研究者詳細 - 堀田　耕司

論文 - 堀田　耕司

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Neural depolarization triggers Mg²⁺ influx in rat hippocampal neurons

Yamanaka R., Shindo Y., Karube T., Hotta K., Suzuki K., Oka K.

Neuroscience 310 731 - 741 2015年12月

ISSN 0306-4522

Homeostasis of magnesium ion (Mg²⁺) plays key roles in healthy neuronal functions, and deficiency of Mg²⁺ is involved in various neuronal diseases. In neurons, we have reported that excitotoxicity induced by excitatory neurotransmitter glutamate increases intracellular Mg²⁺ concentration ([Mg²⁺]_i). However, it has not been revealed whether neuronal activity under physiological condition modulates [Mg²⁺]_i. The aim of this study is to explore the direct relationship between neural activity and [Mg²⁺]_i dynamics. In rat primary-dissociated hippocampal neurons, the [Mg²⁺]_i and [Ca²⁺]_i dynamics were simultaneously visualized with a highly selective fluorescent Mg²⁺ probe, KMG-104, and a fluorescent Ca²⁺ probe, Fura Red, respectively. [Mg²⁺]_i increase concomitant with neural activity by direct current stimulation was observed in neurons plated on an indium-tin oxide (ITO) glass electrode, which enables fluorescent imaging during neural stimulation. The neural activity-dependent [Mg²⁺]_i increase was also detected in neurons whose excitability was enhanced by the treatment of a voltage-gated K⁺ channel blocker, tetraethylammonium (TEA) at the timings of spontaneous Ca²⁺ increase. Furthermore, the [Mg²⁺]_i increase was abolished in Mg²⁺-free extracellular medium, indicating [Mg²⁺]_i increase is due to Mg²⁺ influx induced by neural activity. The direct neuronal depolarization by veratridine, a Na⁺ channel opener, induced [Mg²⁺]_i increase, and this [Mg²⁺]_i increase was suppressed by the pretreatment of a non-specific Mg²⁺ channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Overall, activity-dependent [Mg²⁺]_i increase results from Mg²⁺ influx through 2-APB-sensitive channels in rat hippocampal neurons.

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Intracellular magnesium level determines cell viability in the MPP⁺ model of Parkinson's disease

Shindo Yutaka, Yamanaka Ryu, Suzuki Koji, Hotta Kohji, Oka Kotaro

Biochimica et Biophysica Acta - Molecular Cell Research 1853 （ 12 ） 3182 - 3191 2015年12月

ISSN 0167-4889

Parkinson's disease (PD) is a neurodegenerative disorder resulting from mitochondrial dysfunction in dopaminergic neurons. Mitochondria are believed to be responsible for cellular Mg²⁺ homeostasis. Mg²⁺ is indispensable for maintaining ordinal cellular functions, hence perturbation of the cellular Mg²⁺ homeostasis may be responsible for the disorders of physiological functions and diseases including PD. However, the changes in intracellular Mg²⁺ concentration ([Mg²⁺]_i) and the role of Mg²⁺ in PD have still been obscure. In this study, we investigated [Mg²⁺]_i and its effect on neurodegeneration in the 1-methyl-4-phenylpyridinium (MPP⁺) model of PD in differentiated PC12 cells. Application of MPP⁺ induced an increase in [Mg²⁺]_i immediately via two different pathways: Mg²⁺ release from mitochondria and Mg²⁺ influx across cell membrane, and the increased [Mg²⁺]_i sustained for more than 16h after MPP⁺ application. Suppression of Mg²⁺ influx decreased the viability of the cells exposed to MPP⁺. The cell viability correlated highly with [Mg²⁺]_i. In the PC12 cells with suppressed Mg²⁺ influx, ATP concentration decreased and the amount of reactive oxygen species (ROS) increased after an 8h exposure to MPP⁺. Our results indicate that the increase in [Mg²⁺]_i inhibited cellular ROS generation and maintained ATP production, which resulted in the protection from MPP⁺ toxicity.

