with the TGN, which was distributed in a fragmented and dispersed pattern in the cells induced into the lytic cycle. In contrast, limited colocalization was observed between gp350/220 and endosomal markers, such as a multi-vesicular bodies marker, CD63, a recycling endosome marker, Rab11, and a regulatory secretion vesicles marker, Rab27a. Finally, we observed that treatment of cells with brefeldin A, an inhibitor of vesicle trafficking between the endoplasmic reticulum and Golgi apparatus, resulted in the perinuclear accumulation of gp350/220 and inhibition of its distribution to the plasma membrane. Brefeldin A also inhibited the release of infectious EBV. Taken together, our findings support a model in which EBV acquires its final envelope in intracellular compartments containing markers of Golgi apparatus, providing new insights into how EBV matures.

The Role of Transforming Growth Factor β in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission.
Asuka Nanbo, Makoto Ohashi, Hironori Yoshiyama, Yusuke Ohba
Frontiers in microbiology, 9, 984, 984, 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, Infection of Epstein-Barr virus (EBV), a ubiquitous human gamma herpesvirus, is closely linked to various lymphoid and epithelial malignancies. Previous studies demonstrated that the efficiency of EBV infection in epithelial cells is significantly enhanced by coculturing them with latently infected B cells relative to cell-free infection, suggesting that cell-to-cell contact-mediated viral transmission is the dominant mode of infection by EBV in epithelial cells. However, a detailed mechanism underlying this process has not been fully understood. In the present study, we assessed the role of transforming growth factor β (TGF-β), which is known to induce EBV's lytic cycle by upregulation of EBV's latent-lytic switch BZLF1 gene. We have found that 5 days of cocultivation facilitated cell-to-cell contact-mediated EBV transmission. Replication of EBV was induced in cocultured B cells both with and without a direct cell contact in a time-dependent manner. Treatment of a blocking antibody for TGF-β suppressed both induction of the lytic cycle in cocultured B cells and subsequent viral transmission. Cocultivation with epithelial cells facilitated expression of TGF-β receptors in B cells and increased their susceptibility to TGF-β. Finally, we confirmed the spontaneous secretion of TGF-β from epithelial cells, which was not affected by cell-contact. In contrast, the extracellular microvesicles, exosomes derived from cocultured cells partly contributed to cell-to-cell contact-mediated viral transmission. Taken together, our findings support a role for TGF-β derived from epithelial cells in efficient viral transmission, which fosters induction of the viral lytic cycle in the donor B cells.

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes
Shunsuke Kon, Kojiro Ishibashi, Hiroto Katoh, Sho Kitamoto, Takanobu Shirai, Shinya Tanaka, Mihoko Kajita, Susumu Ishikawa, Hajime Yamauchi, Yuta Yako, Tomoko Kamasaki, Tomohiro Matsumoto, Hirotaka Watanabe, Riku Egami, Ayana Sasaki, Atsuko Nishikawa, Ikumi Kameda, Takeshi Maruyama, Rika Narumi, Tomoko Morita, Yoshiteru Sasaki, Ryosuke Enoki, Sato Honma, Hiromi Imamura, Masanobu Oshima, Tomoyoshi Soga, Jun-ichi Miyazaki, Michael R. Duchen, Jin-Min Nam, Yasuhito Onodera, Shingo Yoshioka, Junichi Kikuta, Masaru Ishii, Masamichi Imajo, Eisuke Nishida, Yoichiro Fujioka, Yusuke Ohba, Toshiro Sato, Yasuyuki Fujita
NATURE CELL BIOLOGY, 19, 5, 530, +, May 2017, [Peer-reviewed]
English, Scientific journal

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells
Sayaka Saitoh, Takeshi Maruyama, Yuta Yako, Mihoko Kajita, Yoichiro Fujioka, Yusuke Ohba, Nobuhiro Kasai, Natsu Sugama, Shunsuke Kon, Susumu Ishikawa, Takashi Hayashi, Tomohiro Yamazaki, Masazumi Tada, Yasuyuki Fujita
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 114, 12, E2327, E2336, Mar. 2017, [Peer-reviewed]
English, Scientific journal

Leukemogenic kinase FIP1L1-PDGFRA and a small ubiquitin-like modifier E3 ligase, PIAS1, form a positive cross-talk through their enzymatic activities
Makoto Ibata, Junko Iwasaki, Yoichiro Fujioka, Koji Nakagawa, Stephanie Darmanin, Masahiro Onozawa, Daigo Hashimoto, Yusuke Ohba, Shigetsugu Hatakeyama, Takanori Teshima, Takeshi Kondo
CANCER SCIENCE, 108, 2, 200, 207, Feb. 2017, [Peer-reviewed]
English, Scientific journal

Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells
Mika Horiguchi, Mari Fujioka, Takeshi Kondo, Yoichiro Fujioka, Xinxin Li, Kosui Horiuchi, Aya O. Satoh, Prabha Nepal, Shinya Nishide, Asuka Nanbo, Takanori Teshima, Yusuke Ohba
CELL STRUCTURE AND FUNCTION, 42, 1, 15, 26, 2017, [Peer-reviewed]
English, Scientific journal

SH2 domain-based FRET biosensor for measuring BCR-ABL activity in living CML cells
Mari Fujioka, Yumi Asano, Shigeyuki Nakada, Yusuke Ohba
Methods in Molecular Biology, 1555, 513, 534, Humana Press Inc., 2017, [Peer-reviewed]
English, In book

Fluorescence bioimaging of intracellular signaling and its clinical application
Ohba Yusuke, Fujioka Yoichiro
Journal of Oral Biosciences, 58, 4, 113, 119, Nov. 2016, [Peer-reviewed]

Epstein-Barr virus exploits host endocytic machinery for cell-to-cell viral transmission rather than a virological synapse
Asuka Nanbo, Kunihiro Kachi, Hironori Yoshiyama, Yusuke Ohba
JOURNAL OF GENERAL VIROLOGY, 97, 11, 2989, 3006, Nov. 2016, [Peer-reviewed]
English, Scientific journal

Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan
Nako Maishi, Yusuke Ohba, Kosuke Akiyama, Noritaka Ohga, Jun-ichi Hamada, Hiroko Nagao-Kitamoto, Mohammad Towfik Alam, Kazuyuki Yamamoto, Taisuke Kawamoto, Nobuo Inoue, Akinobu Taketomi, Masanobu Shindoh, Yasuhiro Hida, Kyoko Hida
SCIENTIFIC REPORTS, 6, 28039, Jun. 2016, [Peer-reviewed]
English, Scientific journal

Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia
Ryu Okumura, Takashi Kurakawa, Takashi Nakano, Hisako Kayama, Makoto Kinoshita, Daisuke Motooka, Kazuyoshi Gotoh, Taishi Kimura, Naganori Kamiyama, Takashi Kusu, Yoshiyasu Ueda, Hong Wu, Hideki Iijima, Soumik Barman, Hideki Osawa, Hiroshi Matsuno, Junichi Nishimura, Yusuke Ohba, Shota Nakamura, Tetsuya Iida, Masahiro Yamamoto, Eiji Umemoto, Koichi Sano, Kiyoshi Takeda
NATURE, 532, 7597, 117, +, Apr. 2016, [Peer-reviewed]
English, Scientific journal

Receptor activator of NF-kappa B ligand induces cell adhesion and integrin alpha 2 expression via NF-kappa B in head and neck cancers
Tamaki Yamada, Masumi Tsuda, Takanori Wagatsuma, Yoichiro Fujioka, Mari Fujioka, Aya O. Satoh, Kosui Horiuchi, Shinya Nishide, Asuka Nanbo, Yasunori Totsuka, Hisashi Haga, Shinya Tanaka, Masanobu Shindoh, Yusuke Ohba
SCIENTIFIC REPORTS, 6, 23545, Mar. 2016, [Peer-reviewed]
English, Scientific journal

A role of the sphingosine-1-phosphate (S1P)-S1P receptor 2 pathway in epithelial defense against cancer (EDAC).
Sayaka Yamamoto, Yuta Yako, Yoichiro Fujioka, Mihoko Kajita, Takeshi Kameyama, Shunsuke Kon, Susumu Ishikawa, Yusuke Ohba, Yusuke Ohno, Akio Kihara, Yasuyuki Fujita
Molecular biology of the cell, 27, 3, 491, 9, 01 Feb. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal, At the initial step of carcinogenesis, transformation occurs in single cells within epithelia, where the newly emerging transformed cells are surrounded by normal epithelial cells. A recent study revealed that normal epithelial cells have an ability to sense and actively eliminate the neighboring transformed cells, a process named epithelial defense against cancer (EDAC). However, the molecular mechanism of this tumor-suppressive activity is largely unknown. In this study, we investigated a role for the sphingosine-1-phosphate (S1P)-S1P receptor 2 (S1PR2) pathway in EDAC. First, we show that addition of the S1PR2 inhibitor significantly suppresses apical extrusion of RasV12-transformed cells that are surrounded by normal cells. In addition, knockdown of S1PR2 in normal cells induces the same effect, indicating that S1PR2 in the surrounding normal cells plays a positive role in the apical elimination of the transformed cells. Of importance, not endogenous S1P but exogenous S1P is involved in this process. By using FRET analyses, we demonstrate that S1PR2 mediates Rho activation in normal cells neighboring RasV12-transformed cells, thereby promoting accumulation of filamin, a crucial regulator of EDAC. Collectively these data indicate that S1P is a key extrinsic factor that affects the outcome of cell competition between normal and transformed epithelial cells.

Attenuation of ligand-induced activation of angiotensin II type 1 receptor signaling by the type 2 receptor via protein kinase C
Takayuki Inuzuka, Yoichiro Fujioka, Masumi Tsuda, Mari Fujioka, Aya O. Satoh, Kosui Horiuchi, Shinya Nishide, Asuka Nanbo, Shinya Tanaka, Yusuke Ohba
SCIENTIFIC REPORTS, 6, 21613, Feb. 2016, [Peer-reviewed]
English, Scientific journal

腫瘍血管内皮細胞はbiglycanの分泌を介してがんの転移を促進する　　　　　　　　　　　　　　　
間石 奈湖, 大場 雄介, 秋山 廣輔, 大賀 則孝, 浜田 淳一, 北本 宗子[永尾], Mohammad Alam T., 進藤 正信, 樋田 泰浩, 樋田 京子
日本癌学会総会記事, 74回, E, 1030, 日本癌学会, Oct. 2015, [Peer-reviewed]
English

Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation
Yoshinori Makino, Masumi Tsuda, Yusuke Ohba, Hiroshi Nishihara, Hirofumi Sawa, Kazuo Nagashima, Shinya Tanaka
CELL COMMUNICATION AND SIGNALING, 13, 35, Jul. 2015, [Peer-reviewed]
English, Scientific journal

Adaptor protein CRK induces epithelial-mesenchymal transition and metastasis of bladder cancer cells through HGF/c-Met feedback loop.
Ryuji Matsumoto, Masumi Tsuda, Lei Wang, Nako Maishi, Takashige Abe, Taichi Kimura, Mishie Tanino, Hiroshi Nishihara, Kyoko Hida, Yusuke Ohba, Nobuo Shinohara, Katsuya Nonomura, Shinya Tanaka
Cancer science, 106, 6, 709, 17, Jun. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal, We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial-mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial-mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues.

Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling
Tadasuke Tsukiyama, Akimasa Fukui, Sayuri Terai, Yoichiro Fujioka, Keisuke Shinada, Hidehisa Takahashi, Terry P. Yamaguchi, Yusuke Ohba, Shigetsugu Hatakeyama
MOLECULAR AND CELLULAR BIOLOGY, 35, 11, 2007, 2023, Jun. 2015, [Peer-reviewed]
English, Scientific journal

Fluorescent Protein-based Biosensors to Visualize Signal Transduction beneath the Plasma Membrane
Yoichiro Fujioka, Asuka Nanbo, Shin-ya Nishide, Yusuke Ohba
ANALYTICAL SCIENCES, 31, 4, 267, 274, Apr. 2015, [Peer-reviewed]
English

Inhibition of Multidrug Transporter in Tumor Endothelial Cells Enhances Antiangiogenic Effects of Low-Dose Metronomic Paclitaxel
Kosuke Akiyama, Nako Maishi, Noritaka Ohga, Yasuhiro Hida, Yusuke Ohba, Mohammad Towfik Alam, Taisuke Kawamoto, Hitomi Ohmura, Kenji Yamada, Chisaho Torii, Masanobu Shindoh, Kyoko Hida
AMERICAN JOURNAL OF PATHOLOGY, 185, 2, 572, 580, Feb. 2015, [Peer-reviewed]
English, Scientific journal

[The Ras-PI3K signaling is involved in the regulation of endocytosis and virus internalization].
Fujioka Y, Ohba Y
Seikagaku. The Journal of Japanese Biochemical Society, 87, 1, 91, 100, Feb. 2015, [Peer-reviewed]

P18/Stathmin1 is regulated by miR-31 in ovarian cancer in response to taxane.
Mohamed Kamel Hassan, Hidemichi Watari, Takashi Mitamura, Zainab Mohamed, Sherif F El-Khamisy, Yusuke Ohba, Noriaki Sakuragi
Oncoscience, 2, 3, 294, 308, 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal, MicroRNAs (miRNAs) have been reported to regulate the development of chemoresistance in many tumors. Stathmin 1 (STMN1) is a microtubule-depolymerizing molecule, involved in chemo-response; however, the mechanism of its regulation is unknown. Herein, the immunohistochemical study indicated significant upregulation of the STMN1 in the ovarian cancer tissues defined as resistant tumors compared with those defined as responsive tumors. STMN1 level elevated in the chemoresistant ovarian cancer cells, KF-TX, compared with the parental, KF, ones. Targeting STMN1 by siRNA restored taxane-sensitivity of KF-TX cells. Screening miRNA profiles from KF/KF-TX cellular set followed by bioinformatics-based prediction, revealed that miR-31 could be a possible regulator of STMN1. Down-modulation of miR-31 was verified by quantitative RT-PCR in the cellular set used. Overexpression of miR-31 in KF-TX cells (KF-TX-miR-31) significantly restored chemo-response and reduced STMN1 expression as well. STMN1 reduction-associated cellular characteristics such as enhanced microtubule polymerization and stability, as indicated by acetylated tubulin quantification, confocal visualization, and G2 phase delay, were observed in KF-TX-miR-31 cells, indicating the functional reduction of STMN1. miR-31 suppressed the luciferase activity in reporter construct containing the STMN1 3'-untranslated region (3'-UTR), confirming that miR-31 directly targets STMN1. miR-31 has therapeutic potency when introduced into ovarian cancer, in combination with taxane.

