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Bourguignon T.,National University of Singapore | Bourguignon T.,Czech University of Life Sciences | Lo N.,University of Sydney | Cameron S.L.,Queensland University of Technology | And 7 more authors.
Molecular biology and evolution | Year: 2015

Termites have colonized many habitats and are among the most abundant animals in tropical ecosystems, which they modify considerably through their actions. The timing of their rise in abundance and of the dispersal events that gave rise to modern termite lineages is not well understood. To shed light on termite origins and diversification, we sequenced the mitochondrial genome of 48 termite species and combined them with 18 previously sequenced termite mitochondrial genomes for phylogenetic and molecular clock analyses using multiple fossil calibrations. The 66 genomes represent most major clades of termites. Unlike previous phylogenetic studies based on fewer molecular data, our phylogenetic tree is fully resolved for the lower termites. The phylogenetic positions of Macrotermitinae and Apicotermitinae are also resolved as the basal groups in the higher termites, but in the crown termitid groups, including Termitinae + Syntermitinae + Nasutitermitinae + Cubitermitinae, the position of some nodes remains uncertain. Our molecular clock tree indicates that the lineages leading to termites and Cryptocercus roaches diverged 170 Ma (153-196 Ma 95% confidence interval [CI]), that modern Termitidae arose 54 Ma (46-66 Ma 95% CI), and that the crown termitid group arose 40 Ma (35-49 Ma 95% CI). This indicates that the distribution of basal termite clades was influenced by the final stages of the breakup of Pangaea. Our inference of ancestral geographic ranges shows that the Termitidae, which includes more than 75% of extant termite species, most likely originated in Africa or Asia, and acquired their pantropical distribution after a series of dispersal and subsequent diversification events. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Takayama Y.,Okazaki Institute for Integrative Bioscience | Uta D.,University of Toyama | Furue H.,National Institute for Physiological science | Furue H.,Graduate University for Advanced Studies | And 2 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) is activated by various noxious stimuli, and the stimuli are converted into electrical signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization, leading to the activation of voltage-gated sodium channels, followed by the generation of action potential. Here we report that the capsaicin-evoked action potential could be induced by two components: a cation influx-mediated depolarization caused by TRPV1 activation and a subsequent anion efflux-mediated depolarization via activation of anoctamin 1 (ANO1), a calcium-activated chloride channel, resulting from the entry of calcium through TRPV1. The interaction between TRPV1 and ANO1 is based on their physical binding. Capsaicin activated the chloride currents in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV1 and ANO1. Similarly, in mouse dorsal root ganglion neurons, capsaicin-activated inward currents were inhibited significantly by a specific ANO1 antagonist, T16Ainh-A01 (A01), in the presence of a high concentration of EGTA but not in the presence of BAPTA [1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid]. The generation of a capsaicin-evoked action potential also was inhibited by A01. Furthermore, pain-related behaviors in mice treated with capsaicin, but not with αβ-methylene ATP, were reduced significantly by the concomitant administration of A01. These results indicate that TRPV1-ANO1 interaction is a significant pain-enhancing mechanism in the peripheral nervous system. © 2015, National Academy of Sciences. All rights reseved.