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Spatiotemporal quantification of subcellular ATP levels in a single HeLa cell during changes in morphology

Suzuki Rika, Hotta Kohji, Oka Kotaro

Scientific Reports 5 2015年11月

The quantitative relationship between change in cell shape and ATP consumption is an unsolved problem in cell biology. In this study, a simultaneous imaging and image processing analysis allowed us to observe and quantify these relationships under physiological conditions, for the first time. We focused on two marginal regions of cells: the microtubule-rich € lamella € and the actin-rich € peripheral structure €. Simultaneous imaging and correlation analysis revealed that microtubule dynamics cause lamellar shape change accompanying an increase in ATP level. Also, image processing and spatiotemporal quantification enabled to visualize a chronological change of the relationships between the protrusion length and ATP levels, and it suggested they are influencing each other. Furthermore, inhibition of microtubule dynamics diminished motility in the peripheral structure and the range of fluctuation of ATP level in the lamella. This work clearly demonstrates that cellular motility and morphology are regulated by ATP-related cooperative function between microtubule and actin dynamics.

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Electrical stimulation of cultured neurons using a simply patterned indium-tin-oxide (ITO) glass electrode

Tanamoto Ryo, Shindo Yutaka, Miki Norihisa, Matsumoto Yoshinori, Hotta Kohji, Oka Kotaro

Journal of Neuroscience Methods 253 272 - 278 2015年09月

ISSN 0165-0270

Background: Indium-tin-oxide (ITO) glass electrodes possess the properties of optical transparency and high electrical conductivity, which enables the electrical stimulation of cultured cells to be performed whilst also measuring the responses with fluorescent imaging techniques. However, the quantitative relationship between the intensity of the stimulating current and the cell response is unclear when using conventional methods that employ a separated configuration of counter and stimulation electrodes. New method: A quantitative electrical current stimulation device without the use of a counter electrode was fabricated. Results: Nerve growth factor (NGF)-induced differentiated PC12 cells were cultured on an ITO single glass electrode, and the Ca2+ response to electrical stimuli was measured using fluorescent Ca2+ imaging. ITO electrode devices with a width less than 0.1mm were found to evoke a Ca2+ response in the PC12 cells. Subsequent variation in the length of the device in the range of 2-10mm was found to have little influence on the efficiency of the electric stimulus. We found that the stimulation of the cells was dependent on the electrical current, when greater than 60μA, rather than on the Joule heat, regardless of the width and length of the conductive area. Comparison with existing method(s): Because of the cells directly in contact with the electrode, our device enables to stimulate the cells specifically, comparing with previous devices with the counter electrode. Conclusions: The ITO device without the use of a counter electrode is a useful tool for evaluating the quantitative neural excitability of cultured neurons.

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Evaluation of drug toxicity profiles based on the phenotypes of ascidian Ciona intestinalis

Mizotani Yuji, Itoh Shun, Hotta Kohji, Tashiro Etsu, Oka Kotaro, Imoto Masaya

Biochemical and Biophysical Research Communications 463 （ 4 ） 656 - 660 2015年05月

ISSN 0006-291X

In vivo toxicity evaluation using model organisms is an important step for the development of new drugs. Here, we report that Ciona intestinalis, a chordate invertebrate, is beneficial to drug toxicity evaluation for the following reasons: rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, and low risk of ethical issues. The dynamic phenotypic change of Ciona larvae during metamorphosis prompted us to examine the effect of cytotoxic drugs on its development by quantifying six toxicity endpoints: degenerated tail size, ampulla length, rotation of body axis, stomach size, heart rate, and body size. As a result, mitochondrial respiratory inhibitors, tubulin polymerization/depolymerization inhibitors, or DNA/RNA synthesis inhibitors showed distinct toxicity profiles against these six endpoints, but drugs with the same targets showed a similar toxicity profile in Ciona. Our results suggest Ciona is an effective animal model for profiling drug toxicity and exploring the mechanisms of drugs with unknown targets.

