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  • 1
    Online Resource
    Online Resource
    Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos
    Proceedings of the National Academy of Sciences ; 2012
    In:  Proceedings of the National Academy of Sciences Vol. 109, No. 27 ( 2012-07-03), p. 10915-10920
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 27 ( 2012-07-03), p. 10915-10920
    Abstract: To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus . We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A -histone H2B – GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4 , which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1202768109
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2012
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  • 2
    Online Resource
    Online Resource
    Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice
    Proceedings of the National Academy of Sciences ; 2004
    In:  Proceedings of the National Academy of Sciences Vol. 101, No. 23 ( 2004-06-08), p. 8687-8692
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 101, No. 23 ( 2004-06-08), p. 8687-8692
    Abstract: Overexpression of transforming growth factor β (TGF-β) has been shown to play pathogenic roles in progression of renal fibrosis, and the severity of tubulointerstitial fibrosis correlates better with renal function than the severity of glomerulosclerosis. Smad proteins are signaling transducers downstream from TGF-β receptors. Three families of Smad proteins have been identified: receptor-regulated Smad2 and Smad3, common partner Smad4, and inhibitory Smad7 (part of a negative-feedback loop). We investigated Smad-mediated TGF-β signaling pathway and regulatory mechanisms of inhibitory Smad7 in unilateral ureteral obstruction (UUO) kidneys in mice, a model of progressive tubulointerstitial fibrosis. Compared with sham-operated kidneys, the level of Smad7 protein, but not mRNA, decreased progressively in UUO kidneys, whereas immunoreactivity for nuclear phosphorylated Smad2 and Smad3 and renal fibrosis were inversely increased. Furthermore, we demonstrated that both the degradation and ubiquitination activity of Smad7 protein were increased markedly in UUO kidneys compared with sham-operated ones. We also found that both Smurf1 and Smurf2 (Smad ubiquitination regulatory factors), which are E3 ubiquitin ligases for Smad7, were increased and that they interacted with Smad7 in UUO kidneys. Our results suggest that the reduction of Smad7 protein resulting from enhanced ubiquitin-dependent degradation plays a pathogenic role in progression of tubulointerstitial fibrosis.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0400035101
    RVK:
    TA 1000
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    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2004
    detail.hit.zdb_id: 209104-5
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  • 3
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    Online Resource
    Visualization of Ca 2+ Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells
    Society for Neuroscience ; 2015
    In:  The Journal of Neuroscience Vol. 35, No. 48 ( 2015-12-02), p. 15837-15846
    Details
    In: The Journal of Neuroscience, Society for Neuroscience, Vol. 35, No. 48 ( 2015-12-02), p. 15837-15846
    Abstract: The endoplasmic reticulum (ER) plays crucial roles in intracellular Ca 2+ signaling, serving as both a source and sink of Ca 2+ , and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca 2+ dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca 2+ dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca 2+ indicator, G-CEPIA1 er . We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca 2+ concentration ([Ca 2+ ] ER ) in dendrites and spines. In this experimental system, the recovery of [Ca 2+ ] ER takes several seconds, and recovery half-time depends on the extent of ER Ca 2+ depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca 2+ , rather than Ca 2+ reuptake, is the dominant mechanism for the replenishment of the local [Ca 2+ ] ER depletion immediately following the stimulation. In spines, the ER filled almost simultaneously with parent dendrites, suggesting that the ER within the spine neck does not represent a significant barrier to Ca 2+ diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca 2+ spikes in PCs, cumulatively increased [Ca 2+ ] ER . These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca 2+ within the neurons, and as a leaky integrator of Ca 2+ spike-inducing synaptic inputs. SIGNIFICANCE STATEMENT Ca 2+ is a key messenger that regulates neuronal functions in the brain. Although the endoplasmic reticulum (ER) plays indispensable roles as a source and sink of Ca 2+ , technical difficulties have impeded the analysis of Ca 2+ dynamics within the ER. In this study, we have used a genetically encoded ER Ca 2+ indicator to visualize Ca 2+ dynamics within the neuronal ER. We found that Ca 2+ -mobilizing synaptic inputs locally decreased the ER Ca 2+ concentration, followed by Ca 2+ replenishment by intraluminal Ca 2+ diffusion throughout the ER of dendrites and spines. Furthermore, Ca 2+ spike-inducing synaptic inputs cumulatively increased the Ca 2+ content of the ER. Thus, our study indicates that the ER functions both as a tunnel to redistribute stored Ca 2+ and as a leaky integrator of synaptic inputs.