Calcium signaling mediated influenza virus entry into host cells.
Y. Fujioka, M. Tsuda, A. Nanbo, T. Hattori, J. Sasaki, T. Sasaki, T. Miyazaki, Y. Ohba
MOLECULAR BIOLOGY OF THE CELL, 25, Dec. 2014, [Peer-reviewed]
English

Agonist-promoted Ubiquitination Differentially Regulates Receptor Trafficking of Endothelin Type A and Type B Receptors
Koji Terada, Takahiro Horinouchi, Yoichiro Fujioka, Tsunehito Higashi, Prabha Nepal, Mika Horiguchi, Sarita Karki, Chizuru Hatate, Akimasa Hoshi, Takuya Harada, Yosuke Mai, Yusuke Ohba, Soichi Miwa
JOURNAL OF BIOLOGICAL CHEMISTRY, 289, 51, 35283, 35295, Dec. 2014, [Peer-reviewed]
English, Scientific journal

Sustained elevation of Snail promotes glial-mesenchymal transition after irradiation in malignant glioma
Roshan Mahabir, Mishie Tanino, Aiman Elmansuri, Lei Wang, Taichi Kimura, Tamio Itoh, Yusuke Ohba, Hiroshi Nishihara, Hiroki Shirato, Masumi Tsuda, Shinya Tanaka
NEURO-ONCOLOGY, 16, 5, 671, 685, May 2014, [Peer-reviewed]
English, Scientific journal

Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection
Mohamed K. Hassan, Hidemichi Watari, Alaa-eldin Salah-eldin, Ahmed S. Sultan, Zainab Mohamed, Yoichiro Fujioka, Yusuke Ohba, Noriaki Sakuragi
PLOS ONE, 9, 4, e94213, Apr. 2014, [Peer-reviewed]
English, Scientific journal

Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy
Mami Matsuda-Lennikov, Futoshi Suizu, Noriyuki Hirata, Manabu Hashimoto, Kohki Kimura, Tadashi Nagamine, Yoichiro Fujioka, Yusuke Ohba, Toshihiko Iwanaga, Masayuki Noguchi
PLOS ONE, 9, 1, e79795, Jan. 2014, [Peer-reviewed]
English, Scientific journal

Apoptosis and Molecular Targeting Therapy in Cancer
Mohamed Hassan, Hidemichi Watari, Ali AbuAlmaaty, Yusuke Ohba, Noriaki Sakuragi
BIOMED RESEARCH INTERNATIONAL, 2014, 150845, 2014, [Peer-reviewed]
English

Beneficial innate signaling interference for antibacterial responses by a Toll-like receptor-mediated enhancement of the MKP-IRF3 axis
Hideo Negishi, Kosuke Matsuki, Nobuyasu Endo, Hana Sarashina, Shoji Miki, Atsushi Matsuda, Keiko Fukazawa, Naoko Taguchi-Atarashi, Hiroaki Ikushima, Hideyuki Yanai, Junko Nishio, Kenya Honda, Yoichiro Fujioka, Yusuke Ohba, Tetsuo Noda, Shun'ichiro Taniguchi, Eisuke Nishida, Yongliang Zhang, Hongbo Chi, Richard A. Flavell, Tadatsugu Taniguchi
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 110, 49, 19884, 19889, Dec. 2013, [Peer-reviewed]
English, Scientific journal

A Ca2+-dependent signalling circuit regulates influenza A virus internalization and infection
Yoichiro Fujioka, Masumi Tsuda, Asuka Nanbo, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba
NATURE COMMUNICATIONS, 4, 2763, Nov. 2013, [Peer-reviewed]
English, Scientific journal

血管内皮細胞によるがん転移促進(Endothelial cells promote tumor metastasis)　　　　　　　　　　　　　　　
間石 奈湖, 大場 雄介, 大賀 則孝, 秋山 廣輔, 山本 和幸, 浜田 淳一, 川本 泰輔, アラム・モハメド・トウフィック, 大村 瞳, 進藤 正信, 樋田 泰浩, 樋田 京子
日本癌学会総会記事, 72回, 127, 127, 日本癌学会, Oct. 2013, [Peer-reviewed]
English

P-gp阻害剤はメトロノミックケモセラピーの血管新生阻害効果を強める(P-gp inhibitor enhance antiangiogenic activity in metronomic chemotherapy)　　　　　　　　　　　　　　　
秋山 廣輔, 大賀 則孝, 樋田 泰浩, 間石 奈湖, ムハンマド・トフィック, 川本 泰輔, 大村 瞳, 山田 健司, 鳥居 ちさほ, 進藤 正信, 大場 雄介, 樋田 京子
日本癌学会総会記事, 72回, 127, 127, 日本癌学会, Oct. 2013, [Peer-reviewed]
English

Inhibition of influenza A virus infection by Galectin-9
Tomoe Hattori, Tomohiro Arikawa, Yoichiro Fujioka, Junki Maruyama, Yousuke Nakayama, Yusuke Ohba, Toshiro Niki, Tadaaki Miyazaki, Mitsuomi Hirashima, Hiroshi Kida
JAPANESE JOURNAL OF VETERINARY RESEARCH, 61, 1-2, 5, 18, May 2013, [Peer-reviewed]
English, Scientific journal

All members of the EPI64 subfamily of TBC/RabGAPs also have GAP activities towards Ras
Hiroyuki Nagai, Sayaka Yasuda, Yusuke Ohba, Mitsunori Fukuda, Takeshi Nakamura
Journal of Biochemistry, 153, 3, 283, 288, Mar. 2013, [Peer-reviewed]
English, Scientific journal

Fluorescent protein-based biosensors and their clinical applications
Yusuke Ohba, Yoichiro Fujioka, Shigeyuki Nakada, Masumi Tsuda
Progress in Molecular Biology and Translational Science, 113, 313, 348, 2013, [Peer-reviewed]
English, Scientific journal

Simultaneous inhibition of Src and Aurora kinases by SU6656 induces therapeutic synergy in human synovial sarcoma growth, invasion and angiogenesis in vivo
Ryuta Arai, Masumi Tsuda, Takuya Watanabe, Toyoyuki Ose, Chikashi Obuse, Katsumi Maenaka, Akio Minami, Yusuke Ohba
EUROPEAN JOURNAL OF CANCER, 48, 15, 2417, 2430, Oct. 2012, [Peer-reviewed]
English, Scientific journal

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1
Shigeki Chiba, Muhammad Baghdadi, Hisaya Akiba, Hironori Yoshiyama, Ichiro Kinoshita, Hirotoshi Dosaka-Akita, Yoichiro Fujioka, Yusuke Ohba, Jacob V. Gorman, John D. Colgan, Mitsuomi Hirashima, Toshimitsu Uede, Akinori Takaoka, Hideo Yagita, Masahisa Jinushi
NATURE IMMUNOLOGY, 13, 9, 832, 842, Sep. 2012, [Peer-reviewed]
English, Scientific journal

がん転移における腫瘍血管内皮細胞の役割(The role of tumor endothelial cells in tumor metastasis)　　　　　　　　　　　　　　　
間石 奈湖, 大場 雄介, 大賀 則孝, 秋山 廣輔, 山本 和幸, 浜田 淳一, 川本 泰輔, 大澤 崇宏, 近藤 美弥子, 大村 瞳, 進藤 正信, 樋田 泰浩, 樋田 京子
日本癌学会総会記事, 71回, 85, 85, 日本癌学会, Aug. 2012, [Peer-reviewed]
English

細胞内シグナル伝達の可視化技術と分子標的治療薬の耐性判定への応用
大場雄介, 津田真寿美
生化学, 84, 5, 359, 365, 日本生化学会, 25 May 2012
Japanese

Biosensors for BCR-ABL activity and their application to cancer　　　　　　　　　　　　　　　
Yusuke Ohba, Stephanie Darmanin, Tatsuaki Mizutani, Masumi Tsuda, Takeshi Kondo
Biosensors and Cancer, 268, 283, CRC Press, 01 Jan. 2012
English, In book

Visualization of intracellular signaling and its application to assessment of response to molecular target drugs
Yusuke Ohba, Masumi Tsuda
Seikagaku, 84, 5, 359, 365, 5, 2012, [Peer-reviewed]
Japanese, Scientific journal

FRETバイオセンサーによるCML細胞のチロシンキナーゼ阻害薬感受性試験~臨床検体での有用性の検討
近藤健, 金安顕子, 盛暁生, 入江達朗, 津田真寿美, 森岡正信, 今村雅寛, 大場雄介
臨床血液, 52, 9, 1133, 30 Sep. 2011
Japanese

DJ-1 associates with synaptic membranes
Yukiko Usami, Taku Hatano, Satoshi Imai, Shin-ichiro Kubo, Shigeto Sato, Shinji Saiki, Yoichiro Fujioka, Yusuke Ohba, Fumiaki Sato, Manabu Funayama, Hiroto Eguchi, Kaori Shiba, Hiroyoshi Ariga, Jie Shen, Nobutaka Hattori
NEUROBIOLOGY OF DISEASE, 43, 3, 651, 662, Sep. 2011, [Peer-reviewed]
English, Scientific journal

腫瘍血管内皮細胞とがん転移との相互作用解析(Analysis of interaction between tumor endothelial cells and tumor metastasis)　　　　　　　　　　　　　　　
間石 奈湖, 大賀 則孝, 樋田 泰浩, 大場 雄介, 浜田 淳一, 秋山 廣輔, 山本 和幸, 大澤 崇宏, 近藤 美弥子, 川本 泰輔, 進藤 正信, 井上 農夫男, 樋田 京子
日本癌学会総会記事, 70回, 430, 430, 日本癌学会, Sep. 2011, [Peer-reviewed]
English

[Visualization of cellular signaling by fluorescent proteins].
Ohba Y, Tsuda M
Nihon yakurigaku zasshi. Folia pharmacologica Japonica, 138, 1, 13, 17, 1, Jul. 2011, [Peer-reviewed]
Japanese, 下村脩博士によって，Aequorea victoria の発光器官から緑色蛍光タンパク質GFP（green fluorescent protein）が発見され，1992年にそのcDNAが単離されて以来，生細胞イメージングは生物学研究の必須ツールになっている．GFPはcDNAの細胞導入のみで，生理的環境下での目的タンパク質の局在や局在変化を可視化し，種々のカラーバリアントが入手可能な現在では複数のタンパク質の挙動の同時観察も可能である．また，フェルスター共鳴エネルギー移動（FRET: Förster resonance energy transfer）や蛍光タンパク質再構成法（BiFC: bimolecular fluorescence complementation）等の技術を用いることで，個々のタンパク質の局在や動態のみならずタンパク質の質的変化，つまりタンパク質間相互作用・構造変化等の時間的・空間的な変化の解析も可能である．これらの手法は細胞内シグナル伝達のダイナミクスを解析するために，最も適したツールと言っても過言ではない．本稿では，蛍光イメージングの基礎や応用例の紹介と各実験系が持つ得失を比較し，それぞれの実験系が何を可視化するのに適しているかを議論したい．

RANKL Expression Specifically Observed in Vivo Promotes Epithelial Mesenchymal Transition and Tumor Progression
Tamaki Yamada, Masumi Tsuda, Tomomi Takahashi, Yasunori Totsuka, Masanobu Shindoh, Yusuke Ohba
AMERICAN JOURNAL OF PATHOLOGY, 178, 6, 2845, 2856, Jun. 2011, [Peer-reviewed]
English, Scientific journal

IRF3 regulates cardiac fibrosis but not hypertrophy in mice during angiotensin II-induced hypertension
Kensuke Tsushima, Tomoko Osawa, Hideyuki Yanai, Akira Nakajima, Akinori Takaoka, Ichiro Manabe, Yusuke Ohba, Yasushi Imai, Tadatsugu Taniguchi, Ryozo Nagai
FASEB JOURNAL, 25, 5, 1531, 1543, May 2011, [Peer-reviewed]
English, Scientific journal

ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses
Sumio Hayakawa, Souichi Shiratori, Hiroaki Yamato, Takeshi Kameyama, Chihiro Kitatsuji, Fumi Kashigi, Showhey Goto, Shoichiro Kameoka, Daisuke Fujikura, Taisho Yamada, Tatsuaki Mizutani, Mika Kazumata, Maiko Sato, Junji Tanaka, Masahiro Asaka, Yusuke Ohba, Tadaaki Miyazaki, Masahiro Imamura, Akinori Takaoka
NATURE IMMUNOLOGY, 12, 1, 37, U56, Jan. 2011
English, Scientific journal

Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species
Koji Atarashi, Takeshi Tanoue, Tatsuichiro Shima, Akemi Imaoka, Tomomi Kuwahara, Yoshika Momose, Genhong Cheng, Sho Yamasaki, Takashi Saito, Yusuke Ohba, Tadatsugu Taniguchi, Kiyoshi Takeda, Shohei Hori, Ivaylo I. Ivanov, Yoshinori Umesaki, Kikuji Itoh, Kenya Honda
SCIENCE, 331, 6015, 337, 341, Jan. 2011, [Peer-reviewed]
English, Scientific journal

The Ras-PI3K Signaling Pathway Is Involved in Clathrin-Independent Endocytosis and the Internalization of Influenza Viruses
Yoichiro Fujioka, Masumi Tsuda, Tomoe Hattori, Junko Sasaki, Takehiko Sasaki, Tadaaki Miyazaki, Yusuke Ohba
PLOS ONE, 6, 1, e16324, Jan. 2011, [Peer-reviewed]
English, Scientific journal

ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses
Sumio Hayakawa, Souichi Shiratori, Hiroaki Yamato, Takeshi Kameyama, Chihiro Kitatsuji, Fumi Kashigi, Showhey Goto, Shoichiro Kameoka, Daisuke Fujikura, Taisho Yamada, Tatsuaki Mizutani, Mika Kazumata, Maiko Sato, Junji Tanaka, Masahiro Asaka, Yusuke Ohba, Tadaaki Miyazaki, Masahiro Imamura, Akinori Takaoka
NATURE IMMUNOLOGY, 12, 1, 37, U56, Jan. 2011, [Peer-reviewed]
English, Scientific journal

Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species
Atarashi Koji, Tanoue Takeshi, Shima Tatsuichiro, Imaoka Akemi, Kuwahara Tomomi, Momose Yoshika, Cheng Genhong, Yamasaki Sho, Saito Takashi, Ohba Yusuke, Taniguchi Tadatsugu, Takeda Kiyoshi, Hori Shohei, Ivanov Ivaylo I, Umesaki Yoshinori, Itoh Kikuji, Honda Kenya
Science, 1, 5, 23 Dec. 2010
English, <p>CD4⁺ T regulatory cells (Tregs), expressing the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, Tregs were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota-particularly clusters IV and XIVa of the genus Clostridium, promoted Treg cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-β (TGF-β) and affected Foxp3⁺ Treg number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic IgE responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.</p>

Conversion of Helicobacter pylori CagA from senescence inducer to oncogenic driver through polarity-dependent regulation of p21
Yasuhiro Saito, Naoko Murata-Kamiya, Toshiya Hirayama, Yusuke Ohba, Masanori Hatakeyama
JOURNAL OF EXPERIMENTAL MEDICINE, 207, 10, 2157, 2174, Sep. 2010, [Peer-reviewed]
English, Scientific journal

A Novel FRET-Based Biosensor for the Measurement of BCR-ABL Activity and Its Response to Drugs in Living Cells
Tatsuaki Mizutani, Takeshi Kondo, Stephanie Darmanin, Masumi Tsuda, Shinya Tanaka, Minoru Tobiume, Masahiro Asaka, Yusuke Ohba
CLINICAL CANCER RESEARCH, 16, 15, 3964, 3975, Aug. 2010, [Peer-reviewed]
English, Scientific journal

腫瘍血管内皮細胞と腫瘍細胞との相互作用解析(Analysis of interaction between tumor endothelial cells and tumor cells)　　　　　　　　　　　　　　　
間石 奈湖, 大賀 則孝, 秋山 廣輔, 北山 和子, 近藤 美弥子, 川本 泰輔, 大澤 崇宏, 山本 和幸, 樋田 泰浩, 大場 雄介, 進藤 正信, 樋田 京子
日本癌学会総会記事, 69回, 103, 103, 日本癌学会, Aug. 2010, [Peer-reviewed]
English

Visualization of Ras-PI3K interaction in the endosome using BiFC
Kaori Tsutsumi, Yoichiro Fujioka, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba
CELLULAR SIGNALLING, 21, 11, 1672, 1679, Nov. 2009, [Peer-reviewed]
English, Scientific journal

Basics of bioimaging and its applications
津田 真寿美, 大場 雄介
The Cell, 41, 11, 462, 465, ニューサイエンス社, Oct. 2009
Japanese

Adaptor Protein Crk Induces Src-Dependent Activation of p38 MAPK in Regulation of Synovial Sarcoma Cell Proliferation
Takuya Watanabe, Masumi Tsuda, Shinya Tanaka, Yusuke Ohba, Hideaki Kawaguchi, Tokifumi Majima, Hirofumi Sawa, Akio Minami
MOLECULAR CANCER RESEARCH, 7, 9, 1582, 1592, Sep. 2009, [Peer-reviewed]
English, Scientific journal

Helicobacter pylori CagA Causes Mitotic Impairment and Induces Chromosomal Instability
Mayumi Umeda, Naoko Murata-Kamiya, Yasuhiro Saito, Yusuke Ohba, Masayuki Takahashi, Masanori Hatakeyama
JOURNAL OF BIOLOGICAL CHEMISTRY, 284, 33, 22166, 22172, Aug. 2009, [Peer-reviewed]
English, Scientific journal

Parkin stabilizes PINK1 through direct interaction
Kahori Shiba, Takeo Arai, Shigeto Sato, Shin-ichiro Kubo, Yusuke Ohba, Yoshikuni Mizuno, Nobutaka Hattori
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 383, 3, 331, 335, Jun. 2009, [Peer-reviewed]
English, Scientific journal

Transcription factor 8 activates R-Ras to regulate angiogenesis
Takayuki Inuzuka, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 379, 2, 510, 513, Feb. 2009, [Peer-reviewed]
English, Scientific journal