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Comprehensive morphological analysis of individual peripheral neuron dendritic arbors in ascidian larvae using the photoconvertible protein kaede

Yokoyama Takatoshi D., Hotta Kohji, Oka Kotaro

Developmental Dynamics 243 （ 10 ） 1362 - 1373 2014年10月

ISSN 1058-8388

Background: Ascidian larval epidermal sensory neurons (ESNs) each extend a single cilium into the outer body structure, or tunic, to form a unique external sensory network, the ASNET (ascidian dendritic network in tunic). The functions of ESNs and the ASNET are unknown, though it has been suggested that they play important roles in swimming larvae. In this study, we used transgenic larva expressing the photoconvertible protein Kaede pan-neuronally to identify the orientation and morphology of single ESN external processes within the ASNET. Results: When individual ESN cell bodies were UV-irradiated, the photoconverted Kaede protein diffused into the external processes that projected to the edge of the tunic, indicating that these processes are cytoplasmically connected to the cell bodies of ESNs. We were, therefore, able to comprehensively catalog the morphology and orientation of each ESN external process. Most trunk ESNs appeared to extend neurites to the palp, but no processes were observed to emanate from palp neurons. ESN processes differed systematically in their morphology and orientation, suggesting that the ASNET is formed in a regulated, non-random fashion. Conclusions: This study reveals the organization of ESN sensory fields in the ascidian larval tunic.

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Identification of oscillatory firing neurons associated with locomotion in the earthworm through synapse imaging

Shimoi T., Mizutani K., Kojima D., Kitamura Y., Hotta K., Ogawa H., Oka K.

Neuroscience 268 149 - 158 2014年05月

ISSN 0306-4522

We used FM imaging to identify neurons that receive sensory feedback from the body wall in a circuit for octopamine (OA)-evoked rhythmic locomotion in the earthworm, Eisenia fetida. We visualized synapses in which postsynaptic neurons receive the sensory feedback, by using FM1-43 dye to label the synapses of both motor and sensory pathways that are associated with locomotion, then clearing the motor pathway synapse labeling, and finally identifying the target synapses by distinguishing physiologically functional synapses through destaining using a high-K⁺ solution. A pair of synaptic regions associated with the sensory feedback was found to be located two or three cell body-widths away from the midline, between the anterior parts of the roots of the second lateral nerves (LNs) at the segmental ganglia (SGs). Using conventional intracellular recording and dye loading of the cell bodies surrounding these synaptic regions, we identified a pair of bilateral neurons with cell bodies larger than those of other cells in these regions, and named them "Oscillatory firing neurons Projecting to Peripheral nerves" (OPPs). These had a bipolar shape and projected neurites to the ipsilateral first and third LNs, fired rhythmically, and had a burst timing synchronized with the motor pattern bursts from the ipsilateral first LNs. Current injection into an OPP caused firing in the ipsilateral first LNs, supporting the hypothesis that OPPs functionally project to the peripheral nerves. OPPs also sent neurites to the adjacent anterior and posterior SGs, suggesting connections with the adjacent segments. We conclude that FM imaging can be used to identify neurons involved in specific functions, and that OPPs are the first neurons to be associated with OA-induced locomotion in the earthworm. © 2014 .

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Ontology for the asexual development and anatomy of the colonial chordate botryllus schlosseri

Manni Lucia, Gasparini Fabio, Hotta Kohji, Ishizuka Katherine J., Ricci Lorenzo, Tiozzo Stefano, Voskoboynik Ayelet, Dauga Delphine

PLoS One 9 （ 5 ） 2014年05月

Ontologies provide an important resource to integrate information. For developmental biology and comparative anatomy studies, ontologies of a species are used to formalize and annotate data that are related to anatomical structures, their lineage and timing of development. Here, we have constructed the first ontology for anatomy and asexual development (blastogenesis) of a bilaterian, the colonial tunicate Botryllus schlosseri. Tunicates, like Botryllus schlosseri, are non-vertebrates and the only chordate taxon species that reproduce both sexually and asexually. Their tadpole larval stage possesses structures characteristic of all chordates, i.e. a notochord, a dorsal neural tube, and gill slits. Larvae settle and metamorphose into individuals that are either solitary or colonial. The latter reproduce both sexually and asexually and these two reproductive modes lead to essentially the same adult body plan. The Botryllus schlosseri Ontology of Development and Anatomy (BODA) will facilitate the comparison between both types of development. BODA uses the rules defined by the Open Biomedical Ontologies Foundry. It is based on studies that investigate the anatomy, blastogenesis and regeneration of this organism. BODA features allow the users to easily search and identify anatomical structures in the colony, to define the developmental stage, and to follow the morphogenetic events of a tissue and/or organ of interest throughout asexual development. We invite the scientific community to use this resource as a reference for the anatomy and developmental ontology of B. schlosseri and encourage recommendations for updates and improvements. © 2014 Manni et al.

