Publications by authors named "Akihiro Shibata"

23 Publications

  • Page 1 of 1

Photoactivatable CaMKII induces synaptic plasticity in single synapses.

Nat Commun 2021 02 2;12(1):751. Epub 2021 Feb 2.

Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan.

Optogenetic approaches for studying neuronal functions have proven their utility in the neurosciences. However, optogenetic tools capable of inducing synaptic plasticity at the level of single synapses have been lacking. Here, we engineered a photoactivatable (pa)CaMKII by fusing a light-sensitive domain, LOV2, to CaMKIIα. Blue light or two-photon excitation reversibly activated paCaMKII. Activation in single spines was sufficient to induce structural long-term potentiation (sLTP) in vitro and in vivo. paCaMKII activation was also sufficient for the recruitment of AMPA receptors and functional LTP in single spines. By combining paCaMKII with protein activity imaging by 2-photon FLIM-FRET, we demonstrate that paCaMKII activation in clustered spines induces robust sLTP via a mechanism that involves the actin-regulatory small GTPase, Cdc42. This optogenetic tool for dissecting the function of CaMKII activation (i.e., the sufficiency of CaMKII rather than necessity) and for manipulating synaptic plasticity will find many applications in neuroscience and other fields.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21025-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854602PMC
February 2021

Efficacy of a Cap-Dependent Endonuclease Inhibitor and Neuraminidase Inhibitors against H7N9 Highly Pathogenic Avian Influenza Virus Causing Severe Viral Pneumonia in Cynomolgus Macaques.

Antimicrob Agents Chemother 2021 02 17;65(3). Epub 2021 Feb 17.

Division of Pathogenesis and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan

H7N9 highly pathogenic avian influenza virus (HPAIV) infection in a human was first reported in 2017. A/duck/Japan/AQ-HE29-22/2017 (H7N9) (Dk/HE29-22), found in imported duck meat at an airport in Japan, possesses a hemagglutinin with a multibasic cleavage site, indicating high pathogenicity in chickens, as in the case of other H7 HPAIVs. In the present study, we examined the pathogenicity of Dk/HE29-22 and the effectiveness of a cap-dependent endonuclease inhibitor (baloxavir) and neuraminidase inhibitors (oseltamivir and zanamivir) against infection with this strain in a macaque model ( = 3 for each group). All of the macaques infected with Dk/HE29-22 showed severe signs of disease and pneumonia even after the virus had disappeared from lung samples. Virus titers in macaques treated with baloxavir were significantly lower than those in the other treated groups. After infection, levels of interferon alpha and beta (IFN-α and IFN-β) in the blood of macaques in the baloxavir group were the highest among the groups, whereas levels of tumor necrosis factor alpha (TNF-α) and interleukin 13 (IL-13) were slightly increased in the untreated group. In addition, immune checkpoint proteins, including programmed death 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT), were expressed at high levels in the untreated group, especially in one macaque that showed severe signs of disease, indicating that negative feedback responses against vigorous inflammation may contribute to disease progression. In the group treated with baloxavir, the percentages of PD-1-, CTLA-4-, and TIGIT-positive T lymphocytes were lower than those in the untreated group, indicating that reduction in virus titers may prevent expression of immune checkpoint molecules from downregulation of T cell responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/AAC.01825-20DOI Listing
February 2021

Ultrafast internal modification of glass by selective absorption of a continuous-wave laser into excited electrons.

Opt Lett 2020 Jun;45(11):3171-3174

The internal modification of glass using ultrashort pulse lasers has been attracting attention in a wide range of applications. However, the remarkably low processing speed has impeded its use in the industry. In this study, we achieved ultrafast internal modification of glass by coaxially focusing a single-pulse femtosecond laser and continuous-wave (CW) laser with the wavelength that is transparent to the glass. Compared with the conventional method, the processing speed increased by a factor of 500. The observation of high-speed phenomena revealed that the CW laser was absorbed by the seed electrons that were generated by the femtosecond laser pulse. This technique may help expand the applications of femtosecond lasers in the industry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1364/OL.394952DOI Listing
June 2020

Detection of avian influenza virus: a comparative study of the in silico and in vitro performances of current RT-qPCR assays.