Integral Role of Transcription Factor 8 in the Negative Regulation of Tumor Angiogenesis
Takayuki Inuzuka, Masumi Tsuda, Shinya Tanaka, Hideaki Kawaguchi, Yujiro Higashi, Yusuke Ohba
CANCER RESEARCH, 69, 4, 1678, 1684, Feb. 2009, [Peer-reviewed]
English, Scientific journal

Increased Motility and Invasiveness in Tumor Cells That Survive 10 Gy Irradiation
Kaori Tsutsumi, Masumi Tsuda, Natsuka Yazawa, Hirotaka Nakamura, Seiichiro Ishihara, Hisashi Haga, Motoaki Yasuda, Rie Yamazaki, Hiroki Shirato, Hideaki Kawaguchi, Takeshi Nishioka, Yusuke Ohba
CELL STRUCTURE AND FUNCTION, 34, 2, 89, 96, 2009, [Peer-reviewed]
English, Scientific journal

Clinicopathological significance of expression of p-c-Jun, TCF4 and beta-Catenin in colorectal tumors
Kayoko Takeda, Ichiro Kinoshita, Yasushi Shimizu, Yusuke Ohba, Tomoo Itoh, Yoshihiro Matsuno, Toshiaki Shichinohe, Hirotoshi Dosaka-Akita
BMC CANCER, 8, 328, Nov. 2008, [Peer-reviewed]
English, Scientific journal

シグナル伝達研究から創薬開発へ 2.細胞内シグナル伝達の可視化とその創薬への応用
大場雄介, 津田真寿美
実験医学, 26, 15, 2506, 2513, 15 Sep. 2008
Japanese

PTHrP promotes malignancy of human oral cancer cell downstream of the EGFR signaling
Tamaki Yamada, Masumi Tsuda, Yusuke Ohba, Hideaki Kawaguchi, Yasunori Totsuka, Masanobu Shindoh
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 368, 3, 575, 581, Apr. 2008
English, Scientific journal

PTHrP promotes malignancy of human oral cancer cell downstream of the EGFR signaling
Tamaki Yamada, Masumi Tsuda, Yusuke Ohba, Hideaki Kawaguchi, Yasunori Totsuka, Masanobu Shindoh
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 368, 3, 575, 581, Apr. 2008, [Peer-reviewed]
English, Scientific journal

Weekly paclitaxel/5-fluorouracil followed by platinum retreatment for patients with recurrent ovarian cancer: a single institution experience
H. Watari, M. Hosaka, T. Mitamura, M. Moriwaki, Y. Ohba, Y. Todo, M. Takeda, Y. Ebina, N. Sakuragi
EUROPEAN JOURNAL OF GYNAECOLOGICAL ONCOLOGY, 29, 6, 573, 577, 2008, [Peer-reviewed]
English, Scientific journal

Homeostatic erythropoiesis by the transcription factor IRF2 through attenuation of type I interferon signaling
Tatsuaki Mizutani, Kohichiro Tsuji, Yasuhiro Ebihara, Shinsuke Taki, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda
EXPERIMENTAL HEMATOLOGY, 36, 3, 255, 264, 2008, [Peer-reviewed]
English, Scientific journal

Spatiotemporal mobilization of Toll/IL-1 receptor domain-containing adaptor molecule-1 in response to dsRNA
Kenji Funami, Miwa Sasai, Yusuke Ohba, Hiroyuki Oshiumi, Tsukasa Seya, Misako Matsumoto
JOURNAL OF IMMUNOLOGY, 179, 10, 6867, 6872, Nov. 2007, [Peer-reviewed]
English, Scientific journal

Oligodendrocyte lineage transcription factor 2 inhibits the motility of a human glial tumor cell line by activating RhoA
Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa
MOLECULAR CANCER RESEARCH, 5, 10, 1099, 1109, Oct. 2007
English, Scientific journal

Oligodendrocyte lineage transcription factor 2 inhibits the motility of a human glial tumor cell line by activating RhoA
Kouichi Tabu, Yusuke Ohba, Tadaki Suzuki, Yoshinori Makino, Taichi Kimura, Akiko Ohnishi, Mieko Sakai, Takuya Watanabe, Shinya Tanaka, Hirofurni Sawa
MOLECULAR CANCER RESEARCH, 5, 10, 1099, 1109, Oct. 2007, [Peer-reviewed]
English, Scientific journal

Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in arabidopsis
Yoichiro Fujioka, Maki Utsumi, Yusuke Ohba, Yuichiro Watanabe
PLANT AND CELL PHYSIOLOGY, 48, 9, 1243, 1253, Sep. 2007
English, Scientific journal

Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in arabidopsis
Yoichiro Fujioka, Maki Utsumi, Yusuke Ohba, Yuichiro Watanabe
PLANT AND CELL PHYSIOLOGY, 48, 9, 1243, 1253, Sep. 2007, [Peer-reviewed]
English, Scientific journal

DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response
Akinori Takaoka, ZhiChao Wang, Myoung Kwon Choi, Hideyuki Yanai, Hideo Negishi, Tatsuma Ban, Yan Lu, Makoto Miyagishi, Tatsuhiko Kodama, Kenya Honda, Yusuke Ohba, Tadatsugu Taniguchi
NATURE, 448, 7152, 501, U14, Jul. 2007
English, Scientific journal

Oleamide derivatives suppress the spontaneous metastasis by inhibiting connexin 26
Yusuke Ohba, Yukiko Kanao, Nobuyoshi Morita, Eri Fujii, Mai Hohrai, Mayuko Takatsuji, Hideki Hirose, Daisaku Miura, Akihiro Watari, Masuo Yutsudo, Hanjun Zhao, Norikazu Yabuta, Akihiko Ito, Yasuyuki Kita, Hiroshi Nojima
International Journal of Cancer, 121, 1, 47, 54, 01 Jul. 2007, [Peer-reviewed]
English, Scientific journal

Synthesis of N-functionalized oleamide derivatives
Yusuke Ohba, Yukiko Kanao, Mayuko Takatsuji, Motoki Ito, Norikazu Yabuta, Hiroshi Nojima, Yasuyuki Kita
Tetrahedron, 63, 18, 3754, 3761, 30 Apr. 2007, [Peer-reviewed]
English, Scientific journal

Differential ras activation between caveolae/raft and non-raft Microdomains
Takashi Fukano, Asako Sawano, Yusuke Ohba, Michiyuki Matsuda, Atsushi Miyawaki
CELL STRUCTURE AND FUNCTION, 32, 1, 9, 15, 2007
English, Scientific journal

Spatiotemporal Mobilization of Toll/IL-1 Receptor Domain-Containing Adaptor Molecule-1 in Response to dsRNA.　　　　　　　　　　　　　　　
J. Immunol., 179, 10, 6867, 6872, 2007

Differential ras activation between caveolae/raft and non-raft Microdomains
Takashi Fukano, Asako Sawano, Yusuke Ohba, Michiyuki Matsuda, Atsushi Miyawaki
CELL STRUCTURE AND FUNCTION, 32, 1, 9, 15, 2007, [Peer-reviewed]
English, Scientific journal

ウイルス感染と宿主応答・宿主側の要因 MyD88依存的TLR下流シグナル経路におけるIRF-1の役割　　　　　　　　　　　　　　　
根岸 英雄, 柳井 秀元, 坂口 信也, 藤田 靖之, 篠原 正浩, 高柳 広, 大場 雄介, 谷口 維紹, 本田 賢也
日本免疫学会総会・学術集会記録, 36, 207, 207, (NPO)日本免疫学会, Nov. 2006
English

Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program
Hideo Negishi, Yasuyuki Fujita, Hideyuki Yanai, Shinya Sakaguchi, Xinshou Ouyang, Masahiro Shinohara, Hiroshi Takayanagi, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 103, 41, 15136, 15141, Oct. 2006
English, Scientific journal

Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways
Tsukasa Shibue, Saori Suzuki, Hideaki Okamoto, Hiroki Yoshida, Yusuke Ohba, Akinori Takaoka, Tadatsugu Taniguchi
EMBO JOURNAL, 25, 20, 4952, 4962, Oct. 2006
English, Scientific journal

Evidence for licensing of IFN-gamma-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program
Hideo Negishi, Yasuyuki Fujita, Hideyuki Yanai, Shinya Sakaguchi, Xinshou Ouyang, Masahiro Shinohara, Hiroshi Takayanagi, Yusuke Ohba, Tadatsugu Taniguchi, Kenya Honda
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 103, 41, 15136, 15141, Oct. 2006, [Peer-reviewed]
English, Scientific journal

Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways
Tsukasa Shibue, Saori Suzuki, Hideaki Okamoto, Hiroki Yoshida, Yusuke Ohba, Akinori Takaoka, Tadatsugu Taniguchi
EMBO JOURNAL, 25, 20, 4952, 4962, Oct. 2006, [Peer-reviewed]
English, Scientific journal

Negative regulation of Toll-like-receptor signaling by IRF-4
H Negishi, Y Ohba, H Yanai, A Takaoka, K Honma, K Yui, T Matsuyama, T Taniguchi, K Honda
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102, 44, 15989, 15994, Nov. 2005, [Peer-reviewed]
English, Scientific journal

IRF-7 is the master regulator of type-I interferon-dependent immune responses
K Honda, H Yanai, H Negishi, M Asagiri, M Sato, T Mizutani, N Shimada, Y Ohba, A Takaoka, N Yoshida, T Taniguchi
NATURE, 434, 7034, 772, 777, Apr. 2005
English, Scientific journal

Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction
K Honda, Y Ohba, H Yanai, H Negishi, T Mizutani, A Takaoka, C Taya, T Taniguchi
NATURE, 434, 7036, 1035, 1040, Apr. 2005
English, Scientific journal

IRF-7 is the master regulator of type-I interferon-dependent immune responses
K Honda, H Yanai, H Negishi, M Asagiri, M Sato, T Mizutani, N Shimada, Y Ohba, A Takaoka, N Yoshida, T Taniguchi
NATURE, 434, 7034, 772, 777, Apr. 2005, [Peer-reviewed]
English, Scientific journal

Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction
K Honda, Y Ohba, H Yanai, H Negishi, T Mizutani, A Takaoka, C Taya, T Taniguchi
NATURE, 434, 7036, 1035, 1040, Apr. 2005, [Peer-reviewed]
English, Scientific journal

Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors
A Takaoka, H Yanai, S Kondo, G Duncan, H Negishi, T Mizutani, S Kano, K Honda, Y Ohba, TW Mak, T Taniguchi
NATURE, 434, 7030, 243, 249, Mar. 2005
English, Scientific journal

Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors
A Takaoka, H Yanai, S Kondo, G Duncan, H Negishi, T Mizutani, S Kano, K Honda, Y Ohba, TW Mak, T Taniguchi
NATURE, 434, 7030, 243, 249, Mar. 2005, [Peer-reviewed]
English, Scientific journal

Negative regulation of Toll-like receptor signaling by IRF-4.
NEGISHI Hideo, OHBA Yusuke, YANAI Hideyuki, TAKAOKA Akinori, HONMA Kiri, YUI Katsuyuki, MATSUYAMA Toshifumi, TANIGUCHI Tadatsugu, HONDA Kenya
Natl. Acad. Sci. U.S.A., 102, 44, 15989, 15994, 2005

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling
K Honda, H Yanai, T Mizutani, H Negishi, N Shimada, N Suzuki, Y Ohba, A Takaoka, WC Yeh, T Taniguchi
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 101, 43, 15416, 15421, Oct. 2004
English, Scientific journal

Cell type-specific regulation of RhoA activity during cytokinesis
H Yoshizaki, Y Ohba, MC Parrini, NG Dulyaninova, AR Bresnick, N Mochizuki, M Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 279, 43, 44756, 44762, Oct. 2004
English, Scientific journal

Cell type-specific regulation of RhoA activity during cytokinesis
H Yoshizaki, Y Ohba, MC Parrini, NG Dulyaninova, AR Bresnick, N Mochizuki, M Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 279, 43, 44756, 44762, Oct. 2004, [Peer-reviewed]
English, Scientific journal

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling
K Honda, H Yanai, T Mizutani, H Negishi, N Shimada, N Suzuki, Y Ohba, A Takaoka, WC Yeh, T Taniguchi
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 101, 43, 15416, 15421, Oct. 2004, [Peer-reviewed]
English, Scientific journal

STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension
C Miyagi, S Yamashita, Y Ohba, H Yoshizaki, M Matsuda, T Hirano
JOURNAL OF CELL BIOLOGY, 166, 7, 975, 981, Sep. 2004
English, Scientific journal

STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension
C Miyagi, S Yamashita, Y Ohba, H Yoshizaki, M Matsuda, T Hirano
JOURNAL OF CELL BIOLOGY, 166, 7, 975, 981, Sep. 2004, [Peer-reviewed]
English, Scientific journal

RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney Cells
A Takaya, Y Ohba, K Kurokawa, M Matsuda
MOLECULAR BIOLOGY OF THE CELL, 15, 6, 2549, 2557, Jun. 2004
English, Scientific journal

RalA activation at nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and migrating Madin-Darby canine kidney Cells
A Takaya, Y Ohba, K Kurokawa, M Matsuda
MOLECULAR BIOLOGY OF THE CELL, 15, 6, 2549, 2557, Jun. 2004, [Peer-reviewed]
English, Scientific journal

Coactivation of Rad1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor
K Kurokawa, RE Itoh, H Yoshizaki, Y Ohba, T Nakamura, M Matsuda
MOLECULAR BIOLOGY OF THE CELL, 15, 3, 1003, 1010, Mar. 2004
English, Scientific journal

Visualization of signal transduction of Ras
Y Ohba, M Matsuda
SEIKAGAKU, 76, 1, 16, 28, Jan. 2004, [Peer-reviewed]
Japanese

Activity of Rho-family GTPases during cell division as visualized with FRET-based probes
H Yoshizaki, Y Ohba, K Kurokawa, RE Itoh, T Nakamura, N Mochizuki, K Nagashima, M Matsuda
JOURNAL OF CELL BIOLOGY, 162, 2, 223, 232, Jul. 2003
English, Scientific journal

Activity of Rho-family GTPases during cell division as visualized with FRET-based probes
H Yoshizaki, Y Ohba, K Kurokawa, RE Itoh, T Nakamura, N Mochizuki, K Nagashima, M Matsuda
JOURNAL OF CELL BIOLOGY, 162, 2, 223, 232, Jul. 2003, [Peer-reviewed]
English, Scientific journal

Mechanism of the spatio-temporal regulation of Ras and Rap1
Y Ohba, K Kurokawa, M Matsuda
EMBO JOURNAL, 22, 4, 859, 869, Feb. 2003
English, Scientific journal

Mechanism of the spatio-temporal regulation of Ras and Rap1
Y Ohba, K Kurokawa, M Matsuda
EMBO JOURNAL, 22, 4, 859, 869, Feb. 2003, [Peer-reviewed]
English, Scientific journal

Novel function of Chat in controlling cell adhesion via Cas-Crk-C3G-pathway-mediated Rap1 activation
A Sakakibara, Y Ohba, K Kurokawa, M Matsuda, S Hattori
JOURNAL OF CELL SCIENCE, 115, 24, 4915, 4924, Dec. 2002
English, Scientific journal

Novel function of Chat in controlling cell adhesion via Cas-Crk-C3G-pathway-mediated Rap1 activation
A Sakakibara, Y Ohba, K Kurokawa, M Matsuda, S Hattori
JOURNAL OF CELL SCIENCE, 115, 24, 4915, 4924, Dec. 2002, [Peer-reviewed]
English, Scientific journal

Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells
RE Itoh, K Kurokawa, Y Ohba, H Yoshizaki, N Mochizuki, M Matsuda
MOLECULAR AND CELLULAR BIOLOGY, 22, 18, 6582, 6591, Sep. 2002
English, Scientific journal

Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells
RE Itoh, K Kurokawa, Y Ohba, H Yoshizaki, N Mochizuki, M Matsuda
MOLECULAR AND CELLULAR BIOLOGY, 22, 18, 6582, 6591, Sep. 2002, [Peer-reviewed]
English, Scientific journal

A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo
K Kurokawa, N Mochizuki, Y Ohba, H Mizuno, A Miyawaki, M Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 33, 31305, 31310, Aug. 2001, [Peer-reviewed]
English, Scientific journal

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis
Y Ohba, K Ikuta, A Ogura, J Matsuda, N Mochizuki, K Nagashima, K Kurokawa, BJ Mayer, K Maki, J Miyazaki, M Matsuda
EMBO JOURNAL, 20, 13, 3333, 3341, Jul. 2001
English, Scientific journal