    Type of Medium: Online Resource
    ISSN: 0270-6474 , 1529-2401
    URL: Article
    DOI: 10.1523/JNEUROSCI.3487-15.2015
    Language: English
    Publisher: Society for Neuroscience
    Publication Date: 2015
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  • 4
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    Online Resource
    A Mesoscale Iron Enrichment in the Western Subarctic Pacific Induces a Large Centric Diatom Bloom
    American Association for the Advancement of Science (AAAS) ; 2003
    In:  Science Vol. 300, No. 5621 ( 2003-05-09), p. 958-961
    Details
    In: Science, American Association for the Advancement of Science (AAAS), Vol. 300, No. 5621 ( 2003-05-09), p. 958-961
    Abstract: We have performed an in situ test of the iron limitation hypothesis in the subarctic North Pacific Ocean. A single enrichment of dissolved iron caused a large increase in phytoplankton standing stock and decreases in macronutrients and dissolved carbon dioxide. The dominant phytoplankton species shifted after the iron addition from pennate diatoms to a centric diatom, Chaetoceros debilis , that showed a very high growth rate, 2.6 doublings per day. We conclude that the bioavailability of iron regulates the magnitude of the phytoplankton biomass and the key phytoplankton species that determine the biogeochemical sensitivity to iron supply of high-nitrate, low-chlorophyll waters.
    Type of Medium: Online Resource
    ISSN: 0036-8075 , 1095-9203
    URL: Article
    DOI: 10.1126/science.1082000
    RVK:
    TA 1000
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    WA 15000
    Language: English
    Publisher: American Association for the Advancement of Science (AAAS)
    Publication Date: 2003
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  • 5
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    Online Resource
    Formation of a One-Dimensional Array of Oxygen in a Microporous Metal-Organic Solid
    American Association for the Advancement of Science (AAAS) ; 2002
    In:  Science Vol. 298, No. 5602 ( 2002-12-20), p. 2358-2361
    Details
    In: Science, American Association for the Advancement of Science (AAAS), Vol. 298, No. 5602 ( 2002-12-20), p. 2358-2361
    Abstract: We report the direct observation of dioxygen molecules physisorbed in the nanochannels of a microporous copper coordination polymer by the MEM (maximum entropy method)/Rietveld method, using in situ high-resolution synchrotron x-ray powder diffraction measurements. The obtained MEM electron density revealed that van der Waals dimers of physisorbed O 2 locate in the middle of nanochannels and form a one-dimensional ladder structure aligned to the host channel structure. The observed O–O stretching Raman band and magnetic susceptibilities are characteristic of the confined O 2 molecules in one-dimensional nanochannels of CPL-1 (coordination polymer 1 with pillared layer structure).
    Type of Medium: Online Resource
    ISSN: 0036-8075 , 1095-9203
    URL: Article
    DOI: 10.1126/science.1078481
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: American Association for the Advancement of Science (AAAS)
    Publication Date: 2002
    detail.hit.zdb_id: 128410-1
    detail.hit.zdb_id: 2066996-3
    detail.hit.zdb_id: 2060783-0
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  • 6
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    Online Resource
    Neural Basis of the Retrieval of People's Names: Evidence from Brain-Damaged Patients and fMRI
    MIT Press ; 2002
    In:  Journal of Cognitive Neuroscience Vol. 14, No. 6 ( 2002-08-15), p. 922-937
    Details
    In: Journal of Cognitive Neuroscience, MIT Press, Vol. 14, No. 6 ( 2002-08-15), p. 922-937
    Abstract: The aim of this study was to identify the neuroanatomical basis of the retrieval of people's names. Lesion data showed that patients with language-dominant temporal lobectomy had impairments in their ability to retrieve familiar and newly learned people's names, whereas patients with language-nondominant temporal lobectomy had difficulty retrieving newly learned people's names. Functional magnetic resonance imaging experiments revealed activations in the left temporal polar region during the retrieval of familiar and newly learned people's names, and in the right superior temporal and bilateral prefrontal cortices during the retrieval of newly learned information from face cues. These data provide new evidence that the left anterior temporal region is crucial for the retrieval of people's names irrespective of their familiarity and that the right superior temporal and bilateral prefrontal areas are crucial for the process of associating newly learned people's faces and names.
    Type of Medium: Online Resource
    ISSN: 0898-929X , 1530-8898
    URL: Article
    DOI: 10.1162/089892902760191144
    Language: English
    Publisher: MIT Press
    Publication Date: 2002
    SSG: 5,2
    SSG: 7,11
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  • 7
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    Online Resource
    Control of Cortical Axon Elongation by a GABA-Driven Ca 2+ /Calmodulin-Dependent Protein Kinase Cascade
    Society for Neuroscience ; 2009
    In:  The Journal of Neuroscience Vol. 29, No. 43 ( 2009-10-28), p. 13720-13729
    Details
    In: The Journal of Neuroscience, Society for Neuroscience, Vol. 29, No. 43 ( 2009-10-28), p. 13720-13729
    Abstract: Ca 2+ signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca 2+ rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK)–CaMKI cascades, CaMKK–CaMKIα and CaMKK–CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABA A receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK–CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIα significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK–CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.