Sci Rep 2020 05 21;10(1):8441. Epub 2020 May 21.

Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università, 10, Legnaro, Padova, 35020, Italy.

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-64003-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242438PMC
May 2020

Characterization of H7N9 avian influenza viruses isolated from duck meat products.

Transbound Emerg Dis 2020 Mar 6;67(2):792-798. Epub 2019 Nov 6.

Division of Virology, Department of Microbiology and Immunoslogy, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi-basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ-HE29-22/2017 (HE29-22) and A/duck/Japan/AQ-HE29-52/2017 (HE29-52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ-HE28-3/2016 (HE28-3)] in mice and ferrets. In mice, HE29-52 was more pathogenic than HE29-22 and HE28-3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tbed.13398DOI Listing
March 2020

A novel H7N3 reassortant originating from the zoonotic H7N9 highly pathogenic avian influenza viruses that has adapted to ducks.

Transbound Emerg Dis 2019 Nov 4;66(6):2342-2352. Epub 2019 Aug 4.

Division of Transboundary Animal Disease, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Japan.

The first human case of zoonotic H7N9 avian influenza virus (AIV) infection was reported in March 2013 in China. This virus continues to circulate in poultry in China while mutating to highly pathogenic AIVs (HPAIVs). Through monitoring at airports in Japan, a novel H7N3 reassortant of the zoonotic H7N9 HPAIVs, A/duck/Japan/AQ-HE30-1/2018 (HE30-1), was detected in a poultry meat product illegally brought by a passenger from China into Japan. We analysed the genetic, pathogenic and antigenic characteristics of HE30-1 by comparing it with previous zoonotic H7N9 AIVs and their reassortants. Phylogenetic analysis of the entire HE30-1 genomic sequence revealed that it comprised at least three different sources; the HA (H7), PB1, PA, NP, M and NS segments of HE30-1 were directly derived from H7N9 AIVs, whereas the NA (N3) and PB2 segments of HE30-1 were unrelated to zoonotic H7N9. Experimental infection revealed that HE30-1 was lethal in chickens but not in domestic or mallard ducks. HE30-1 was shed from and replicated in domestic and mallard ducks and chickens, whereas previous zoonotic H7N9 AIVs have not adapted well to ducks. This finding suggests the possibility that HE30-1 may disseminate to remote area by wild bird migration once it establishes in wild bird population. A haemagglutination-inhibition assay indicated that antigenic drift has occurred among the reassortants of zoonotic H7N9 AIVs; HE30-1 showed similar antigenicity to some of those H7N9 AIVs, suggesting it might be prevented by the H5/H7 inactivated vaccine that was introduced in China in 2017. Our study reports the emergence of a new reassortant of zoonotic H7N9 AIVs with novel viral characteristics and warns of the challenge we still face to control the zoonotic H7N9 AIVs and their reassortants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tbed.13291DOI Listing
November 2019

Characterization of a novel reassortant H7N3 highly pathogenic avian influenza virus isolated from a poultry meat product taken on a passenger flight to Japan.

J Vet Med Sci 2019 Mar 24;81(3):444-448. Epub 2019 Jan 24.

Exotic Disease Inspection Division, Laboratory Department, Animal Quarantine Service, Ministry of Agriculture, Forestry and Fisheries, Tokoname, Aichi 479-0881, Japan.

A new reassortant H7N3 avian influenza virus (AIV) was isolated from a duck meat product that was illegally taken on board a passenger flight from China to Japan in March 2018. Sequencing analysis revealed that the H7N3 isolate, A/duck/Japan/AQ-HE30-1/2018 (Dk/HE30-1) (H7N3), was a reassortant highly pathogenic avian influenza virus (HPAIV) that contained the haemagglutinin (HA) gene of Chinese H7N9 HPAIV. Dk/HE30-1 (H7N3) possessed a novel polybasic sequence motif PEVPKRRRTAR/GLF at the HA cleavage site that has never previously been reported in H7 HPAIVs. The HA antigenicity of Dk/HE30-1 (H7N3) slightly differed from that of H7N9 HPAIVs previously reported. These findings will help further our knowledge of the circulation and genetic evolution of emerging AIVs in endemic areas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1292/jvms.18-0628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451897PMC
March 2019

Repeated detection of H7N9 avian influenza viruses in raw poultry meat illegally brought to Japan by international flight passengers.