Spatio-temporal images of growth-factor-induced activation of Ras and Rap1
N Mochizuki, S Yamashita, K Kurokawa, Y Ohba, T Nagai, A Miyawaki, M Matsuda
NATURE, 411, 6841, 1065, 1068, Jun. 2001
English, Scientific journal

CalDAG-GEFIII activation of Ras, R-Ras, and Rap1
S Yamashita, N Mochizuki, Y Ohba, M Tobiume, Y Okada, H Sawa, K Nagashima, M Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 275, 33, 25488, 25493, Aug. 2000
English, Scientific journal

Rap2 as a slowly responding molecular switch in the Rap1 signaling cascade
Y Ohba, N Mochizuki, K Matsuo, S Yamashita, M Nakaya, Y Hashimoto, M Hamaguchi, T Kurata, K Nagashima, M Matsuda
MOLECULAR AND CELLULAR BIOLOGY, 20, 16, 6074, 6083, Aug. 2000
English, Scientific journal

Regulatory proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3
Y Ohba, N Mochizuki, S Yamashita, AM Chan, JW Schrader, S Hattori, K Nagashima, M Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 275, 26, 20020, 20026, Jun. 2000
English, Scientific journal

Crk activation of JNK via C3G and R-Ras
N Mochizuki, Y Ohba, S Kobayashi, N Otsuka, AM Graybiel, S Tanaka, M Matsuda
JOURNAL OF BIOLOGICAL CHEMISTRY, 275, 17, 12667, 12671, Apr. 2000
English, Scientific journal

Eosinophilic inclusions in ependymoma represent microlumina: a light and electron microscopic study
N Kawano, Y Ohba, K Nagashima
ACTA NEUROPATHOLOGICA, 99, 2, 214, 218, Feb. 2000
English, Scientific journal

A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo.　　　　　　　　　　　　　　　
J. Biol. Chem., 276, 33, 6074, 6083, 2000

Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i)
N Mochizuki, Y Ohba, E Kiyokawa, T Kurata, T Murakami, T Ozaki, A Kitabatake, K Nagashima, M Matsuda
NATURE, 400, 6747, 891, 894, Aug. 1999
English, Scientific journal

Melanotic peritoneal sarcomatosis originating from clear cell sarcoma
Yusuke Ohba, Hiroaki Suzuki, Hiroaki Hiraga, Tomoo Ito, Hirofumi Sawa, Makoto Nagai, Shin-Ichi Satoh, Hiroyuki Iwaki, Kazuo Nagashima
Pathology International, 49, 7, 653, 657, 1999
English, Scientific journal

血管内皮細胞におけるSARS-CoV-2侵入機構の解析
桜井優弥, 間石奈湖, 藤岡容一朗, 大場雄介, 武田遼, 佐々木道仁, 大場靖子, 樋田泰浩, 澤洋文, 澤洋文, 樋田京子, 日本血管生物医学会学術集会プログラム・抄録集, 31st (CD-ROM), 2023

アレナウイルスZ蛋白の自己重合を検出するFRETバイオセンサーの開発と,それを利用した抗ウイルス薬スクリーニング法の確立
水谷龍明, 大場雄介, 水田賢志, 浦田秀造, 日本蛋白質科学会年会プログラム・要旨集, 23rd (CD-ROM), 2023

フラビウイルスの粒子産生における分泌型NS1タンパク質の役割
田村友和, 田村友和, 鳥居志保, 鳥居志保, 梶原健太郎, 安齋樹, 藤岡容一朗, 野田暉翔, 田鍬修平, 森岡佑平, 森岡佑平, 鈴木理滋, YUZY Fauzyah, 小野慎子, 小野慎子, 大場雄介, 岡田雅人, 福原崇介, 福原崇介, 松浦善治, 松浦善治, 日本ウイルス学会学術集会プログラム・予稿集(Web), 69th, 2022

Visualizing virus entry into host cells
藤岡容一朗, 天野麻穂, 大場雄介, 医学のあゆみ, 280, 9, 2022

Pathogen dynamics in host cells
藤岡容一朗, 天野麻穂, 大場雄介, 実験医学, 39, 2, 2021

エボラウイルスエンベロープへのホスファチジルセリン集積に関与する宿主スクランブラーゼの同定　　　　　　　　　　　　　　　
南保 明日香, 丸山 隼輝, 今井 正樹, 氏江 美智子, 高田 礼人, 大場 雄介, 河岡 義裕, 日本女性科学者の会学術誌, 20, 1, 65, 66, Mar. 2020
(一社)日本女性科学者の会, Japanese

抗ラッサウイルス作用を有するリード阻害剤の創出
水谷龍明, 浦田秀造, 水田賢志, 大場雄介, 長崎大学熱帯医学研究拠点共同研究報告集, 2018, 2019

【タンパク質・核酸の分子修飾】細胞質/オルガネラでの分子修飾 細胞内シグナル リン酸化(チロシン)
笹島 仁, 大場 雄介, 生体の科学, 69, 5, 428, 429, Oct. 2018
(公財)金原一郎記念医学医療振興財団, Japanese

少数性生物学ってなんだ?少数の要素を計測する―ウイルス何個で“感染”するか?
藤岡容一朗, 田端和仁, 田端和仁, 大場雄介, 野地博行, 野地博行, 実験医学, 35, 19, 3197‐3203, 01 Dec. 2017
Japanese

RANKLによるインテグリンα2の発現亢進はインテグリンβ2の細胞内輸送を介して細胞接着を亢進する
大場雄介, 山田珠希, 山田珠希, 山田珠希, 津田真寿美, 藤岡容一朗, 藤岡真理, 堀内浩水, 堀口美香, 佐藤絢, ネパール ブラバ, 王せい, 柏木彩花, 西出真也, 南保明日香, 芳賀永, 田中伸哉, 進藤正信, 日本生理学雑誌(Web), 79, 2, 49 (WEB ONLY), 49, May 2017
(一社)日本生理学会, Japanese

インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定　　　　　　　　　　　　　　　
藤岡 容一朗, 西出 真也, 尾瀬 農之, 加藤 いづみ, 福原 秀雄, 藤岡 真理, 堀内 浩水, 佐藤 絢, Nepal Prabha, 柏木 彩花, Wang Jing, 堀口 美香, Paudel Sarad, 南保 明日香, 宮崎 忠昭, 前仲 勝実, 大場 雄介, 日本細胞生物学会大会講演要旨集, 69回, 61, 61, May 2017
(一社)日本細胞生物学会, Japanese

インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定
藤岡容一朗, 西出真也, 尾瀬農之, 加藤いづみ, 福原秀雄, 藤岡真理, 堀内浩水, 佐藤絢, NEPAL Prabha, 柏木彩花, WANG Jing, 堀口美香, PAUDEL Sarad, 南保明日香, 宮崎忠昭, 前仲勝実, 前仲勝実, 大場雄介, 日本細胞生物学会大会(Web), 69th, ROMBUNNO.T8‐04(P2‐028) (WEB ONLY), 61, May 2017
(一社)日本細胞生物学会, Japanese

高速原子間力顕微鏡を用いた細胞表面膜構造動態のライブセルイメージング
吉田藍子, 吉田藍子, 酒井信明, 植草良嗣, 粂田昌宏, 吉村成弘, 大場雄介, 日本生化学会大会(Web), 90th, 2017

Fluorescence bioimaging of intracellular signaling and its clinical application
Yusuke Ohba, Yoichiro Fujioka, Journal of Oral Biosciences, 58, 4, 113, 119, 01 Nov. 2016
Japanese Association for Oral Biology, English, Book review

CRKアダプター蛋白質はHGF/c-Metフィードバックループを介して膀胱癌のEMTと転移を誘導する　　　　　　　　　　　　　　　
王 磊, 松本 隆児, 津田 真寿美, 間石 奈湖, 安部 崇重, 木村 太一, 谷野 美智枝, 西原 広史, 樋田 京子, 大場 雄介, 篠原 信雄, 田中 伸哉, 日本癌学会総会記事, 74回, J, 1142, Oct. 2015
日本癌学会, English

Tumor endothelial cells promote metastasis via biglycan secretion
N. Maishi, Y. Ohba, K. Akiyama, N. Ohga, J. I. Hamada, M. T. Alam, M. Shindoh, Y. Hida, K. Hida, EUROPEAN JOURNAL OF CANCER, 51, S20, S20, Sep. 2015
English, Summary international conference

Lysosomal interaction of Akt with Phafin2: A critical step in the induction of autophagy
Masayuki Noguchi, Futoshi Suizu, Mami Lennikov-Matsuda, Bala Jyoti, Yusuke Ohba, Noriyuki Hirata, MOLECULAR CANCER THERAPEUTICS, 14, 7, Jul. 2015
English, Summary international conference

IDENTIFICATION OF FIP1L1-PDGFRA ASSOCIATING MOLECULE THAT LOCATES IN THE NUCLEUS AND AUGMENTS THE ACTIVITY OF FIP1L1-PDGFRA
T. Kondo, M. Ibata, J. Iwasaki, Y. Fujioka, K. Nakagawa, S. Darmanin, M. Onozawa, D. Hashimoto, Y. Ohba, S. Hatakeyama, T. Teshima, HAEMATOLOGICA, 100, 327, 327, Jun. 2015
English, Summary international conference

MULTICENTER CLINICAL STUDY TO EVALUATE A UTILITY OF FRET-BASED DRUG SENRITIVITY TEST THAT PREDICTS EARLY MOLECULAR RESPONSE IN NEWLY DIAGNOSED CML PATIENTS TREATED WITH NILOTINIB
T. Kondo, Y. Ohba, S. I. Fujisawa, T. Miyagishima, A. Mori, H. Iwasaki, M. Shindo, Y. Kakinoki, N. Kobayashi, S. Yamamoto, Y. Haseyama, S. Ando, N. Fujimoto, M. Nishio, M. Kurosawa, Y. Kohgo, T. Teshima, HAEMATOLOGICA, 99, 340, 340, Jun. 2014
English, Summary international conference

インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構　　　　　　　　　　　　　　　
藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介, 日本細胞生物学会大会講演要旨集, 66回, 103, 103, May 2014
(一社)日本細胞生物学会, Japanese

インフルエンザウイルスのCa2+シグナルを介した宿主細胞侵入機構　　　　　　　　　　　　　　　
藤岡 容一朗, 津田 真寿美, 南保 明日香, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介, 日本細胞生物学会大会講演要旨集, 66回, 146, 146, May 2014
(一社)日本細胞生物学会, Japanese

腫瘍血管内皮細胞のがん転移促進
間石奈湖, 大場雄介, 大賀則孝, 大賀則孝, 秋山廣輔, 浜田淳一, 山本和幸, 川本泰輔, 進藤正信, 樋田泰浩, 樋田京子, 日本病理学会会誌, 103, 1, 209, 209, Mar. 2014
(一社)日本病理学会, Japanese

Ras-PI3Kシグナルが制御する外来因子取込み機構の解析　　　　　　　　　　　　　　　
藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介, 日本生理学雑誌, 76, 2, 81, 81, Mar. 2014
(一社)日本生理学会, Japanese

腫瘍血管内皮細胞のがん転移への関わり
間石奈湖, 大場雄介, 樋田泰浩, 浜田淳一, 山本和幸, 大賀則孝, 秋山廣輔, 川本泰輔, 大澤崇宏, 近藤美弥子, 大村瞳, 井上農夫男, 進藤正信, 樋田京子, 日本口腔科学会雑誌, 63, 1, 135, 135, Jan. 2014
(NPO)日本口腔科学会, Japanese

Ras-PI3Kシグナルが制御する外来因子取込み機構の解析　　　　　　　　　　　　　　　
藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 大場 雄介, 日本細胞生物学会大会講演要旨集, 65回, 153, 153, May 2013
(一社)日本細胞生物学会, Japanese

腫瘍血管内皮細胞のがん転移への関与
間石奈湖, 大場雄介, 山本和幸, 大賀則孝, 浜田淳一, 秋山廣輔, 川本泰輔, 大澤崇宏, 大村瞳, 樋田泰浩, 進藤正信, 樋田京子, 日本病理学会会誌, 102, 1, 437, 437, Apr. 2013
(一社)日本病理学会, Japanese

SrcとAuroraキナーゼの2重阻害による滑膜肉腫の相乗的抗腫瘍効果
新井 隆太, 渡部 琢哉, 三浪 明男, 津田 真寿美, 大場 雄介, 北海道整形災害外科学会, 54, 2, 250, Mar. 2013
Japanese, Summary national conference

がん転移における腫瘍血管内皮細胞の役割
間石奈湖, 大場雄介, 大賀則孝, 秋山廣輔, 山本和幸, 浜田淳一, 川本泰輔, ALAM Mohammad T, 樋田泰浩, 樋田京子, 日本血管生物医学会学術集会プログラム・抄録集, 21st, 134, 2013
Japanese

がん微小環境における腫瘍血管の異常性獲得機構の解明
樋田京子, 大賀則孝, 秋山廣輔, 間石奈湖, 川本泰輔, 大場雄介, 樋田泰浩, がん研究分野の特性等を踏まえた支援活動公開シンポジウムプログラム・抄録集 平成24年度, 32, 2013
Japanese

がん転移における腫瘍血管内皮細胞の役割
間石奈湖, 大場雄介, 大賀則孝, 秋山廣輔, 山本和幸, 浜田淳一, 川本泰輔, ALAM MOHAMMAD T, 大村瞳, 樋田泰浩, 樋田京子, 日本がん転移学会学術集会・総会プログラム抄録集, 22nd, 79, 2013
Japanese

SU6656はSrcとオーロラキナーゼの二重阻害によってin vivo滑膜肉腫に対する有効な抗腫瘍効果を示す
新井 隆太, 渡部 琢哉, 津田 真寿美, 大場 雄介, 三浪 明男, 日本整形外科学会雑誌, 86, 8, S1101, Aug. 2012
Japanese, Summary national conference

抗ウイルス応答において活性化されるRIG-Iの強力な調節因子ZAPSの同定
白鳥 聡一, 早川 清雄, 大和 弘明, 亀山 武志, 北辻 千展, 樫木 芙美, 後藤 翔平, 亀岡 章一郎, 藤倉 大輔, 山田 大翔, 水谷 龍明, 数馬田 美香, 佐藤 麻衣子, 田中 淳司, 浅香 正博, 大場 雄介, 宮崎 忠昭, 今村 雅寛, 高岡 晃教, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 87, 4, 194, 194, 01 Aug. 2012
Japanese

Galectin-9は細胞およびマウスにおけるインフルエンザAウイルスの感染を抑制する
服部ともえ, 服部ともえ, 丸山隼輝, 藤岡容一朗, 中山洋佑, 大場雄介, 仁木敏郎, 有川智博, 宮崎忠昭, 平島光臣, 喜田宏, 喜田宏, 日本ウイルス学会学術集会プログラム・抄録集, 60th, 2012

RANKLはインテグリンα2の発現とエンドサイトーシスを介したインテグリンの細胞内輸送を亢進する
我妻孝則, 津田真寿美, 山田珠希, 藤岡容一朗, 芳賀永, 戸塚靖則, 進藤正信, 大場雄介, 日本分子生物学会年会プログラム・要旨集(Web), 35th, 3P-0363 (WEB ONLY), 2012
Japanese

腫瘍血管内皮細胞のがん転移への関わり
間石奈湖, 大場雄介, 山本和幸, 大賀則孝, 浜田淳一, 秋山廣輔, 川本泰輔, 大澤崇宏, 近藤美弥子, 大村瞳, 樋田泰浩, 樋田京子, 日本がん転移学会学術集会・総会プログラム抄録集, 21st, 104, 2012
Japanese

DJ-1はシナプス小胞に局在する　　　　　　　　　　　　　　　
波田野 琢, 宇佐見 由希子, 久保 紳一郎, 大場 雄介, 有賀 寛芳, 斉木 臣二, 佐藤 栄人, 服部 信孝, Dementia Japan, 25, 3, 424, 424, Oct. 2011
(一社)日本認知症学会, Japanese

SrcおよびAuroraキナーゼ2重阻害による滑膜肉腫の相乗的in vivo抗腫瘍効果(Dual inhibition of Src and Aurora kinases abrogates tumor growth, invasion, and angiogenesis of synovial sarcoma in vivo)　　　　　　　　　　　　　　　
津田 真寿美, 新井 隆太, 渡部 琢哉, 尾瀬 農之, 小布施 力史, 前仲 勝実, 三浪 明男, 大場 雄介, 日本癌学会総会記事, 70回, 274, 274, Sep. 2011
日本癌学会, English