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Design and synthesis of a FlAsH-type Mg²⁺ fluorescent probe for specific protein labeling

Fujii Tomohiko, Shindo Yutaka, Hotta Kohji, Citterio Daniel, Nishiyama Shigeru, Suzuki Koji, Oka Kotaro

Journal of the American Chemical Society 136 （ 6 ） 2374 - 2381 2014年02月

ISSN 0002-7863

Although the magnesium ion (Mg²⁺) is one of the most abundant divalent cations in cells and is known to play critical roles in many physiological processes, its mobilization and underlying mechanisms are still unknown. Here, we describe a novel fluorescent Mg²⁺ probe, "KMG-104-AsH", composed of a highly selective fluorescent Mg ²⁺ probe, "KMG-104", and a fluorescence-recoverable probe, "FlAsH", bound specifically to a tetracysteine peptide tag (TCtag), which can be genetically incorporated into any protein. This probe was developed for molecular imaging of local changes in intracellular Mg²⁺ concentration. KMG-104-AsH was synthesized, and its optical properties were investigated in solution. The fluorescence intensity of KMG-104-AsH (at λ_em/max = 540 nm) increases by more than 10-fold by binding to both the TCtag peptide and Mg²⁺, and the probe is highly selective for Mg²⁺ (K_d/Mg = 1.7 mM, K_d/Ca 100 mM). Application of the probe for imaging of Mg²⁺ in HeLa cells showed that this FlAsH-type Mg²⁺ sensing probe is membrane-permeable and binds specifically to tagged proteins, such as TCtag-actin and mKeima-TCtag targeted to the cytoplasm and the mitochondrial intermembrane space. KMG-104-AsH bound to TCtag responded to an increase in intracellular Mg²⁺ concentration caused by the release of Mg²⁺ from mitochondria induced by FCCP, a protonophore that eliminates the inner membrane potential of mitochondria. This probe is expected to be a strong tool for elucidating the dynamics and mechanisms of intracellular localization of Mg²⁺. © 2014 American Chemical Society.

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A method for selective ablation of neurons in C. elegans using the phototoxic fluorescent protein, KillerRed

Kobayashi Junya, Shidara Hisashi, Morisawa Yuma, Kawakami Maki, Tanahashi Yuta, Hotta Kohji, Oka Kotaro

Neuroscience Letters 548 261 - 264 2013年08月

ISSN 0304-3940

Specific neuron ablation with laser microbeam has been used in behavioral analysis of Caenorhabditis elegans. However, this method is hard to acquire many ablated worms, and is unable to compare behavioral changes just before and after ablation. Here, we developed an ablation method by using genetically encoded photosensitizer protein, KillerRed, which produces reactive oxygen species by green light irradiation. Ablation of AWA sensory neurons abolished the chemotaxis to AWA specific sensitive attractant, diacetyl, and no functional effect on the other sensory neuron, AWC, which senses benzaldehyde. This ablation method can be useful for analyzing neural in situ. © 2013 Elsevier Ireland Ltd.

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Raman Spectroscopic Imaging of the Whole Ciona intestinalis Embryo during Development

Nakamura Mitsuru J., Hotta Kohji, Oka Kotaro

PLoS One 8 （ 8 ） 2013年08月

Intracellular composition and the distribution of bio-molecules play central roles in the specification of cell fates and morphogenesis during embryogenesis. Consequently, investigation of changes in the expression and distribution of bio-molecules, especially mRNAs and proteins, is an important challenge in developmental biology. Raman spectroscopic imaging, a non-invasive and label-free technique, allows simultaneous imaging of the intracellular composition and distribution of multiple bio-molecules. In this study, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm^-1, respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis. © 2013 Nakamura et al.