Virology 2018 11 20;524:10-17. Epub 2018 Aug 20.

Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060-0818, Japan; Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, Hokkaido 001-0020, Japan. Electronic address:

H7N9 highly and low pathogenic avian influenza viruses (HPAIV and LPAIV, respectively) have been isolated from duck meat products that were brought illegally into Japan by flight passengers in their hand luggage. These H7N9 virus isolates were phylogenetically closely related to those prevailing in China. Antigenic analysis revealed that the hemagglutinin of the H7N9 HPAIV isolate was slightly different from those of the H7N9 LPAIV and older H7 strains. These meat products contaminated with AIVs repeatedly brought into Japan lead to increased risks of poultry and public health. Continuous border disease control based on the detection and culling of infected poultry and meat products is, thus, essential for the prevention of introduction and spread of AIVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2018.08.001DOI Listing
November 2018

Rap2 and TNIK control Plexin-dependent tiled synaptic innervation in .

Elife 2018 07 31;7. Epub 2018 Jul 31.

Department of Zoology, The University of British Columbia, Vancouver, Canada.

During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase () and its effector, TNIK (), act genetically downstream of Plexin () to restrict presynaptic assembly and to form tiled synaptic innervation in . Both constitutively GTP- and GDP-forms of mutants exhibit synaptic tiling defects as mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mutants causes a severe synaptic tiling defect. Conversely, overexpression of strongly inhibited synapse formation, suggesting that is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.38801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6067881PMC
July 2018

Cortical actin nodes: Their dynamics and recruitment of podosomal proteins as revealed by super-resolution and single-molecule microscopy.

PLoS One 2017 30;12(11):e0188778. Epub 2017 Nov 30.

Center for Meso-Bio Single-Molecule Imaging (CeMI), Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan.

Electron tomography of the plasma membrane (PM) identified several layers of cortical actin meshwork running parallel to the PM cytoplasmic surface throughout the PM. Here, cortical actin structures and dynamics were examined in living cells, using super-resolution microscopy, with (x,y)- and z-resolutions of ~140 and ~400 nm, respectively, and single-molecule imaging. The super-resolution microscopy identified sub-micron-sized actin clusters that appeared identical by both phalloidin post-fixation staining and Lifeact-mGFP expression followed by fixation, and therefore, these actin clusters were named "actin-pl-clusters". In live cells, the actin-pl-clusters visualized by Lifeact-mGFP linked two or more actin filaments in the fine actin meshwork, acting as a node of the meshwork, and dynamically moved on/along the meshwork in a myosin II-dependent manner. Their formation depended on the Arp2/3 activities, suggesting that the movements could involve both the myosin motor activity and actin polymerization-depolymerization. The actin-pl-clusters differ from the actin nodes/asters found previously after latrunculin treatments, since myosin II and filamin A were not colocalized with the actin-pl-clusters, and the actin-pl-clusters were much smaller than the previously reported nodes/asters. The Lifeact linked to a fluorescently-labeled transmembrane peptide from syntaxin4 (Lifeact-TM) expressed in the PM exhibited temporary immobilization in the PM regions on which actin-pl-clusters and stress fibers were projected, showing that ≥66% of actin-pl-clusters and 89% of stress fibers were located in close proximity (within 3.5 nm) to the PM cytoplasmic surface. Podosome-associated cytoplasmic proteins, Tks4, Tks5, cortactin, and N-WASP, were transiently recruited to actin-pl-clusters, and thus, we propose that actin-pl-clusters also represent "actin podosome-like clusters".
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188778PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708734PMC
December 2017

ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement.

Sci Rep 2017 07 28;7(1):6791. Epub 2017 Jul 28.

Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan.

Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities such as protein-protein interactions and conformational changes. Here, using saturation mutagenesis, we developed a dark yellow fluorescent protein named ShadowY that can serve as an acceptor for FLIM-FRET. ShadowY is spectrally similar to the previously reported dark YFP but has a much smaller quantum yield, greater extinction coefficient, and superior folding property. When ShadowY was paired with mEGFP or a Clover mutant (Clover) and applied to a single-molecule FRET sensor to monitor a light-dependent conformational change of the light-oxygen-voltage domain 2 (LOV2) in HeLa cells, we observed a large FRET signal change with low cell-to-cell variability, allowing for precise measurement of individual cell responses. In addition, an application of ShadowY to a separate-type Ras FRET sensor revealed an EGF-dependent large FRET signal increase. Thus, ShadowY in combination with mEGFP or Clover is a promising FLIM-FRET acceptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-07002-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533704PMC
July 2017

Dynamic Meso-Scale Anchorage of GPI-Anchored Receptors in the Plasma Membrane: Prion Protein vs. Thy1.

Cell Biochem Biophys 2017 Dec 24;75(3-4):399-412. Epub 2017 Jun 24.

Center for Meso-Bio Single-Molecule Imaging (CeMI), Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, 606-8507, Japan.

The central mechanism for the transmission of the prion protein misfolding is the structural conversion of the normal cellular prion protein to the pathogenic misfolded prion protein, by the interaction with misfolded prion protein. This process might be enhanced due to the homo-dimerization/oligomerization of normal prion protein. However, the behaviors of normal prion protein in the plasma membrane have remained largely unknown. Here, using single fluorescent-molecule imaging, we found that both prion protein and Thy1, a control glycosylphosphatidylinositol-anchored protein, exhibited very similar intermittent transient immobilizations lasting for a few seconds within an area of 24.2 and 3.5 nm in diameter in CHO-K1 and hippocampal neurons cultured for 1- and 2-weeks, respectively. Prion protein molecules were immobile during 72% of the time, approximately 1.4× more than Thy1, due to prion protein's higher immobilization frequency. When mobile, prion protein diffused 1.7× slower than Thy1. Prion protein's slower diffusion might be caused by its transient interaction with other prion protein molecules, whereas its brief immobilization might be due to temporary association with prion protein clusters. Prion protein molecules might be newly recruited to prion protein clusters all the time, and simultaneously, prion protein molecules in the cluster might be departing continuously. Such dynamic interactions of normal prion protein molecules would strongly enhance the spreading of misfolded prion protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12013-017-0808-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691105PMC
December 2017

Kinetics of Endogenous CaMKII Required for Synaptic Plasticity Revealed by Optogenetic Kinase Inhibitor.

Neuron 2017 Apr 16;94(1):37-47.e5. Epub 2017 Mar 16.

Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA; Max Planck Florida Institute for Neuroscience, Jupiter, FL 33458, USA. Electronic address:

Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1-2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2017.02.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425291PMC
April 2017

A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer.

Sci Rep 2015 Oct 15;5:15334. Epub 2015 Oct 15.

Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8585, Japan.

Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep15334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606784PMC
October 2015

Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.

PLoS One 2015 23;10(3):e0121109. Epub 2015 Mar 23.

Supportive Center for Brain Research, National Institute for Physiological Science, Okazaki, Aichi, Japan; Department of Physiological Sciences, The Graduate University for Advanced Studies, Okazaki, Aichi, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.

Genetically encoded fluorescence resonance energy transfer (FRET) biosensors have been successfully used to visualize protein activity in living cells. The sensitivity and accuracy of FRET measurements directly depend on biosensor folding efficiency, expression pattern, sensitivity, and dynamic range. Here, to improve the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor, we amplified the association domain of the CaMKIIα gene using error-prone polymerase chain reaction (PCR) and fused it to the N-terminus of mCherry in a bacterial expression vector. We also created an Escherichia coli expression library based on a previously reported fluorescent protein folding reporter method, and found a bright red fluorescent colony that contained the association domain with four mutations (F394L, I419V, A430T, and I434T). In vitro assays using the purified mutant protein confirmed improved folding kinetics of the downstream fluorescent protein, but not of the association domain itself. Furthermore, we introduced these mutations into the previously reported CaMKIIα FRET sensor and monitored its Ca2+/calmodulin-dependent activation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM), and found that the expression pattern and signal reproducibility of the mutant sensor were greatly improved without affecting the autophosphorylation function and incorporation into oligomeric CaMKIIα. We believe that our improved CaMKIIα FRET sensor would be useful in various types of cells and tissues, providing data with high accuracy and reproducibility. In addition, the method described here may also be applicable for improving the performance of all currently available FRET sensors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121109PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370617PMC
February 2016

Light composite scalar in twelve-flavor QCD on the lattice.