慢性骨髄白血病に対する分子標的治療薬の反応性・抵抗性判定試験(Seeing response and resistance to BCR-ABL inhibition in chronic myeloid leukemia)　　　　　　　　　　　　　　　
大場 雄介, 金安 顕子, 水谷 龍明, ダルマニン・ステファニー, 近藤 健, 津田 真寿美, 日本癌学会総会記事, 70回, 242, 242, Sep. 2011
日本癌学会, English

RANKLは口腔癌細胞のインテグリンα2の発現と細胞接着を亢進する(RANKL upregulates integrin α2 expression and cell adhesion in oral cancer cells)　　　　　　　　　　　　　　　
大場 雄介, 山田 珠希, 藤岡 容一朗, 甲斐原 拓真, 戸塚 泰則, 進藤 正信, 津田 真寿美, 日本細胞生物学会大会講演要旨集, 63回, 156, 156, May 2011
(一社)日本細胞生物学会, English

SU6656 disturbs G2/M progression via inhibition of Aurora kinases
Tsuda Masumi, Arai Ryuta, Ohba Yusuke, 日本細胞生物学会大会要旨集, 63rd, 159, May 2011
Summary national conference

染色体・核・遺伝子発現・シグナル伝達 インフルエンザウイルスは、カルシウムシグナル伝達によって、Ras-PI3K-仲介エンドサイトーシスを活性化する(Signal transduction/Chromosome/Cell nucleus/Gene expression Influenza viruses activate Ras-PI3K-mediated endocytosis via calcium signaling)　　　　　　　　　　　　　　　
藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介, 日本細胞生物学会大会講演要旨集, 63回, 112, 112, May 2011
(一社)日本細胞生物学会, English

Analysis of interaction between tumor endothelial cells and tumor cells
Nako Maishi, Noritaka Ohga, Yasuhiro Hida, Yusuke Ohba, Taisuke Kawamoto, Kosuke Akiyama, Kazuko Kitayama, Miyako Kondoh, Takahiro Osawa, Kazuyuki Yamamoto, Nobuo Inoue, Masanobu Shindoh, Kyoko Hida, CANCER RESEARCH, 71, Apr. 2011
English, Summary international conference

SU6656 suppresses human synovial sarcoma progression through Src and Aurora kinase inhibition
Arai Ryuta, Tsuda Masumi, Watanabe Takuya, Ose Toyoyuki, Obuse Chikashi, Maenaka Katsumi, Minami Akio, Ohba Yusuke, 日本分子生物学会年会プログラム・要旨集(Web), 34th, WEB ONLY 2P-0690, 2011
Summary national conference

自然免疫系における核酸認識受容体を介したシグナルの新規調節因子と抗ウイルス作用
亀山武志, 早川清雄, 白鳥聡一, 大和弘明, 北辻千展, 樫木芙美, 後藤翔平, 亀岡章一郎, 藤倉大輔, 水谷龍明, 数馬田美香, 佐藤麻衣子, 今村雅寛, 浅香正博, 大場雄介, 宮崎忠昭, 高岡晃教, 日本ウイルス学会学術集会プログラム・抄録集, 58th, 220, 15 Oct. 2010
Japanese

RANKL発現は腫瘍形成とEMTを亢進する(RANKL expression promotes tumorigenesis and epithelial mesenchymal transition)　　　　　　　　　　　　　　　
山田 珠希, 津田 真寿美, 戸塚 靖則, 進藤 正信, 大場 雄介, 日本癌学会総会記事, 69回, 494, 494, Aug. 2010
日本癌学会, English

Srcファミリーキナーゼ阻害薬はin vivo滑膜肉腫の増殖・浸潤・血管新生を抑制する
新井 隆太, 渡部 琢哉, 津田 真寿美, 川口 秀明, 大場 雄介, 三浪 明男, 日本整形外科学会雑誌, 84, 8, S1244, Aug. 2010
Japanese, Summary national conference

RANKLは微小環境特異的に発現し細胞接着・EMT・腫瘍形成を促進する生体内特異的機能マーカーである(RANKJL expression specifically observed in vivo promotes cell adhesion, epithelial mesenchymal transition, and tumor progression)　　　　　　　　　　　　　　　
山田 珠希, 津田 真寿美, 進藤 正信, 戸塚 泰則, 大場 雄介, 日本細胞生物学会大会講演要旨集, 62回, 196, 196, May 2010
(一社)日本細胞生物学会, English

メンブレントラフィック Ras-PI3Kシグナル経路がエンドサイトーシスとインフルエンザ感染を調節する(Membrane traffic The Ras-PI3K signaling pathway mediates endocytosis and the incorporation of influenza viruses)　　　　　　　　　　　　　　　
藤岡 容一朗, 津田 真寿美, 服部 ともえ, 佐々木 純子, 佐々木 雄彦, 宮崎 忠昭, 大場 雄介, 日本細胞生物学会大会講演要旨集, 62回, 117, 117, May 2010
(一社)日本細胞生物学会, English

生体内腫瘍形成における腫瘍微小環境誘発性のRANKL発現の必要性(Requirement for tumor microenvironment-induced RANKL expression in the tumorigenesis in vivo)　　　　　　　　　　　　　　　
山田 珠希, 津田 真寿美, 戸塚 靖則, 進藤 正信, 大場 雄介, 日本癌学会総会記事, 68回, 47, 47, Aug. 2009
日本癌学会, English

FRETバイオセンサーを用いたCML分子標的薬剤の薬効評価系の構築(A FRET-based biosensor for the evaluation of the efficacy of molecular targeted drugs for chronic myeloid leukemia)　　　　　　　　　　　　　　　
水谷 龍明, 近藤 健, ダルマニン・ステファニー, 津田 真寿美, 田中 伸哉, 浅香 正博, 大場 雄介, 日本癌学会総会記事, 68回, 457, 457, Aug. 2009
日本癌学会, English

悪性胸膜中皮腫におけるシグナル伝達アダプター分子CRKを介したRac活性の可視化
谷野美智枝, 王磊, 津田真寿美, 西原広史, 大場雄介, 矢野聖二, 曽根三郎, 田中伸哉, 日本呼吸器学会雑誌, 47, 237, 10 May 2009
Japanese, Others

Receptor activator of NF‐kB ligand (RANKL)発現口腔癌細胞株の悪性形質に関する機能解析
山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信, 日本病理学会会誌, 98, 1, 250, 250, 20 Mar. 2009
(一社)日本病理学会, Japanese

慢性骨髄性白血病に対する分子標的治療薬の新たな薬効評価系の構築
MIZUTANI TATSUAKI, KONDO TAKESHI, STEPHANIE DARMANIN, TSUDA MASUMI, KAWAGUCHI HIDEAKI, ASAKA MASAHIRO, OBA YUSUKE, 日本病理学会会誌, 98, 1, 278, 20 Mar. 2009
Japanese

Cell Motility and Invasion of Surviving Tumor Cells after 10 Gy Irradiation
K. Tsutsumi, M. Tsuda, N. Yazawa, H. Nakamura, M. Yasuda, R. Yamazaki, H. Shirato, H. Kawaguchi, Y. Ohba, T. Nishioka, INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS, 75, 3, S538, S539, 2009
English, Summary international conference

Effect of Src family kinase inhibitor on proliferation and invasion in human synovial sarcoma in vivo
Arai Ryuta, Tsuda Masumi, Watanabe Takuya, Kawaguchi Hideaki, Ohba Yusuke, Minami Akio, 日本分子生物学会年会講演要旨集, 32nd, Vol.1, 231, 2009
Summary national conference

Negative regulation of angiogenesis by zinc-finger transcription factor TCF8
Takayuki Inuzuka, Masumi Tsuda, Shinya Tanaka, Yujiro Higashi, Hideaki Kawaguchi, Yusuke Ohba, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 45, S11, S12, Oct. 2008
English, Summary international conference

上皮細胞増殖因子(EGF)依存的なPTHrPの発現制御と口腔癌細胞の増殖能・局所浸潤能との関連性
山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信, 補体シンポジウム講演集, 45th, 180, 180, Jul. 2008
(一社)日本補体学会, Japanese

口腔癌細胞における上皮細胞増殖因子受容体(EGFR)依存的なPTHrP発現制御と局所浸潤能との関連性
山田珠希, 津田真寿美, 大場雄介, 戸塚靖則, 進藤正信, 川口秀明, 適応医学, 12, 1, 25, 25, 20 May 2008
日本適応医学会, Japanese

Visualization of an interaction between Ras and Ras-binding domain in living cells by bimolecular fluorescence complementation
Kaori Tsutsumi, Masumi Tsuda, Hideaki Kawaguchi, Yusuke Ohba, FASEB JOURNAL, 22, Apr. 2008
English, Summary international conference

口腔癌細胞の接着に関連する分子の機能解析
山田珠希, 津田真寿美, 大場雄介, 川口秀明, 戸塚靖則, 進藤正信, 生化学, 2P-1143, 2008
Japanese

慢性骨髄性白血病に対する分子標的治療薬の新たな薬効評価系の構築
MIZUTANI TATSUAKI, KONDO TAKESHI, DARMANIN STEPHANIE, TSUDA MASUMI, KAIHARA TAKUMA, KAWAGUCHI HIDEAKI, ASAKA MASAHIRO, OBA YUSUKE, 生化学, 4P-1171, 2008
Japanese

ParkinによるPINK1の安定化機構(平成18年度老人性疾患病態・治療研究センター研究発表会)
柴 佳保里, 新井 健夫, 久保 紳一郎, 大場 雄介, 水野 美〓, 服部 信孝, 順天堂医学, 53, 3, 521, 522, Sep. 2007
順天堂大学, Japanese

EGFシグナル伝達はPTHrPの発現を誘導し、口腔癌細胞の悪性化に関与する(EGF signaling regulates PTHrP expression and malignancies in oral cancer cells)　　　　　　　　　　　　　　　
山田 珠希, 津田 真寿美, 大場 雄介, 戸塚 靖則, 進藤 正信, 日本癌学会総会記事, 66回, 501, 501, Aug. 2007
日本癌学会, English

植物におけるsmall RNA生成/制御の場の解析
藤岡容一朗, 内海真希, 岩崎信太郎, 大場雄介, 渡辺雄一郎, 日本RNA学会年会要旨集, 9th, 138, 28 Jul. 2007
Japanese

慢性骨髄性白血病に対する新たな診断技術の開発
MIZUTANI TATSUAKI, TSUDA MASUMI, KAWAGUCHI HIDEAKI, OBA YUSUKE, 生化学, 4P-0935, 2007
Japanese

(345) Formulation of in planta FRET System for Analysis of Viral Movement(Abstract of the Paper Presented at the 2006 Annual Meeting in Sapporo)
Fujioka Y., Ohba Y., Watanabe Y., Annals of the Phytopathological Society of Japan, 72, 4, 294, 294, 25 Nov. 2006
The Phytopathological Society of Japan (PSJ), Japanese

免疫2006 7章 病気と免疫 病原体認識受容体シグナルにおけるIRFファミリー転写因子の役割
高岡晃教, 柳井秀元, 本田賢也, 大場雄介, 谷口維紹, Mol Med, 42, 308, 317, 15 Dec. 2005
Japanese

MyD88‐IRF7シグナル伝達系の時空間制御によるIFN産生機構
大場雄介, 本田賢也, 柳井秀元, 高岡晃教, 谷口維紹, 日本癌学会学術総会記事, 64th, 523, 15 Aug. 2005
Japanese

I型インターフェロン依存性免疫応答における主要な調節因子としてのIRF‐7の役割
柳井秀元, 本田賢也, 大場雄介, 高岡晃教, 吉田進昭, 谷口維紹, 日本癌学会学術総会記事, 64th, 523, 15 Aug. 2005
Japanese

樹状細胞群の分化と活性化の分子機構
HONDA KEN'YA, YANAI HIROYUKI, MIZUTANI TATSUAKI, NEGISHI HIDEO, OBA YUSUKE, TAKAOKA AKINORI, TANIGUCHI TADATSUGU, 日本分子生物学会年会プログラム・講演要旨集, 27th, 345, 25 Nov. 2004
Japanese

樹状細胞群におけるIRFファミリー転写因子の役割
YANAI HIDEYUKI, HONDA KEN'YA, ASAGIRI MASATAKA, NEGISHI HIDEO, MIZUTANI TATSUAKI, SHIMADA NAOYA, YOSHIDA NOBUAKI, OBA YUSUKE, TAKAOKA AKINORI, 日本分子生物学会年会プログラム・講演要旨集, 27th, 593, 25 Nov. 2004
Japanese

RalA activation at nascent lamellipodia of EGF-stimulated Cos7 cells and migrating MDCK cells
Akiyuki Takaya, Yusuke Ohba, Kazuo Kurokawa, Michiyuki Matsuda, CELL STRUCTURE AND FUNCTION, 29, 57, 57, May 2004
English, Summary international conference

Regulation of a Small GTPase Rap2
OHBA Yusuke, 北海道醫學雜誌 = Acta medica Hokkaidonensia, 76, 1, 13, 20, 01 Jan. 2001
Japanese

Activation of the ERK/MAPK pathway by an isoform of Rap1gap associated with G alpha I
N Mochizuki, Y Ohba, K Nagashima, A Endo, M Matsuda, CIRCULATION, 102, 18, 353, 353, Oct. 2000
English, Summary international conference

Oral Cancer　　　　　　　　　　　　　　　
IN TECH, 2012

Bio Science 新用語ライブラリー免疫　　　　　　　　　　　　　　　
羊土社, 2000

TKI抵抗性CMLに対するエキソーム解析とFRET解析の統合による個別化治療の可能性
科学研究費助成事業
01 Apr. 2024 - 31 Mar. 2027
高橋 直人, 横山 和明, 三浦 昌朋, 大場 雄介
日本学術振興会, 基盤研究(C), 秋田大学, 24K11508

Visualization of symbiotic interfaces and cellular responses
Grants-in-Aid for Scientific Research
19 Nov. 2020 - 31 Mar. 2025
大場 雄介
研究で実施する、細胞表面で生じるマテリアルと細胞表面タンパク質の「弱い相互作用」が生じている現場である細胞膜の動態観察を実現するため、高速ライブセル原子間力顕微鏡（atomic force microscopy，AFM；以下、高速ライブセルAFM）および蛍光顕微鏡による相関イメージング（correlative light and acomic force microscopy, CLAM）を実装した。また、領域内の測定拠点としての活動が円滑に行えるように、体制整備および予備実験が完了した。これによりマテリアルー生体の「弱い相互作用」の現場の「まるごと」観察を実現した。
【AFM】ウイルスや種々の外来因子を用いて、柔らかい細胞膜上の6.0 μm × 4.5 μm ×1.0 μmの範囲において、xyzすべての方向に対して10 nmの等方性空間分解能、また0.5フレーム/秒（2秒に1枚の撮像）以上の時間分解能を両立した観察系を複数セットアップした。これらの装置の性能について確認し、予定通りのスペックが出 ていることを確認している。
【蛍光顕微鏡】分子同定や細胞応答の可視化に欠かせない蛍光イメージングについては、その分解能を向上するために超解像技術を導入した。また、薄層斜光証明法（highly inclined and laminated optical sheet microscopy，HILO）により細胞膜頂端膜の局所励起と一分子イメージングが可能な照明・観察光学系を、AFMが実装済みの顕微鏡にインストールした。
さらに、「弱い相互作用」をCLAM上でどのように可視化するかについて予備的検討を、ウイルス粒子と細胞膜との関係をモデルに行った。その結果、相互作用がない場合には粒子は細胞膜上にとどまることができないこと、相互作用の強弱により側方拡散係数（lateral diffusion coefficient）が異なることを新たに見出した。これによりマテリアル一つの解像度で「弱い相互作用」を定量解析する準備が整った。
Japan Society for the Promotion of Science, Grant-in-Aid for Transformative Research Areas (A), Hokkaido University, 20H05872