News Article | December 23, 2015
Site: phys.org

Some reptiles such as crocodilians and some turtles are known to display temperature-dependent sex determination (TSD), where the ambient temperature of the developing eggs determines the individual's sex. For example in the American alligator's eggs, incubation at 33 ºC produces mostly males, while incubation at 30 ºC produces mostly females. An international joint research team between Japan and the US have determined that the thermosensor protein TRPV4 is associated with TSD in the American alligator. The research has been published in Scientific Reports. The research team headed by Professor Taisen Iguchi of the National Institute for Basic Biology (Okazaki Institute for Integrative Bioscience) and PhD student Ryohei Yatsu of SOKENDAI (The Graduate University for Advanced Studies), in collaboration with Professor Makoto Tominaga of the National Institute for Physiological Sciences (Okazaki Institute for Integrative Bioscience) and Professor Louis J. Guillette Jr. and assistant professor Satomi Kohno of the Medical University of South Carolina, have investigated the molecular mechanism of how temperature determines sex. In their research using American alligators, they found that a thermosensitive protein called TRPV4 is present within the developing alligator gonad inside the egg. Alligator TRPV4 is responsive to warm temperatures near mid-30s, and can activate cell signaling by inducing calcium ion influx. The current study also demonstrates that by specific pharmacological inhibition of TPRV4 protein function in the developing egg, genes important for male development (for example, genes encoding anti-Müllerian hormone and SOX9) are influenced, and partial feminization at male producing temperatures have been observed. From these results the authors demonstrate that TRPV4 may significantly influence the male gonadal sex determination pathway at a molecular level during TSD in the alligator. This is the first demonstrated report of a biomolecule associated with regulation of the very unique temperature-dependent sex determination mechanism. PhD student Ryohei Yatsu said, "Reptiles can be difficult to study at times, but we were delighted to obtain such an interesting result and elucidate part of the alligator TSD mechanism. We still have much to research, but we are interested in how our results relate with other TSD species diversity and evolution". Professor Taisen Iguchi said, "Organisms that have adopted TSD systems may be more susceptible to the risks of environmental change, such as global warming. In future, we would like to know how an unstable environmental factor such as incubation temperature was able to establish itself as a sex determination factor." More information: Ryohei Yatsu et al. TRPV4 associates environmental temperature and sex determination in the American alligator, Scientific Reports (2015). DOI: 10.1038/srep18581

Kim J.H.,Kyungpook National University | Tsukaya H.,University of Tokyo | Tsukaya H.,Okazaki Institute for Integrative Bioscience
Journal of Experimental Botany | Year: 2015

Transcription factors are key regulators of gene expression and play pivotal roles in all aspects of living organisms. Therefore, identifcation and functional characterization of transcription factors is a prerequisite step toward understanding life. This article reviews molecular and biological functions of the two transcription regulator families, GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), which have only recently been recognized. A myriad of experimental evidence clearly illustrates that GRF and GIF are bona fde partner proteins and form a plant-specifc transcriptional complex. One of the most conspicuous outcomes from this research feld is that the GRF-GIF duo endows the primordial cells of vegetative and reproductive organs with a meristematic specifcation state, guaranteeing the supply of cells for organogenesis and successful reproduction. It has recently been shown that GIF1 proteins, also known as ANGUSTIFOLIA3, recruit chromatin remodelling complexes to target genes, and that AtGRF expression is directly activated by the foral identity factors, APETALA1 and SEPALLATA3, providing an important insight into understanding of the action of GRF-GIF. Moreover, GRF genes are extensively subjected to post-transcriptional control by microRNA396, revealing the presence of a complex regulatory circuit in regulation of plant growth and development by the GRF-GIF duo. © 2015 The Author. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Danev R.,Okazaki Institute for Integrative Bioscience | Kanamaru S.,Tokyo Institute of Technology | Marko M.,New York State Department of Health | Nagayama K.,Okazaki Institute for Integrative Bioscience
Journal of Structural Biology | Year: 2010

Cryo-tomography in the electron microscope is unique in its ability to provide high-resolution, three-dimensional structural information about cells, organelles and macromolecules in a nearly native, frozen-hydrated state. However, the phase-contrast imaging method used in conventional cryo-electron tomography fails to faithfully represent the full range of structural features in such specimens. Only certain features are recorded with adequate contrast, and overall contrast is low. The recently developed Zernike phase contrast method has the potential to solve this problem, and here we apply it for the first time to cryo-electron tomography. The new method has uniform transfer characteristics for a wide range of spatial frequencies, leading to improved overall signal-to-noise ratio and raising the prospects of higher resolution and quantitative representation of specimen densities in the reconstructed tomograms. © 2010 Elsevier Inc.

Danev R.,Okazaki Institute for Integrative Bioscience | Nagayama K.,Okazaki Institute for Integrative Bioscience
Ultramicroscopy | Year: 2011

Images acquired with a phase plate often exhibit fringing and/or contrast reversal artifacts. The two basic parameters controlling the performance of the phase plate are phase shift and cut-on periodicity. We investigate theoretically and numerically the effect of these parameters on the image quality. The analysis covers not just the typical negative phase shift phase plates but also positive phase shift ones. The theoretical study derives formulas for calculating the optimal phase plate phase shift and for the maximum achievable contrast with a given specimen. Two figures of merit - fidelity and contrast - were defined and used to quantify the numerical results. Larger cut-on periodicities provide better performance with higher contrast and less artifacts in the images. Both, the theoretical results and the simulations indicate that positive phase shift phase plates generate higher contrast with better linearity and are free from contrast reversal artifacts. However, with such phase plates the amplitude and the phase contrast components are opposed to each other and the simulations show stronger fringing outside of objects. Based on these results it is difficult to predict if and to what extent the positive phase shift phase plates will be advantageous in practice. Two methods for reduction of fringing artifacts were compared-tapered phase plate and low-frequency amplification software filter. Overall the software solution produced better results and is much easier to implement than modifying the hardware of the phase plate to realize the taper. © 2011 Elsevier B.V.

