教員・研究者詳細

Temperature is critical for the survival and proliferation of animals, which must be adapted to cope with environmental temperature changes. In this study, we demonstrated natural variations in the phenotypes of temperature tolerance and temperature acclimation of the nematode Caenorhabditis elegans, and we decoded whole genome sequence of six natural variations, which enabled us to map responsible gene polymorphisms onto specific chromosomal regions. The C. elegans laboratory strain, N2, survives at 2 A degrees C after cultivation at 15 A degrees C but is unable to survive at 2 A degrees C after cultivation at 20 or 25 A degrees C. This cultivation-temperature-dependent cold tolerance occurs within a few hours after the temperature shift and is termed cold acclimation. We measured the cold tolerance and cold acclimation phenotypes of many natural variants isolated from various areas. CB4854 showed weaker cold tolerance associated with gene polymorphisms on the sex chromosome decoded by whole genome sequencing. Variable cold acclimation phenotypes were exhibited in twelve natural isolates and the large difference was seen between CB4856 and AB1 strains. CB4856, isolated from Hawaii, acclimated slowly to a new temperature, whereas AB1, isolated from Australia, acclimated rapidly. By the whole genome sequencing analysis, two different polymorphisms responsible for the accelerated cold acclimation in AB1 were mapped to specific chromosomal regions.

DOI： 10.1007/s00360-016-1011-3

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Caenorhabditis elegans homologue of Prox1/Prospero is expressed in the glia and is required for sensory behavior and cold tolerance 査読あり

Eriko Kage-Nakadai, Akane Ohta, Tomoyo Ujisawa, Simo Sun, Yoshikazu Nishikawa, Atsushi Kuhara, Shohei Mitani

Genes to Cells 21 ( 9 ) 936 - 948 2016年9月

出版者・発行元：WILEY-BLACKWELL

The Caenorhabditis elegans (C.elegans) amphid sensory organ contains only 4 glia-like cells and 24 sensory neurons, providing a simple model for analyzing glia or neuron-glia interactions. To better characterize glial development and function, we carried out RNA interference screening for transcription factors that regulate the expression of an amphid sheath glial cell marker and identified pros-1, which encodes a homeodomain transcription factor homologous to Drosophila prospero/mammalian Prox1, as a positive regulator. The functional PROS-1::EGFP fusion protein was localized in the nuclei of the glia and the excretory cell but not in the amphid sensory neurons. pros-1 deletion mutants exhibited larval lethality, and rescue experiments showed that pros-1 and human Prox1 transgenes were able to rescue the larval lethal phenotype, suggesting that pros-1 is a functional homologue of mammalian Prox1, at least partially. We further found that the structure and functions of sensory neurons, such as the morphology of sensory endings, sensory behavior and sensory-mediated cold tolerance, appeared to be affected by the pros-1 RNAi. Together, our results show that the C.elegans PROS-1 is a transcriptional regulator in the glia but is involved not only in sensory behavior but also in sensory-mediated physiological tolerance.

DOI： 10.1111/gtc.12394

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Cold tolerance assay for studying cultivation-temperature-dependent cold habituation in C. elegans

Ujisawa T, Ohta, A, Okahata M, Sonoda S, Kuhara, A

Protocol Exchange 2014年

担当区分：筆頭著者,　責任著者

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Long-term calcium imaging of ASJ sensoryneuron controlling cold tolerance in C. elegans

Ujisawa T, Ohta, A, Kuhara, A

Protocol Exchange 2014年

担当区分：責任著者

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Molecular mechanism for trimetric G protein-coupled thermosensation and synaptic regulation in the temperature response circuit of Caenorhabditis elegans 査読あり

Akane Ohta, Atsushi Kuhara

Neuroscience Research 76 ( 3 ) 119 - 124 2013年7月

担当区分：筆頭著者,　責任著者出版者・発行元：ELSEVIER IRELAND LTD

How the nervous system controls the sensation and memory of information from the environment is an essential question. The nematode Caenorhabditis elegans is a useful model for elucidating neural information processing that mediates sensation and memory. The entire nervous system of C. elegans consists of only 302 neurons, and their wiring diagram has been revealed by electron microscopy analysis. Here, we review the molecular and physiological mechanisms responsible for the neural circuit-mediated temperature-seeking behavior (thermotaxis) in C. elegans. Recent molecular biology studies and optogenetic analyses, such as the optical manipulation of neural activity, and neural imaging have revealed the novel concept of neural calculation. Most significantly, trimetric G proteincoupled thermosensation, single sensory neuron-based memory, and the orchestrated synaptic transmission system have been elucidated. (c) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2013.03.008

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Development of a Human Herpesvirus 6 Species-Specific Immunoblotting Assay 査読あり

Yuki Higashimoto, Akane Ohta, Yukihiro Nishiyama, Masaru Ihira, Ken Sugata, Yoshizo Asano, Daniel L. Peterson, Dharam V. Ablashi, Paolo Lusso, Tetsushi Yoshikawa

Journal of Clinical Microbiology 50 ( 4 ) 1245 - 1251 2012年4月

出版者・発行元：AMER SOC MICROBIOLOGY

In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.

DOI： 10.1128/JCM.05834-11

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Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B 査読あり

Masaru Ihira, Akane Ohta, Ken Sugata, Sadao Suga, Yoshizo Asano, Tetsushi Yoshiakwa

Journal of Virological Methods 154 ( 1-2 ) 223 - 225 2008年12月

出版者・発行元：ELSEVIER SCIENCE BV

Genotyping of human herpesvirus 6 (HHV-6) is important clinically, particularly for the diagnosis of neurological diseases. The objective of this study was to establish a rapid HHV-6 genotyping method using the loop-mediated isothermal amplification (LAMP) method. An Accl site is located in the target sequence of HHV-6 B, but not in HHV-6 A. LAMP products were digested with the Accl enzyme and then separated by agarose gel electrophoresis to differentiate the digest pattern of the two variants. The fragment patterns were clearly different between HHV-6 A and B. In order to evaluate the reliability of this HHV-6 genotyping method for use in the clinical laboratory, serum samples from 20 patients with either primary HHV-6 infection or viral reactivation were collected and analyzed. HHV-6 DNA was amplified directly from the serum samples and all 20 LAMP products were positive for HHV-6 B. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jviromet.2008.07.004

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Discriminating between varicella-zoster virus vaccine and wild-type strains by loop-mediated isothermal amplification 査読あり

Yuki Higashimoto, Masaru Ihira, Akane Ohta, Shigeki Inoue, Chie Usui, Yoshizo Asano, Tetsushi Yoshikawa

Journal of Clinical Microbiology 46 ( 8 ) 2665 - 2670 2008年8月

出版者・発行元：AMER SOC MICROBIOLOGY

The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaI for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains.

DOI： 10.1128/JCM.00216-08

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