Phys Rev Lett 2013 Oct 14;111(16):162001. Epub 2013 Oct 14.

Kobayashi-Maskawa Institute for the Origin of Particles and the Universe (KMI), Nagoya University, Nagoya 464-8602, Japan.

On the basis of lattice simulations using highly improved staggered quarks for twelve-flavor QCD with several bare fermion masses, we observe a flavor-singlet scalar state lighter than the pion in the correlators of fermionic interpolating operators. The same state is also investigated using correlators of gluonic interpolating operators. Combined with our previous study that showed twelve-flavor QCD to be consistent with being in the conformal window, we infer that the lightness of the scalar state is due to infrared conformality. This result shed some light on the possibility of a light composite Higgs boson ("technidilaton") in walking technicolor theories.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1103/PhysRevLett.111.162001DOI Listing
October 2013

Rac1 recruitment to the archipelago structure of the focal adhesion through the fluid membrane as revealed by single-molecule analysis.

Cytoskeleton (Hoboken) 2013 Mar 5;70(3):161-77. Epub 2013 Mar 5.

Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan.

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cm.21097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3627312PMC
March 2013

Archipelago architecture of the focal adhesion: membrane molecules freely enter and exit from the focal adhesion zone.

Cytoskeleton (Hoboken) 2012 Jun 4;69(6):380-92. Epub 2012 May 4.

Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Japan; Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane, mechanically linking the extracellular matrix with the termini of actin stress fibers, providing key scaffolds for the cells to migrate in tissues. The FA was considered as a micron-scale, massive assembly of various proteins, although its formation and decomposition occur quickly in several to several 10 s of minutes. The mechanism of rapid FA regulation has been a major mystery in cell biology. Here, using fast single fluorescent-molecule imaging, we found that transferrin receptor and Thy1, non-FA membrane proteins, readily enter the FA zone, diffuse rapidly there, and exit into the bulk plasma membrane. Integrin β3 also readily enters the FA zone, and repeatedly undergoes temporary immobilization and diffusion in the FA zone, whereas approximately one-third of integrin β3 is immobilized there. These results are consistent with the archipelago architecture of the FA, which consists of many integrin islands: the membrane molecules enter the inter-island channels rather freely, and the integrins in the integrin islands can be rapidly exchanged with those in the bulk membrane. Such an archipelago architecture would allow rapid FA formation and disintegration, and might be applicable to other large protein domains in the plasma membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cm.21032DOI Listing
June 2012

Detection of alginate oligosaccharides from mollusks.

Biosci Biotechnol Biochem 2006 Nov 7;70(11):2793-6. Epub 2006 Nov 7.

Central Research Institute, Maruha Group Inc., Tsukuba, Ibaraki, Japan.

We attempted in this study to detect alginate oligosaccharides (AO) from mollusks. The samples used were digestive organs taken from turban shells and abalones which commonly ate brown algae. High-performance liquid chromatography (HPLC) and negative-ion electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) analyses were used to confirm the presence of AO. Samples spiked with AO resulted in observable peaks where the HPLC area was increased. The highest content was estimated to be 401.8 mg/100 g of digestive organ. The product-ion data derived from AO molecular weight were detected at a constant interval by Q-TOF MS/MS analysis. These findings indicate that AO was present in the digestive organs of mollusks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1271/bbb.60313DOI Listing
November 2006

Identification of a novel conjugate in human urine: bile acid acyl galactosides.

Steroids 2005 Mar;70(3):185-92

Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.steroids.2004.12.006DOI Listing
March 2005