Miyanari Y.,Okazaki Institute for Integrative Bioscience
Methods | Year: 2014

The three-dimensional remodeling of chromatin within nucleus is being recognized as determinant for genome regulation. Recent technological advances in live imaging of chromosome loci begun to explore the biological roles of the movement of the chromatin within the nucleus. To facilitate better understanding of the functional relevance and mechanisms regulating genome architecture, we applied transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences in mouse cells. The application, called TAL effector-mediated genome visualization (TGV), allows us to label specific repetitive sequences and trace nuclear remodeling in living cells. Using this system, parental origin of chromosomes was specifically traced by distinction of single-nucleotide polymorphisms (SNPs). This review will present our approaches to monitor nuclear dynamics of target sequences and highlights key properties and potential uses of TGV. © 2014 Elsevier Inc.

Tokuda N.,Nagoya University | Terada T.P.,Nagoya University | Sasai M.,Nagoya University | Sasai M.,Korea Institute for Advanced Study | Sasai M.,Okazaki Institute for Integrative Bioscience
Biophysical Journal | Year: 2012

Eukaryotic genome is organized in a set of chromosomes each of which consists of a chain of DNA and associated proteins. Processes involving DNA such as transcription, duplication, and repair, therefore, should be intrinsically related to the three-dimensional organization of the genome. In this article, we develop a computational model of the three-dimensional organization of the haploid genome of interphase budding yeast by regarding chromosomes as chains moving under the constraints of nuclear structure and chromatin-chromatin interactions. The simulated genome structure largely fluctuates with the diffusive movement of chromosomes. This fluctuation, however, is not completely random, as parts of chromosomes distribute in characteristic ways to form "territories" in the nucleus. By suitably taking account of constraints arising from the data of the chromosome-conformation-capture measurement, the model explains the observed fluorescence data of chromosome distributions and motions. © 2012 Biophysical Society.

Nagayama K.,Okazaki Institute for Integrative Bioscience
Journal of Electron Microscopy | Year: 2011

It has been six decades since the concept of phase-plate electron microscopy was first reported by Boersch, but an experimental report on a phase plate with a theoretically rational performance has only recently been released by a group including the present author. Currently, many laboratories around the world are attempting to develop a wide range of phase plates to enhance the capabilities of transmission electron microscopy. They are reporting not only advantages of their own developments but also a fundamental problem inherent to electron beam devices, namely charging, i.e. the accumulation of electrostatic charge. In this report, we review the 60-year history of phase-plate development, with a particular focus on the fundamental issue of phase-plate charging. Next, we review biological applications of qualified phase plates, which have been successful in avoiding charging to some extent. Finally, we compare and discuss electron microscopic images, taken with or without phase plates, of biological targets such as proteins (GroEL and TRPV4), protein complexes (flagellar motor), viruses (T4 phage, ε-15 phage and herpes simplex virus), bacterial (cyanobacteria) and mammalian (PtK2) cells. © The Author 2011. Published by Oxford University Press [on behalf of Japanese Society of Microscopy]. All rights reserved.

Danev R.,Okazaki Institute for Integrative Bioscience | Nagayama K.,Okazaki Institute for Integrative Bioscience
Methods in Enzymology | Year: 2010

Phase plates are a new technique in the field of cryo-electron microscopy. They provide improved contrast and signal-to-noise ratio in images of radiation sensitive specimens. Thin film phase plates are being tested in biological applications and have demonstrated benefits for single particle analysis and cryo-tomography. There are still unsolved problems, such as reliability of manufacturing and deterioration of performance with time. Several other types of phase plates are currently under development and may become available for cryo-microscopy in near future. Presented is a short overview of the current state of the field as well as ideas for the future directions. Also included is a detailed description of the instrumentation requirements and the experimental procedures for phase plate application. © 2010 Elsevier Inc.

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