    Type of Medium: Online Resource
    ISSN: 0270-6474 , 1529-2401
    URL: Article
    DOI: 10.1523/JNEUROSCI.3018-09.2009
    Language: English
    Publisher: Society for Neuroscience
    Publication Date: 2009
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  • 8
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    Online Resource
    Critical role of PA28γ in hepatitis C virus-associated steatogenesis and hepatocarcinogenesis
    Proceedings of the National Academy of Sciences ; 2007
    In:  Proceedings of the National Academy of Sciences Vol. 104, No. 5 ( 2007-01-30), p. 1661-1666
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 104, No. 5 ( 2007-01-30), p. 1661-1666
    Abstract: Hepatitis C virus (HCV) is a major cause of chronic liver disease that frequently leads to steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCV core protein is not only a component of viral particles but also a multifunctional protein because liver steatosis and HCC are developed in HCV core gene-transgenic (CoreTg) mice. Proteasome activator PA28γ/REGγ regulates host and viral proteins such as nuclear hormone receptors and HCV core protein. Here we show that a knockout of the PA28γ gene induces the accumulation of HCV core protein in the nucleus of hepatocytes of CoreTg mice and disrupts development of both hepatic steatosis and HCC. Furthermore, the genes related to fatty acid biosynthesis and srebp-1c promoter activity were up-regulated by HCV core protein in the cell line and the mouse liver in a PA28γ-dependent manner. Heterodimer composed of liver X receptor α (LXRα) and retinoid X receptor α (RXRα) is known to up-regulate srebp-1c promoter activity. Our data also show that HCV core protein enhances the binding of LXRα/RXRα to LXR-response element in the presence but not the absence of PA28γ. These findings suggest that PA28γ plays a crucial role in the development of liver pathology induced by HCV infection.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.0607312104
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2007
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  • 9
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    Archaeal adaptation to higher temperatures revealed by genomic sequence of Thermoplasma volcanium
    Proceedings of the National Academy of Sciences ; 2000
    In:  Proceedings of the National Academy of Sciences Vol. 97, No. 26 ( 2000-12-19), p. 14257-14262
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 97, No. 26 ( 2000-12-19), p. 14257-14262
    Abstract: The complete genomic sequence of the archaeon Thermoplasma volcanium , possessing optimum growth temperature (OGT) of 60°C, is reported. By systematically comparing this genomic sequence with the other known genomic sequences of archaea, all possessing higher OGT, a number of strong correlations have been identified between characteristics of genomic organization and the OGT. With increasing OGT, in the genomic DNA, frequency of clustering purines and pyrimidines into separate dinucleotides rises (e.g., by often forming AA and TT, whereas avoiding TA and AT). Proteins coded in a genome are divided into two distinct subpopulations possessing isoelectric points in different ranges (i.e., acidic and basic), and with increasing OGT the size of the basic subpopulation becomes larger. At the metabolic level, genes coding for enzymes mediating pathways for synthesizing some coenzymes, such as heme, start missing. These findings provide insights into the design of individual genomic components, as well as principles for coordinating changes in these designs for the adaptation to new environments.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.97.26.14257
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2000
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
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  • 10
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    Effect of exhaust pipe connection on the exhaust noise of an internal combustion engine
    Acoustical Society of America (ASA) ; 1996
    In:  The Journal of the Acoustical Society of America Vol. 100, No. 4_Supplement ( 1996-10-01), p. 2619-2619
    Details
    In: The Journal of the Acoustical Society of America, Acoustical Society of America (ASA), Vol. 100, No. 4_Supplement ( 1996-10-01), p. 2619-2619
    Abstract: It has been considered that the fundamental frequency of the exhaust noise of an internal combustion engine is the inverse of the number of firings per second. However, in most of the data measured with automobiles, there are many frequency components of 1/2, 1/4, or 1/6 of the fundamental frequency and their higher harmonics. An investigation on the generation of such frequency components is reported. It was found that the differences in the lengths of the exhaust pipes generate such frequency components. This paper describes, first, the reduction of the theoretical equation in which the phase modulation in the exhaust noise due to the difference in the length of the exhaust pipe of each cylinder generates such frequency components. The spectra which have the same frequency components as the spectra of the measured exhaust noise are obtained by numerical computation of the equation. The same results are also obtained by computer simulations. The difference in sound quality is felt by hearing the synthesized sound. It is expected that this study is usable in realizing an engine of agreeable exhaust noise quality.
    Type of Medium: Online Resource
    ISSN: 0001-4966 , 1520-8524
    URL: Article
    DOI: 10.1121/1.417688
    RVK:
    EQ 1000
    Language: English
    Publisher: Acoustical Society of America (ASA)
    Publication Date: 1996
    detail.hit.zdb_id: 1461063-2
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