Biophysical Chemistry for Material Symbiosis
Grants-in-Aid for Scientific Research
19 Nov. 2020 - 31 Mar. 2025
山吉 麻子, 前仲 勝実, 荏原 充宏, 望月 慎一, 長谷 耕二, 大場 雄介, 宇都 甲一郎, 白石 貢一, 植畑 拓也, 森 健, 天野 麻穂, 山本 剛史
本研究課題の目的は、学術変革領域「マテリアル・シンバイオシスのための生命物理化学」の研究推進と領域運営である。
【領域会議等の開催】2021年度9月より公募研究班の領域参画が始まった。領域目標を達成するためには、個々の研究の活性化に加え、領域全体が一体となって相互補完的な融合研究を実施することが肝要である。そこで、領域全メンバー参加型の第１回領域会議（2021/11/4-11/5）をハイブリッド開催し、最新の研究成果の共有と共同研究推進の場を提供した。また別途、A01-A03各班での班会議を開催し情報共有を行った。総括班員による班会議も定期開催し、領域体制の最適化に勤めた。
【領域共催のシンポジウムの開催】日本薬剤学会第36年会において、ラウンドテーブル「シンバイオティック・マテリアルの実現と新しい創薬モダリティを考える」を企画・開催した（2021/1/13）。また、日本DDS学会第37年会において、若手ワークショップ「腸内細菌叢の共生原理に学ぶDDS」を企画・開催した（2019/6/29）。領域メンバーはもとより、国内外研究協力者が領域研究に関する研究発表を行い、領域研究を外部に向けて発信した。さらに、男女共同参画推進を目的としたシンポジウムを共催した（「北九州サイエンスガールプロジェクト（北九州市立大学公開講座）」2021/10/2, 10/9, 10/16; 「令和3年度 長崎大学リケジョ育成プログラム 志セミナー」, 2021/12/4）。
【イメージングブートキャンプ】領域班員、若手研究者に、物質共生学研究で重要となる細胞イメージング技術を修得する場を提供することを目的に、イメージングブートキャンプ2021 （2021/9/7-9）を共催した。
【共同研究推進】分野横断的かつ積極的な共同研究を推進するために、総括班よりマテリアル合成支援や超高速AFM解析支援等を提供した。
Japan Society for the Promotion of Science, Grant-in-Aid for Transformative Research Areas (A), Nagasaki University, 20H05871

一次線毛とコロナウイルス感染におけるACE2の役割の解明
科学研究費助成事業
08 Mar. 2023 - 31 Mar. 2024
大場 雄介, PANINA YULIA
本研究は、コロナウイルスの細胞侵入におけるangiotensin converting enzyme 2（ACE2）の役割に焦点を当てて実施した。肺と線維芽細胞において内因性ACE2の局在を特定し、線毛に局在しないことを発見した。ACE2を過剰発現した肺上皮細胞モデルを確立し、ACE2が形態変化を引き起こすことを見出した。そこで、細胞骨格の変化を解析した結果、アクチン細胞骨格が大幅に変化していることがわかった。一方、微小管や中間径フィラメントなどには変化がなかった。ACE2依存性のアクチンの変化は、Wnt/Hedgehog経路非依存性、Hippo-Yes-associated protein（YAP）/transcriptional coactivator with a PDZ-binding domain（TAZ）経路依存性であることが見いだされた。さらに、ウイルス感染におけるACE2の役割を明らかにするために、Hippo-YAP/TAZ経路の詳細な解析を行った。
ウイルス粒子とACE2タンパク質との結合が細胞形態形成に与える影響を解析するため、疑似ウイルス粒子の調製を行った。重症急性呼吸器症候群コロナウイルス-2（severe acute respiratory syndrome Coronavirus 2, SARS-CoV-2）のスパイクタンパク質を発現する口蹄疫ウイルス（vesicular stomatitis virus, VSV）の疑似ウイルス粒子を常法により作製したところ、一部の疑似ウイルス粒子のみにスパイクタンパク質の発現が認められた。適切なコロナウイルススパイクタンパク質の発現レベルを持つ安定細胞株を樹立することで、スパイクタンパク質を均一に持つ疑似ウイルス粒子を生成にも成功した。
日本学術振興会, 特別研究員奨励費, 北海道大学, 22KF0004

一次線毛とコロナウイルス感染におけるACE2の役割の解明
科学研究費助成事業
18 Nov. 2021 - 31 Mar. 2024
大場 雄介, PANINA YULIA
本研究は、SARS-CoV-2受容体ACE2について、基礎的な細胞プロセスやCOVID-19症状の重症度との関連を理解すること目的に、特に一次繊毛との関連性に着目して研究を進めている。最終的には、ACE2が繊毛に局在する分子メカニズムの解明、SARS-CoV-2とACE2の結合が繊毛に与える影響、高血圧・糖尿病病態がこれらにどのような影響を与えるのかの解明を目指す。
本年度、つまり研究開始から6ヶ月間の到達目標は、ACE2の細胞内局在を決定し、ACE2発現細胞と非発現細胞との違いを調べることであった。まず、ACE2の局在を研究するのに適したモデル細胞を決定した。いくつかの細胞株をテストした結果、ヒトBEAS-2B細胞株、マウス退治線維芽細胞株、マウス3T3細胞株が、繊毛の識別性およびACE2抗体に交差性から適したモデルであると判断した。次に、市販のACE2抗体を複数用いて免疫蛍光法を行ったところ、ACE2を検出できるのは一つのみであることが判明した。この抗体を用いて、ACE2が細胞膜に局在していることを確認した。また、ACE2を恒常的に過剰発現させたBEAS-2B細胞株を用い、元のACE2非発現BEAS-2B細胞株と比較したところ、細胞の構造、形状、細胞骨格に大きな変化があることを見いだした。さらに、ACE2の過剰により細胞形態が変化することが確認されたため、細胞接着を担う細胞膜タンパク質を探索し、その局在が変化する接着分子を同定した。一方、生きた細胞での局在を、蛍光タンパク質タグしたACE2で検討しようと試みたが、繊毛局在性は認められなかった。
日本学術振興会, 特別研究員奨励費, 北海道大学, 21F21380

オルガネラバイオロジーの新展開
科学研究費助成事業
09 Jul. 2021 - 31 Mar. 2023
大場 雄介
我々は最近、EGF刺激によりエンドサイトーシスを誘導した時、細胞膜から小胞が形成される初期段階で、初期エンドソームマーカーであるRab5と後期エンドソームマーカーであるRab7が長時間同時に局在する小胞が存在することを見出した。このような遷移状態のオルガネラはこれまでに報告がなく、その形成メカニズムや役割は未知である。そこで、この小胞の特徴や運ばれる物質、形成に関わる因子を調べ、Rab5/7ダブルポジティブ小胞の生理的意義を明らかにすることを目指した。はじめに、Rab5/7ダブルポジティブ小胞に局在する分子を調べ、EEA1およびSNX5が共局在することがわかった。Rab5/7ダブルポジティブ小胞は2-5 μmの大きい小胞であり、また、この小胞と共局在するSNX5はマクロピノソームのマーカー分子として知られていることから、Rab5/7ダブルポジティブ小胞はマクロピノサイトーシスによって形成されるマクロピノソームであることが示唆された。
次に、この小胞によって輸送される物質および輸送経路を調べた。Rab5/7ダブルポジティブ小胞はEGF刺激後に形成されることから、EGF受容体がこの小胞内に含まれているかどうかを調べた。その結果、Rab5/7ダブルポジティブ小胞とRhodamine標識EGFは共局在しており、この小胞はEGF受容体を輸送していることが示唆された。また、Rab5/7ダブルポジティブ小胞の挙動をタイムラプスイメージングで観察したところ、リソソームマーカーのみと共局在したことから、この小胞は分解経路に輸送されることが示唆された。
日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 21K19347

エンドソームとミトコンドリアの連携ゾーンによる細胞生理機能制御機構の解明
科学研究費助成事業
01 Apr. 2020 - 31 Mar. 2022
大場 雄介
Ras-PI3Kシグナルの制御ペプチドRAPEL配列の結合因子探索から同定したミトコンドリア外膜タンパク質voltage-dependent anion chanle 2（VDAC2）の機能解析を行った。これまでに、VDAC2は生化学的にRAPELと結合すること、ノックダウンによりRas-PI3Kのエンドソーム局在、クラスリン非依存性エンドサイトーシス、およびエンドソーム成熟化が抑制されること、過剰発現によりこれらが亢進することを確認した。すなわち、VDAC2はRas-PI3Kによるエンドサイトーシスの制御を正にコントロールする因子であることが明らかになった。 ミトコンドリア外膜タンパク質であるVDAC2が、どのようにしてエンドサイトーシス制御に関与するかを明らかにするため、我々が開発したオルガネラマーカーを用いて、生細胞タイムラプスイメージングを行った。それにより、ミトコンドリアとエンドソームの間には、一過性に繰り返す相互作用が生じることを発見した。この相互作用は、VDAC2のノックダウンで減少した。さら、ミトコンドリアと接触するエンドソーム上では、早期エンドソームマーカーの蛍光強度が減少し、接触過程でエンドソームが成熟化することが示唆された。実際、ミトコンドリアとの接触中にpHが低下することがわかった。さらに、光遺伝学手法を用いてミトコンドリアとエンドソームの接触を誘発したところ、エンドソームの成熟化が進み、VDAC2ノックダウンでその成熟化が抑制された。したがってVDAC2はエンドソーム とミトコンドリアのtethering factorであると同時に、その連携ゾーンにおける働きが重要であることが示唆された。野生型のVDAC2発現はVDACノックダウンを補完できるが、ある変異型VDAC2はそれが不可能であることから、連携ゾーンでのVDAC2の機能解明を目指す。
日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 20H04895

蛍光偏光を用いた等方性分解能顕微鏡の開発と細胞膜動態の可視化　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
07 Oct. 2019 - 31 Mar. 2022
大場 雄介, 藤岡 容一朗, 佐藤 絢
Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Hokkaido University, 19KK0198

細胞膜動態の高分解能計測によるエンドサイトーシス超初期過程の分子基盤の解明　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Challenging Research (Exploratory)
28 Jun. 2019 - 31 Mar. 2021
大場 雄介
Japan Society for the Promotion of Science, Challenging Research (Exploratory), Hokkaido University, 19K22506

インフルエンザウイルス感染におけるシンギュラリティ　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2019 - 31 Mar. 2021
大場 雄介
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 19H05411

Analysis of the communication zone between endosomes and mitochondria
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2018 - 31 Mar. 2020
大場 雄介
本研究は、低分子量GTP結合タンパク質Rasと、その標的因子の1つであるphosphoinositide 3-kinase (PI3K)が、他の標的因子とは異なり上皮増殖因子 (epidermal growth factor, EGF) 刺激依存的にエンドソームでRasと複合体を形成する分子メカニズムを解明することから開始した。
Rasの主な標的因子のRas-binding domain（RBD）のアミノ酸配列を比較にり、PI3K特異的配列を見出し、Ras-PI3K endosomal localization（RAPEL）配列と命名した。RAPEL結合因子を酵母ツーハイブリッド法で探索したところ、ミトコンドリア外膜タンパク質voltage-dependent anion channel 2（VDAC2）が同定された。VDAC2のノックダウンによりRas-PI3K複合体のエンドソーム局在、クラスリン非依存性エンドサイトーシス、エンドソームの成熟化が抑制された。
次に、ミトコンドリアタンパク質がいかにしてエンドサイトーシスの制御に関与するかを調べるため、生細胞内でミトコンドリアとエンドソームのダイナミクスを詳細に観察したところ、両者は一過性に近接していることが明らかになった。この近接は、コントロール細胞ではEGF刺激後で促進されたのに対し、VDAC2ノックダウン細胞では変化が認められなかった。さらに光遺伝学を用いてミトコンドリア―エンドソーム間相互作用を青色光により誘導する系を構築したところ、相互作用依存性にエンドソームの酸性化が促進された。したがって、エンドソームとミトコンドリア間の接触ゾーンの形成がエンドソームとの成熟化を促進すること、RAPELとVDAC2がゾーン形成のtetheringに関与することが示唆された。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 18H04850

エンドソームとミトコンドリアの物理的相互作用と機能連関による細胞機能の調節機構　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2017 - 31 Mar. 2020
大場 雄介
昨年度に引き続き、低分子量Gタンパク質Rasと標的分子複合体のうち、Ras-PI3K複合体のみがエンドソーム移行する分子メカニズムの解明を目指し研究を行った。標的因子のRas-binding domain（RBD）のアミノ酸配列解析により、PI3K-RBDに存在する特徴的な配列を同定し、この配列を欠損させるとRas-PI3Kのエンドソーム局在が抑制されたことから、Ras-PI3K endosomal localization domain（RAPEL domain）と命名した。RAPEL過剰発現によりエンドサイトーシスが抑制され、エンドサイトーシスを介して細胞に侵入するインフルエンザウイルスの感染も抑制された。さらに、RAPELと細胞膜透過性ペプチド（cell penetrating peptide, CPP）との誘導ペプチドを合成した。ペプチドを導入した細胞にインフルエンザウイルスを暴露したところ、感染抑制効果が認められた。さらに、ペプチドによる感染抑制効果はヒト呼吸上皮細胞を用いたin situでも認められた。以上より、RAPELはペプチド療法にも応用可能であることが示された。これらの結果は、学術雑誌に受理された。
一方、上記の結果は、RAPELに結合する因子がRas-PI3K複合体のエンドソーム移行とエンドサイトーシスを制御することを示唆するものである。質量分析法と酵母ツーハイブリッド法によるスクリーニングで得られたRAPEL結合因子計50個のうち、6因子についての解析が進んでいる。うち3因子の解析が終了し論文を作成・投稿準備中である。因子Aについては新規のオルガネラ間相互作用を担うタンパク質であることが判明した。因子Bはイノシトールリン脂質依存的にRas-PI3Kのエンドソーム局在の制御因子であることを突き止めた。因子Cについては上記二つの負の制御因子であった。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 17H04016

Configuration of cell axes for the development of cell authentication system
Grants-in-Aid for Scientific Research Challenging Research (Exploratory)
30 Jun. 2017 - 31 Mar. 2019
Ohba Yusuke
In order to set a "template" for authenticating cells and a "coordinate axis" for describing it, parameters regarding the arrangement of each organelle were obtained. For this purpose, an organelle marker library was constructed using various fluorescent proteins. It was found that the facilitation of the folding of fluorescent proteins might negatively affect the localization of the organelle markers. In addition, by analyzing the arrangement of organelles using the library, it was found that the centroids of the endosomal system formed a certain line in cells.
Japan Society for the Promotion of Science, Challenging Research (Exploratory), Hokkaido University, 17K19507

細胞競合現象の時空間的解析とその操作　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2017 - 31 Mar. 2019
大場 雄介
本研究は、光による細胞競合の誘導系を開発し、細胞競合の発生を時空間的に厳密に制御すること、同時に分子の活性化、分子間相互作用、イオン濃度変化等を蛍光バイオイメージングで観察し、winner、looser間での細胞競合が生じるための時空間的な分子動態を詳細に明らかにすることを目的に行っている。
昨年度までに、488 nmの光で分子間相互作用が誘導されるCry2-CIBの系を用いて、二つのRas活性化法（RasV12とSosを用いるもの）を構築した。この二つの系を用いて、RasV12の系のみで細胞競合が観察され、Sosによる内在性Rasの活性化では細胞競合が誘導されないことを見出した。この結果は、細胞競合において何がwinnerとlooserを決めるかという根本的課題に迫る緒になると期待された。
しかし、この系では青色光の刺激が必要なため、シグナル伝達研究に有効なフェルスター共鳴エネルギー移動（Foerster resonance energy transfer, FRET）や蛍光タンパク質再構成法（bimolecular fluorescence complementation, BiFC）等の青色光を励起光として用いるイメージング手法との併用が難しい。そこで、それらイメージング手法との併用を目指し、遠赤色光受容タンパク質phytochromeB（PhyB）とphytochrome interaction factor 3（PIF3）の結合を遠赤色光により誘導できるオプトジェネティクスの手法を取り入れた。これにより、遠赤色光照射によりSrcを細胞質から細胞膜あるいはエンドソームに移行させることに成功した。また、様々なオルガネラに局在するPhyBとPIF3を作製し、異種オルガネラ間相互作用を誘導する系も併せて開発した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 17H05611