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NO/cGMP/PKG signaling pathway induces magnesium release mediated by mitoK_ATP channel opening in rat hippocampal neurons

Yamanaka Ryu, Shindo Yutaka, Hotta Kohji, Suzuki Koji, Oka Kotaro

FEBS Letters 587 （ 16 ） 2643 - 2648 2013年08月

ISSN 0014-5793

Intracellular Mg²⁺ concentration ([_Mg2+]i) and NO regulate cell survival and death. To reveal the involvement of NO in intracellular Mg²⁺ regulation, we visualized intracellular Mg ²⁺ using the fluorescent Mg²⁺ indicator KMG-104-AM in rat hippocampal neurons. Pharmacological experiments using SNAP, 8-Br-cGMP, diazoxide and several inhibitors revealed that the NO/cGMP/Protein kinsase G (PKG) signaling pathway triggers an increase in [_Mg2+]i, and that Mg²⁺ mobilization is due to Mg²⁺ release from mitochondria induced by mitoK_ATP channel opening. In addition, Mg²⁺ release is potentiated by the positive feedback loop including mitoK_ATP channel opening, mitochondrial depolarization and PKC activation. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Odorant-induced membrane potential depolarization of AIY interneuron in Caenorhabditis elegans

Shidara Hisashi, Kobayashi Junya, Tanamoto Ryo, Hotta Kohji, Oka Kotaro

Neuroscience Letters 541 199 - 203 2013年04月

ISSN 0304-3940

Although some interneurons in C. elegans have been shown to have unusual region-specific Ca²⁺ dynamics, the region-specific Ca²⁺ and membrane potential response properties of these neurons are largely unknown due to technical limitations. In this report, we focused on one of these neurons, AIY interneuron, where Ca²⁺ dynamics have been detected only in neurites, and not the soma, during odor and temperature stimulation to determine whether membrane potential and Ca²⁺ are region-specific dynamics and distinct from one another. To visualize voltage change both in the soma and neurites of AIY, we used voltage-sensitive fluorescent protein (VSFP) 2.42. First, we confirmed that the sensor protein worked correctly in C. elegans by depolarizing AIY interneuron with high concentrations of KCl. Next, we observed membrane potential depolarization during odor (isoamyl alcohol) stimulation in both neurites and the soma. Additionally, depolarization of membrane potential with direct application of high KCl induced a Ca²⁺ increase in the soma. From these results, we conclude that membrane potential behavior and Ca²⁺ dynamics in AIY differ in its subcellular regions and that VSFP2.42 can be a useful tool for studying information processing in single neurons. © 2013 Elsevier Ireland Ltd.

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Odorant-induced membrane potential depolarization of AIY interneuron in Caenorhabditis elegans.

堀田耕司

Neuroscience letters Epub ahead of print 2013年02月

研究論文（学術雑誌）, 共著, 査読有り

Crosstalk between second messengers predicts the motility of the growth cone

Kobayashi Takahiko, Nagase Fumiaki, Hotta Kohji, Oka Kotaro

Scientific Reports 3 2013年

Axon guidance involves multiple second messenger signal transduction pathways. Although each signal transduction pathway has been characterized, only a few studies have examined crosstalk between these cascades. Here, we applied a simultaneous second messenger imaging method to the growth cone and demonstrated correlations between cAMP, cGMP, and Ca 2+. The levels of cAMP and cGMP in non-stimulated freely extending growth cones showed a negative correlation without delay. Although there was no direct correlation between cAMP and Ca 2+, examination of cross correlations using small time windows showed frequent switching behavior from negative to positive and vice versa. Furthermore, spatially asymmetric cAMP and cGMP signals in freely deviating growth cones were visualized directly. These results indicate that we succeed in relating second messenger crosstalk to growth cone deviation and extension, and also indicate the possibility of predicting axon guidance from this second messenger crosstalk.

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Three-dimensional anatomy of the Ciona intestinalis tailbud embryo at single-cell resolution