Drug screening to improve mitochondrial functions
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2016 - 31 Mar. 2018
Imai Yuzuru, SHIBA Kahori, INOSHITA Tsuyoshi, OHBA Yusuke, ARANO Taku
The genes responsible for autosomal recessive early-onset Parkinson’s disease, Parkin and PINK1 encode a ubiquitin ligase and a mitochondrial protein kinase, respectively. A series of studies have revealed that Parkin in collaboration with PINK1 has a role for mitochondrial quality control, impairment of which leads to dopaminergic neurodegeneration. In this study, we aimed at searching drugs to improve mitochondrial functions through moderate activation of PINK1-Parkin signaling.
We developed a cell-based reporter assay to monitor PINK1-Parkin signaling and screened approximately 310-thousands of chemicals. Hit compounds obtained in the screening were further tested for mitochondrial membrane potential and cell viability and three candidates were identified as seed compounds.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Juntendo University, 16K15484

Intracellular signaling machinery that regulates endocytosis and uptake of exogenous factors(Fostering Joint International Research)
Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research)
2016 - 2018
Ohba Yusuke, Tani Tomomi
We have developed a technology to obtain information on "angle" in biological phenomena by measuring the "orientation" of molecules using the properties of polarization and anisotropy of fluorescence. In particular, we established a method to detect the changes in the angle (curvature) of cell membranes by translating it to changes in fluorescence intensity corresponding to alteration of the molecular orientation. With these advantages, the curvature of the plasma membrane and its change, which had been visible only observable with electron microscopy, can be optically observed at a high time resolution (second level), making possible to visualize cell membrane deformation in the very early stage of endocytosis.
Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research), Hokkaido University, 15KK0290

ウイルス感染コンピテンシーの不均一性を決定する宿主因子の同定　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2015 - 31 Mar. 2017
大場 雄介
本研究では、インフルエンザウイルスの感染コンピテンシーを規定する因子の同定をその機能解析を行うことを目的に研究を進めている。これまで我々は、インフルエンザウイルスの細胞内取込において、カルシウムシグナルが重要であることを報告しており、本研究では細胞膜に存在するカルシウム制御に関与する分子に着目して研究を行っている。昨年度までに、ある細胞膜タンパク質がその制御に重要であることを見出した。この因子のノックダウンおよび薬剤での阻害によって、インフルエンザウイルス感染時のカルシウム応答は完全に抑制され、またプラークアッセイを用いた実験においてもインフルエンザ感染を強く抑制した。また、この分子の発現量とウイルス感染効率に強い相関があったことから、当該因子がウイルスが宿主細胞への感染過程において重要な役割と担っているおり、ウイルス感染コンピテンシーの規定因子の1つであるという観点から研究を進めた。
この因子はウイルス粒子のHAタンパク質と結合することが昨年までに明らかとなっていたため、当該因子のC末端細胞表面部位に存在するシアル酸化の標的になるアスパラギンをグルタミンに置換した変異体を作製した。変異体とHAの結合は野生型のそれと比べて減少していた。また、当該因子をノックダウンした細胞に野生型を戻すと、細胞内カルシウムの上昇とインフルエンザウイルス粒子の取り込み、インフルエンザ感染の全てが回復したが、変異型の発現を戻しても回復効果は認められなかった。以上から、シアル酸を介した当該因子とウイルス粒子の結合が、ウイルス感染時のカルシウム動員と粒子の取り込み、その後の感染成立に重要であることが示された。本研究成果は学術雑誌に投稿中である。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 15H01248

Investigation of a physiological role of endosome-mitochondrial interaction by fluorescence bioimaging
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2015 - 31 Mar. 2017
Ohba Yusuke, Nanbo Asuka, Nishide Shinya, Fujioka Yoichiro, Fujioka Mari, Horiuchi Kosui, Satoh Aya O.
We have reported that the complex of small G protein Ras and its target molecule PI3K is localized in the endosomes and controls endocytosis and viral particle internalization. However, the molecular mechanism by which the Ras-PI3K complex is localized is the endosome remains unknown. In this study, the amino acid sequence responsible for the above phenomenon was identified. By screening the binding protein for the sequence, a mitochondrial protein was identified. Knockdown of this mitochondrial protein inhibited the translocation of Ras-PI3K complex to the endosome, endocytosis, and mitochondrial-endosome interaction, suggesting that binding of PI3K and the factor regulates endocytosis through interaction between organelles.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 15K15023

Intracellular signaling machinery controlling endocytosis and uptake of extracellular materials
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2014 - 31 Mar. 2017
Ohba Yusuke, Nanbo Asuka, Nishide Shinya, Fujioka Yoichiro, Fujioka Mari, Horiuchi Kosui, Satoh Aya O.
Endosome is a multifunctional platform through the emission and the regulation of a variety of signal transduction pathways. However, the relationship between signaling dynamics and cell function via the endosomes in living cells has yet to be analyzed in detail. In this study, we demonstrated that angiotensin II (AII) type 2 receptor (AT2R) negatively regulates type 1 receptor (AT1R)-signaling through a series of events including heterodimerization of AT1R and AT2R, internalization via endocytosis of the complex, change in the molecular orientation of receptors in the complex. We also clarified that activation of both receptors and and phosphorylation of serine residues at the C-terminus of AT2R by protein kinase C (PKC) are indispensable for this function.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 26293041

バイオイメージングによるウイルス感染と細胞応答の定量解析　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2014 - 31 Mar. 2016
大場 雄介
本研究においては、細胞への感染成立には、インフルエンザウイルス粒子が何個必要かという命題を解くために、研究を行っている。そのため、ウイルス粒子が細胞内侵入する際に重要なシグナル伝達経路の解析と、ウイルス粒子の精密計測により粒子数とその経路の関係性を検討した。シグナル伝達解析においては感染時のカルシウム上昇に着目し、その責任分子の機能解析を進めた。同定した責任分子は24回膜貫通型の膜タンパク質であり、生化学的解析を行うための可溶化が困難であった。種々の界面活性剤を用いて可溶化条件を検討したところ、凝集せずに単量体として可溶化することに成功した。この条件を用いることで、この責任分子とウイルス粒子の結合を介してカルシウム上昇が引き起こされることが明らかになった。
また、領域内の東京大学工学系研究科・野地先生、田端先生との共同研究により、用いる
ウイルスサンプルの粒子数とMOIの関係を明らかにしたうえで、感染性の評価を行った。その結果、ウイルス感染効率はこれまで考えられていたMOIよりも、粒子数に依存することが明らかになった。また、粒子数が多い時と少ない時では異なる細胞の応答性が観察された。粒子数が多い時には感染にある種の正のフィードバックのようなブースト機構が存在すること、そのブースト機構にカルシウムが重要であることが解った。実際に高い複製が認められ、多くの粒子が取り込まれたと考えられる細胞には、上記の責任分子が多く発言していた。一方で、複製が盛んではない（0ではない）細胞においては上記の責任分子の発現量が低く、カルシウム応答も見られないことから、カルシウム非依存性のメカニズムによりウイルスが感染するものと考えられた。両者の境界は細胞1つあたりウイルス粒子20個程度であり、感染には数10個程度の粒子が必要であることが明らかになった。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 26115701

Study on EGFR-TKI resistance using FRET biosensor
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2014 - 31 Mar. 2016
Akita Hirotoshi, Ohba Yusuke, Kinoshita Ichiro
EGFR-TKI resistant cells were isolated in three non-small cell lung cancer (NSCLC) cell lines, using FRET biosensor and flow cytometer. cDNA microarray analysis showed common gene expression changes among the resistant cells. Annotation analysis revealed three up-regulated pathways and five down-regulated pathways. The ABC transporter system was included in the genes and pathways, which were found to be changed in relation to the acquired resistance, suggesting that this system could be involved in the resistance to EGFR-TKI in NSCLCs.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 26670414

Analyzing the mechanism of TKI-resistance in Ph-positive ALL
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2013 - 31 Mar. 2016
KONDO TAKESHI, OHBA Yusuke, MINAMI YOSUKE
Using the FRET-based drug sensitivity test, we identified and isolated TKI-rafractory cell population in Ph-positive ALL. Analysis of gene expression profile in TKI-refractory cells indicated that so many intracellular pathways were activated. One of them was sumoylation pathway. In vitro analysis, we proved that BCR-ABL is sumoylated. Moreover, inhibition of sumoylation effectively induced apoptosis in Ph+ALL cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 25461404

Multidimendional FRET imaging of intracellular signal transduction
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2010 - 31 Mar. 2016
Matsuda Michiyuki, KIYOKAWA Etsuko, HIRATSUKA Takuya, OHBA Yusuke, AOKI Kazuhiro, NAKAMURA Takeshi
FRET biosensors visualize activities of signaling molecules in living cells. However, the use was limited mostly to the cultured 2D cells. Here, we aimed at developing technologies to apply FRET biosensors in 3D culture cells and in living tissues. Due to high recombination rates, stable expression of FRET biosensors had been a difficult task. To overcome this problem, we employed the transposon-mediated gene transfer. We found either piggcBac transposase or Tol2 trasnpoase worked efficiently for the gene transfer of FRET biosensor cDNAs. In particular, we found tol2-mediated gene transfer worked efficiently to develop transgenic mice highly-expressing FRET biosensors, which we named FRET mice. By using FRET mice, we observed activation of various signaling molecules in living tissues.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Kyoto University, 22113002

Challenge to the accurate detection of minimal drug resistant cells
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2013 - 31 Mar. 2015
OHBA Yusuke
In Philadelphia chromosome-positive leukemia, growth and survival of leukemia cells solely depend on constitutive tyrosine kinase activity of new fusion gene product BCR-ABL formed by chromosome reciprocal translocation between chromosomes 9 and 22. Therefore, the treatment for this disease has been radically improved by the introduction of the specific tyrosine kinase inhibitor of BCR-ABL. However, it is desired to develop new technologies that enable the detection of a minimal number of drug-resistant cells because substantial patients experience drug resistance regardless of the presence or absence of past and current treatment. We have previously developed a novel technique to detect relatively small number of drug resistant cells. Here, we challenged to centuplicate the detection ability by the improvement of the FRET biosensor, the detection method and the analytical method. As a result, we obtained 162-fold improvement throughout this study.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 25670184

Analysis of signal transduction pathways evoked by virus infection
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2011 - 31 Mar. 2014
OHBA Yusuke, TSUDA Masumi
Virus infections are one of the largest threats of humanity in modern society with scientific and technological advances, of which the H1N1 pandemic in 2009 would be a practical example. We have previously reported that PI3K bound to and activated by Ras plays a crucial role in the regulation of endocytosis by deciphering the signal transduction pathways with the best use of fluorescence bioimaging techniques.
In this study, we found that Ras-PI3K signaling is required for influenza virus infection, and identified intracellular calcium as a key molecule to regulate a series of influenza virus infection processes. It might also be expected to lead to a new concept of antivirus therapeutics through application of these results.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Principal investigator, Competitive research funding, 23390090

微小環境の定量解析に資する培養・イメージングシステムの構築
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
01 Apr. 2011 - 31 Mar. 2013
大場 雄介
本研究では、腫瘍微小環境を舞台にがん細胞と間質細胞等が演じるドラマを分子レベルで解明する実験系の提供を第一目的とし、昨年度から研究を実施している。大きく分けて以下の2つの研究項目に取り組み、複雑系システムを理解するために多次元イメージング系の基礎を確立した。
細胞の蛍光ラベル：様々な蛍光タンパク質により細胞内のオルガネラをマークする発現ベクターを構築した。蛍光タンパク質については、Sirius、CFP、YFP、tdTomato、TurboFP650、KeimaおよびEGFPの7色それぞれについて、核(ヒストン)、細胞膜 、細胞膜、アクチン細胞骨格、細胞接着斑、早期/後期エンドソーム、ミトコンドリア、微小管、小胞体、ゴルジ装置、リサイクリングエンドゾームのオルガネラ局在発現ベクターを完成させた （計84種）。
3次元培養系の確立：前項で樹立した多彩な蛍光タンパク質でラベルした細胞を用いて、腫瘍微小環境を模倣した3次元培養系を構築した。 この培養条件下において液性因子や基質種類・混合比を調整しながら細胞の挙動を顕微鏡観察し、そのダイナミクスを時空間的に解析した。実際、扁平上皮癌細胞を用いた癌組織の形成を試験管内で再構成することに成功するとともに、その過程においての細胞の挙動の観察にも成功した。また、以前我々が同定した扁平上皮癌細胞の悪性度制御因子をこの系に用いることにより、高分化型扁平上皮癌から低分化型の癌組織を誘導することにも成功した。このことは、本研究で確立した3次元培養系が生体内の癌組織を反映していることが証明されたことになり、今後腫瘍微小環境内での詳細な分子メカニズムの解明のための協力なツールになると期待できる。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, Principal investigator, Competitive research funding, 23114501

Foundational approach for next-generation personalized cancer therapywith molecular target agents
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
2011 - 2012
OHBA Yusuke
With the use of a biosensor based on the principle of Forster resonance energy transfer, we isolated imatinib-resistant cells of Ph-positive acute lymphoid leukemia and explored their properties. Twenty-four signaling pathways were found activated in the resistant cells, among which inhibition of the mevalonate pathway was revealed effective in preventing repopulation of such resistant cells. These results thereby indicate that the combined application of statin may serve to cure Ph-positive acute lymphoid leukemia as well as imatinib-resistant chronic myeloid leukemia.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, Principal investigator, Competitive research funding, 23659196

Exploitation of gene-targeting therapy based on the cellular and biological features of oral-carcinoma stem cell and tumor microenvironment.
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
2008 - 2011
TOTSUKA Yasunori, SHINDOH Masanobu, HIGASHINO Fumihiro, HIDA Kyoko, KITAMURA Tetsuya, OHBA Yusuke, MATSUMOTO Kenichi, OHIRO Yoichi
We have shown that the tumor endothelial cells which exist in cancer stroma have a different gene background from normal endothelial cells. It has been becoming clear that epithelial-mesenchymal transition (EMT) plays an important role in tumor invasion and metastasis. It is expected that the investigation on the interaction of the oral squamous cell carcinoma cell in epithelium nature and a mesenchymal cell will contribute to the establishment of effective therapeutic method because stromal cells have the ability to supply nutrition and oxygen to a tumor. HuR protein usually exists in the nucleus, and HuR is participating to stabilize AU-rich element (ARE) mRNA. We showed that ARE mRNA is stabilized through HuR by a viral carcinogenesis system that concerns cell transformation. Oral environment has a unique features including distinct organs such as salivary glands. We identified that RANKL, an osteoclast inducer, expression was higher in oral environment. Furthermore, when oral cancer cells were implanted in oral region of nude mice, cancer cells proliferated intentionally, and EMT was observed. Tumor microenvironment is deeply correlated with cancer cell proliferation and invasion/metastasis. We have isolated endothelial cells in tumor stroma, and investigated the biological feature of tumor endothelial cells. As a result, tumor angiogenesis was suppressed by inhibiting Cox-2 known also for the factor inducing inflammation, and the resistance for chemothrapy was acquired by MDR1 upregulatin caused by VEGF signaling
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 20249079

癌細胞の微小環境適応因子の探索
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
2009 - 2010
大場 雄介
前年度に引き続き口腔癌をモデルに癌細胞微小環境適応因子として同定したRANKLの機能解析を行りた。RANKLのmRNAレベルおよびタンパクレベルでの発現をRT-PCRと免疫染色で行ったところ、いずれのヒト口腔癌組織における発現が培養細胞に比べて高く、なかでも低分化型の組織での発現が高かった。各種口腔癌細胞株における低いRANKLの発現量は、樹立した元の腫瘍の組織型が低分化型であっても同様であったが、培養癌細胞をヌードマウスの口腔に移植したところ、RANKLの発現が亢進した。一方で同じ細胞株をマウスの下肢に移植したところ、口腔に移植して形成された腫瘍と比較して有意に小さく、RANKLの発現も低かった。すなわち生体内の周囲組織依存的に発現したRANKLが、腫瘍形成を制御することが明らかになった。さらにRANKLの腫瘍形成における役割を明らかにするために、RANKL過剰発現細胞を樹立しマウスの下肢に接種したところ、口腔環境同様に腫瘍の形成が認められた。また組織学的に、コントロール細胞で形成された腫瘍が高分化型扁平上皮癌の組織型を示したのに対し、RANKL発現細胞によって形成された腫瘍は低分化性であった。以上の結果から、RANKLは生体内特異的に発現し腫瘍形成を制御する環境適応因子であり、低分化型の癌すなわち悪性度の高い癌の形成に関与する因子であることが明らかとなった。またこの組織型の変化は上皮間葉転換EMTによることも解明した。
また、RANKLの下流で誘導される因子としてインテグリンα2を同定し、現在悪性度との関与を解析中である。興味深いことにインテグリンα2の発現上昇は活性化型インテグリンの細胞内輸送を亢進するというデータが得られている。近年、EMT等の癌細胞の形態変化と細胞内膜輸送の関連性が報告されつつあり、我々のデータは新たな分子メカニズムを提供する可能性が示唆される。
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, Principal investigator, Competitive research funding, 21659093

Visualization of molecular dynamics of epithelial-mesenchymal and mesenchymal-epithelial transition
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2008 - 2010
OHBA Yusuke, TSUDA Masumi
We have hereby identified transcription factor 8 (TCF8) as a regulator of epithelial-mesenchymal transition, a critical process in cancer invasion/metastasis and endothelial mobilization to tumor angiogenesis. The molecular mechanisms by which TCF8 governs EMT were also revealed in both cancer cells and endothelial cells. In addition, novel evidence that the difference in the tumor microenvironment yields distinct feature of cancer malignancy is also provided, which might lead to the development of new strategies to cancer development.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Principal investigator, Competitive research funding, 20390107

蛍光イメージング技術の臨床応用　　　　　　　　　　　　　　　
共同研究
2010
Competitive research funding

Clinical application of fluorescence imaging　　　　　　　　　　　　　　　
Cooperative Research
2010
Competitive research funding

Informatory expression system connecting cancer and immunity.
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas
2005 - 2009
TANIGUCHI Tadatsugu, TAKAOKA Akinori, TAMURA Tonohiko, HONDA Kenya, OOBA Yuusuke, YANAI Hideyuki
We investigated biological mechanisms and role of effecter molecules, e.g. interferons, IRFs and Noxa. It revealed that these molecules are involved in transformation, apoptosis induction and metastasis signaling pathways. We also found the cross-talk pathway between TLRs signaling. Moreover, we identified DAI and HMGB1, 2, 3 are important for the nucleic-acid mediated innate immune responses. Since these pathways regulate tumorgenesis and exacerbation, our findings will be proved to be useful for the development of novel tumor therapy in near future.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Priority Areas, The University of Tokyo, 17012005

Regulatory signaling in intestinal dendritic cells preventing inflammatory bowel diseases
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
2007 - 2008
HONDA Kenya, TAKEDA Kiyoshi, TAYA Chouji, OHBA Yusuke
腸管樹状細胞を機能的に細分化し、シグナル伝達機構を明らかにし、炎症性腸疾患との関わりを検討することを目的として研究を推進した。新たにCD70が活性化型と抑制型樹状細胞を区別するバイオマーカーとして利用できることが明らかにした。CD70は消化管粘膜固有層特異的に存在する樹状細胞集団の一部において高発現しており、他の臓器にはそのような細胞を認めなかった。更にCD70陽性粘膜固有層樹状細胞は、細胞外ATPの受容体であるP2XやP2Y受容体を高発現しており、ATP刺激によって、共培養したナイーブCD4T細胞をIL17を高産生するTH17細胞へと分化させることも見出した。即ちCD70陽性粘膜固有層樹状細胞は"活性化型樹状細胞"であると考えられた。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Osaka University, 19390137

口腔がんの顎骨浸潤は抑制できるか?-EGFレセプターリン酸化阻害による効果-
Grants-in-Aid for Scientific Research Grant-in-Aid for Exploratory Research
2007 - 2008
戸塚 靖則, 進藤 正信, 大場 雄介, 北村 哲也, 樋田 京子, 東野 史裕, 津田 真寿美
口腔がんの多くは舌や歯肉などの口腔内組織に発症するが、これらの臓器・組織は解剖学的に顎骨に近接しており、腫瘍の増大により容易に顎骨に浸潤する。この場合は、腫瘍の根治性の点から、顎骨離断術や部分切断術を施行せざるをえず、顔面の審美性や機能性に多大な障害を生じ、治癒後の社会生活やQOLに大きな影響を及ぼす。それ故、腫瘍の顎骨浸潤を抑制することが可能となれば、口腔がん患者にどって大きな福音となり、その開発が真に望まれている。
口腔癌細胞の多くは、epidemal growth factor (EGF) receptorを高頻度に発現しており、また顎下腺並びに唾液中には高濃度のEGFが存在していることが明らかになっている。従って、口腔癌の微小環境中に存在するEGFが、癌細胞に対してパラクライン的に作用し、骨浸潤に関与する形質発現に重要な影響を及ぼしていることが示唆される。
口腔がん細胞株HSC2, HSC3, HSC4, Ca9.22, SASを用いてEGFRの発現をWestern Blotで検討したところ、全てのがん細胞株でEGFRの発現亢進がみられた。これらの細胞をEGFで刺激した際、EGFRのリン酸化の亢進およびMAPKであるERK1/2, p38.JNKの活性化がみられた。ERK1/2はEGF依存的にPTHrPを発現亢進した。HSC2細胞にPTHrPのsiRNAを導入することで細胞増殖、浸潤活性の抑制がみられた。
このような所見は、口腔がん細胞の顎骨浸潤にはEGF/EGFRによるPTHrPの発現が重要な役割を演じていることを示唆するものだった。
Japan Society for the Promotion of Science, Grant-in-Aid for Exploratory Research, Hokkaido University, 19659515

受容体サブタイプのモーダルシフトによるアンギオテンシン感受性調節機構の解明
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas
2007 - 2008
大場 雄介
レニン-アンギオテンシン系の研究は国内外を問わずもっともホットな領域の一つといえる。アンジオテンシンは二つのセルセンサーAT1とAT2を介してシグナルを伝えることが知られているが、AT1については既に多くの知見が得られているのに対し、AT2の機能は未知のままである。我々はAT2の発現がAT1シグナルに制御されているという基礎的データを得ており、細胞はAT1からAT2へセンサーをモーダルシフトすることにより、外界に存在するアンジオテンシンのセンシング機構を制御しているものと考えられる、その分子機序が解明されればインパクトは大きい。
本研究ではAT1がアンジオテンシンをセンスして惹起されるERKの活性化を指標にAT1とAT2の機能連関の解明を試みた。AT2はAT2に対し、ERKのリン酸化に関しては拮抗的に働くが、この作用は他のEGFやLPAによるERKのリン酸化に対しては認められないことから、シグナル伝達の極めて上流でクロストークしていることが示唆された。また、蛍光タグを付加したAT1とAT2を発現させ、蛍光顕微鏡で観察したところ、両者が共発現した場合には互いの局在を変化させ共局在することが明らかになった。そこで、2つのセルセンサーの結合を解析したところ、免疫沈降法および蛍光共鳴エネルギー移動(FRET) 法の双方でAT1とAT2のアンジオテンシン依存的な結合が認められた。特にFRETを用いたイメージングでは2つのセルセンサーが細胞膜上で結合した後に、エンドサイトーシスされ、エンドソーム上で結合することが、両者センサーからのシグナルクロストークに重要であることが明らかとなった。これらの現象には、双方の受容体にリガンドが結合することが必要であり、更にその下流に位置する因子として、あるセリンスレオニンキナーゼの必要性を同定した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Priority Areas, Hokkaido University, Principal investigator, Competitive research funding, 19045003

3分子複合体を生きた細胞で検出する技術の確立
Grants-in-Aid for Scientific Research Grant-in-Aid for Exploratory Research
2007 - 2008
大場 雄介
生きた細胞において多分子複合体が、何時何処で形成されるかを追及することは、生命をシステムと理解し、正常状態や病態発症メカニズムを解明する上で重要であにも関わらず、この重要なメカニズムを検出する実験系そのものが現在のところ確立されていない。本研究課題では、生きた細胞における多分子複合体の検出系を確立し、新時代の細胞内情報伝達研究推進への足がかりを築くことを目的として研究を開始した。まず、淡色で分子間相互作用の観察を可能とする、蛍光蛋白質再構成(BiFC)法を用いて蛋白質間相互作用の動的観察系を世界に先がかけて開発した。この技術を用い、RasとPI3Kによる特異的なエンドゾーム上での結合を画像化し、その動きを追跡することに成功した。更にこの研究の過程で、実際にBiFCと他の蛍光蛋白質および、免疫染色を併用することにより、Ras、PI3Kの結合とその代謝産物PIP_3の産生、エンドゾームマーカーであるEEA1の局在からなる、合計3つの蛋白質と一つの脂質の共局在を可視化することに成功した。それらの分子メカニズムを詳細に解析する過程で、RasによるPI3Kの活性化には二つの細胞内シグナル伝達経路が存在し、ひとつはRas非依存性、もう一つはRas依存性であり、後者がエンドゾームにおけるPI3Kの活性化に必要であることも示した。PI3Kの活性化は細胞の生存に必須であることから、その機能阻害は細胞障害が懸念されるが、今回得られた結果は、PI3Kの活性を時空間的に変調することでその一部の機能を調節1可能であることを示唆しており、エンドサイトーシスを介した種々の物質の取り込みを制御する新たな手法の開発に発展すると期待される。
Japan Society for the Promotion of Science, Grant-in-Aid for Exploratory Research, Hokkaido University, Principal investigator, Competitive research funding, 19659094

腫瘍細胞ー微小環境相互作用の時空間ダイナミクス　　　　　　　　　　　　　　　
科学研究費補助金
2008
Competitive research funding

Spatiotemporal dynamics of tumor cell-microenvironment interaction　　　　　　　　　　　　　　　
Grant-in-Aid for Scientific Research
2008
Competitive research funding

多次元バイオイメージングで解明するがんと免疫の時空間シグナルネットワーク
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)
2005 - 2007
大場 雄介
1.癌における時空間シグナルネットワーク
(1)口腔内は上皮細胞増殖因子(EGF)が多量に存在する環境であるため、口腔癌の悪性化にはEGF受容体下流の情報伝達経路が重要である。口腔癌細胞において、EGF受容体の下流で上皮小体ホルモン関連蛋白質(PTHrP)が誘導され、PTHrPが口腔癌細胞の細胞増殖、運動能及び浸潤能等の悪性度を制御していることを明らかにした。本研究成果はBiochem. Biophys. Res. Commun.誌に発表した。
(2)悪性膠芽腫細胞における浸潤能を制御する分子機構として、転写因子OLIG2の発現と低分子量G蛋白質Rhoの活性化、さらにRhoの活性化の局在との関連を明らかにした。本研究成果はMol. Cancer Res.誌に発表した。
2.免疫におけるシグナルネットワーク
(1)核酸による自然免疫系発動に関する分子動態について明らかにした。DNAをセンスする細胞内因子DAIを同定し、DAIとB型DNAの直接結合を画像化することに成功した。また、その過程が培養細胞でのインターフェロン産生に重要であることを明らかにし、Natureに報告した。また、2重鎖RNAに応答するToll様受容体下流におけるアダプター分子TICAM1の細胞内動態について解析し、J. Immunolo.誌に報告した。
(2)インターフェロン産生における負の制御因子IRF2のノックアウトマウスを用い、過剰なインターフェロンシグナルによって生じる慢性貧血の分子機序を明らかにした。本研究成果は、Exp. Hematol.誌に発表した。
本研究課題3年間での成果について、英文学術雑誌に計13本の報告を行った。
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (A), 東京大学->北海道大学, Principal investigator, Competitive research funding, 17689018

蛍光バイオイメージングを用いた細胞内情報伝達の時空間制御機構の解明　　　　　　　　　　　　　　　
科学研究費補助金
2006
Competitive research funding

Spatiotemporal regulation of signal transduction pathways using fluorescence imaging　　　　　　　　　　　　　　　
Grant-in-Aid for Scientific Research
2006
Competitive research funding

Rasファミリー蛋白質Ralの活生化を生きた細胞内で観察し、その機能を解明する
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
2002 - 2003
大場 雄介
RalはRasファミリーGたんぱく質の一つで、Rasにより活性化される。最近、エンドサイトーシス、エキソサイトーシス、フィロポディアの形成に対するRalの関与が示唆されつつあるものの、詳細な分子機序は未知の点が多い。そこで生きた細胞内でRal活性化の時空間的な制御機構の解析を通じて、Ralの生理的機能を知るために、蛍光共鳴エネルギー移動(FRET)の原理を利用したモニター分子を作製し解析した。
モニター分子の構造はYFP、RalA、RalBP、CFP及び膜局在化配列からなる。この分子を発現する293T細胞可溶化液のFRET効率を蛍光分光光度計で測定したところ、GTP結合率とFRET効率が正の相関を示した。従って作製したモニター分子はRalの活性化をFRET効率の上昇として検出できることが確認できた。次にCOS1細胞において上皮細胞増殖因子(EGF)依存性のRalの活性化を多重蛍光タイムラプス顕微鏡で観察し、FRET効率を画像化して解析したところ、EGFによって生じる葉状仮足に限局してRalが活性化していた。この結果はRalの活生化に関し初めて得られた時空間情報である。
さらに、EGF刺激によるRal活性化の機構を詳細に検討した。Rasの優勢劣性型変異体を発現した細胞では、Ralの活性化が抑制されていることから、EGF依存性のRal活性化にはRasの活性化を必要とすることが確認された。しかし、Rasの活性化は葉状仮足で高いが、必ずしも葉状仮足に限局するわけではないので、葉状仮足の形成もまたRalの活性化に必要であることが示唆された。実際、優勢劣性型変異体のRacを導入して、EGF依存性葉状仮足形成を阻害すると、Ralの活性化は阻害された。すなわち、増殖因子依存性Ralの活性化は複数の経路によって制御されていることが明らかとなった。
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Osaka University, Principal investigator, Competitive research funding, 14770098

低分子量G蛋白が細胞膜で情報を受け取るメカニズムの可視化
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Priority Areas
1999 - 2003
松田 道行, 黒川 量雄, 大場 雄介, 中村 岳史, 望月 直樹
本研究の目的は、細胞内情報伝達の重要なキープレーヤーである低分子量G蛋白の活性化の様子を生細胞でリアルタイムにモニターするプローブを作成し、バーチャル細胞作成のために必要な、細胞内情報伝達の時空間パラメータを獲得することである。本年度は、K-Ras、N-Ras、R-Ras、RalA、RalBのプローブ開発を行った。K-Ras、N-Ras、RalAに関しては感度の高いプローブの開発に成功したが、R-RasおよびRalBのプローブは感度が低く、改良の余地を残している。また、既存のプローブの高感度化をYFPおよびCFPをより明るいものに置換することにより達成した。これにより、画像データのシグナルノイズ比を向上させることに成功した。さらに、共焦点レーザー顕微鏡を用いて、細胞内の膜画分と細胞膜画分とのシグナルを定量的に分ける手法を確立した。
一方、これら新規に開発したプローブを使って様々な生命現象の解明に取り組んだ。特に、これまで機能の不明であったRalAが、細胞増殖因子の刺激により葉状突起で限局して活性化されること、RalAの活性化が増殖因子依存性あるいは細胞運動時の葉状突起の形成に必要であることをさまざまな手法を用いて証明した。RalAは小胞の融合に関与するとうい他グループのデータとあわせて考えると、葉状突起の形成には、細胞骨格系と細胞膜系の双方のダイナミックな変化が必要であり、RhoファミリーG蛋白はその前者に、RalAはその後者に密接に関わることが示唆された。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Priority Areas, 11242209

BCR−ABLチロシンキナーゼ活性測定用試薬
Patent right, 大場 雄介, 近藤 健, 国立大学法人 北海道大学
特願2008-135973, 23 May 2008
特開2009-278942, 03 Dec. 2009
200903026427616457

インターフェロン−α及び/又はβ(IFN−α/β)の発現誘導を促進する補助剤のスクリーニングする方法
Patent right, 谷口 維紹, 本田 賢也, 大場 雄介, 国立大学法人 東京大学
特願2005-101706, 31 Mar. 2005
特開2008-148556, 03 Jul. 2008
200903046155691471

AGENT FOR INHIBITING VIRAL INFECTION AND/OR TREATING INFECTIOUS DISEASE, AND METHOD FOR INHIBITING VIRAL INFECTION AND/OR TREATING INFECTIOUS DISEASE　　　　　　　　　　　　　　　
Patent right
WO/2011/126071

METHOD FOR SCREENING OF SUBSTANCE CAPABLE OF PREVENTING VIRUS INFECTION, AND AGENT FOR PREVENTING VIRUS INFECTION　　　　　　　　　　　　　　　
Patent right
WO/2011/046160

ANTI-ANGIOGENIC AGENT　　　　　　　　　　　　　　　
Patent right
WO 2010/005046

ＢＣＲ－ＡＢＬチロシンキナーゼ活性測定試薬　　　　　　　　　　　　　　　
Patent right
2009-278942