Nakamura Mitsuru J., Terai Jun, Okubo Reiko, Hotta Kohji, Oka Kotaro

Developmental Biology 372 （ 2 ） 274 - 284 2012年12月

ISSN 0012-1606

During embryogenesis, chordates pass through a tailbud stage in which the larval tail is formed. Since acquisition of a tadpole-like tail during tailbud stage is one of the key events in the evolution of chordates, understanding the anatomy of the tailbud stage chordate embryo is of special interest. In this study, to understand comprehensively the anatomy of the tailbud embryo at single-cell-level, real microscopic image stacks of the tailbud embryo in Ciona intestinalis were reconstructed into a 3D computer model. This comprehensive 3D model of the ascidian tailbud embryo was based on real images of confocal laser scanning microscope (CLSM) and therefore, cell shape, location and cell arrangement reflect real geometries of the tailbud embryo. We found that the tailbud embryo consists of 1579 cells, including 836 epidermal cells, 228 cells in the central nervous system, 218 mesenchymal cells, four trunk ventral cells, two B/B^*8.11 cells, 36 muscle cells, 40 notochord cells, four primordial germ cells, and 199 endodermal cells. Moreover, we identified for the first time two populations of previously undefined cells (a total of 12 cells) in Ciona: one located in the lateral trunk and the other located under the tail dorsal epidermis. This information provides a first step for understanding how the body plan of the chordate tailbud embryo formed and evolved. © 2012 Elsevier Inc.

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Three-dimensional anatomy of the Ciona intestinalis tailbud embryo at single-cell resolution.

堀田耕司

Developmental Biology （ 372 ） 274-284 2012年12月

研究論文（学術雑誌）, 共著, 査読有り

Peptidomic analysis of the central nervous system of the protochordate, Ciona intestinalis

Kawada Tsuyoshi, Ogasawara Michio, Sekiguchi Toshio, Aoyama Masato, Hotta Kohji, Oka Kotaro, Satake Honoo

Endocrinology 152 （ 6 ） 2416 - 2427 2011年06月

ISSN 0013-7227

The phylogenetic position of ascidians as the chordate invertebrates closest to vertebrates suggests that they might possess homologs and/or prototypes of vertebrate peptide hormones and neuropeptides as well as ascidian-specific peptides. However, only a small number of peptides have so far been identified in ascidians. In the present study, we have identified various peptides in the ascidian, Ciona intestinalis. Mass spectrometry-based peptidomic analysis detected 33 peptides, including 26 novel peptides, from C. intestinalis. The ascidian peptides are largely classified into three categories: 1) prototypes and homologs of vertebrate peptides, such as galanin/galanin-like peptide, which have never been identified in any invertebrates; 2) peptides partially homologous with vertebrate peptides, including novel neurotesin-like peptides; 3) novel peptides. These results not only provide evidence that C. intestinalis possesses various homologs and prototypes of vertebrate neuropeptides and peptide hormones but also suggest that several of these peptides might have diverged in the ascidian-specific evolutionary lineage. All Ciona peptide genes were expressed in the neural complex, where as several peptide gene transcripts were also distributed in peripheral tissues, including the ovary. Furthermore, a Ciona neurotensin-like peptide, C. intestinalis neurotensin-like peptide 6, was shown to down-regulate growth of Ciona vitellogenic oocytes. These results suggest that the Ciona peptides act not only as neuropeptides in the neural tissue but also as hormones in nonneuronal tissues and that ascidians, unlike other invertebrates, such as nematodes, insects, and sea urchins, established an evolutionary origin of the peptidergic neuroendocrine, endocrine, and nervous systems of vertebrates with certain specific molecular diversity. Copyright © 2011 by The Endocrine Society.

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Chromatophore Activity during Natural Pattern Expression by the Squid Sepioteuthis lessoniana: Contributions of Miniature Oscillation

Mamiko Suzuki, Tetsuya Kimura, Hiroto Ogawa, Kohji Hotta, Kotaro Oka

PLoS One epub 2011年04月

研究論文（学術雑誌）, 共著, 査読有り

CIPRO 2.5

Endo Toshinori, Ueno Keisuke, Yonezawa Kouki, Mineta Katsuhiko, Hotta Kohji, Satou Yutaka, Yamada Lixy, Ogasawara Michio, Takahashi Hiroki, Nakajima Ayako, Nakachi Mia, Nomura Mamoru, Yaguchi Junko, Sasakura Yasunori, Yamasaki Chisato, Sera Miho, Yoshizawa Akiyasu C., Imanishi Tadashi, Taniguchi Hisaaki, Inaba Kazuo

Nucleic Acids Research 39 （ SUPPL. 1 ） 2011年01月

ISSN 0305-1048

The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includesoriginal large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite ofdevelopmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and geneexpression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analysesat various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a communitysupported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http:// cipro.ibio.jp/2.5. © The Author(s) 2010.

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