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Now showing items 33 - 48 of 68

  • Color regulation in the archaebacterial phototaxis receptor phoborhodopsin (sensory rhodopsin II)

    Takahashi, Tetsuo   Yan, Bing   Mazur, Paul   Derguini, Fadila   Nakanishi, Koji   Spudich, John L.  

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  • Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1

    Takahashi, Tetsuo   Honda, Risako   Nishikawa, Yoshihisa  

    The assembly of the lipid-linked oligosaccharide, Glc3Man9GlcNAc2-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene (ALG1), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S.cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.
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  • Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis

    Lu, Jishun   Takahashi, Tetsuo   Ohoka, Atsuko   Nakajima, Kei-ichi   Hashimoto, Ryo   Miura, Nobuaki   Tachikawa, Hiroyuki  

    Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5 degrees C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved alpha-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.
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  • Determination of dioxin concentrations in fish and seafood samples using a highly sensitive reporter cell line, DR-EcoScreen cells

    Kojima, Hiroyuki   Takeuchi, Shinji   Tsutsumi, Tomoaki   Yamaguchi, Katsuyuki   Anezaki, Katsunori   Kubo, Keiko   Iida, Mitsuru   Takahashi, Tetsuo   Kobayashi, Satoshi   Jin, Kazuo   Nagai, Tadanori  

    There is a strong need for the development of relatively rapid and low-cost bioassays for the determination of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in environmental and food samples. In this study, we applied a reporter gene assay using DR-EcoScreen cells (DR-cell assay), which is highly sensitive to dioxins, to the determination of PCDD/Fs and dl-PCBs in fish and seafood samples. The PCDD/Fs and dl-PCBs were extracted from homogenated samples (10 g) of 30 fish and shellfish, purified by clean-up procedure using a multilayered silica gel column and an alumina column, and applied to DR-cell assay. Interestingly, the bioanalytical equivalent (BEQ) values obtained from the DR-cell assay [< 0.1 similar to 5.4 pg BEQ g(-1) wet weight (ww)] were closely correlated with the toxicity equivalent (TEQ) values from conventional high-resolution gas chromatography/high-resolution mass spectrometry (HRGC-HRMS) analysis (r(2) = 0.912), and the slope of regression line was 0.913. Therefore, we multiplied the BEQ values from the DR-cell assay by a conversion coefficient (1.095, the reciprocal of 0.913) to approximate the TEQ values from the HRGC-HRMS analysis. Furthermore, we used this DR-cell assay to perform a prescreening test of PCDD/Fs and dl-PCBs in 16 fish and seafood samples purchased from a supermarket, revealing that a sample from the fatty flesh of a bluefin tuna exceeded 8 pg TEQ g(-1) ww (the European Union-tolerance limit). Taken together, these results suggest that the DR-cell assay might be applicable as a rapid and low-cost prescreening method to determine dioxin levels in fish and seafood samples. (C) 2011 Elsevier Ltd. All rights reserved.
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  • Diversion of the sign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs

    Takahashi, Tetsuo   Kubota, Mamoru   Watanabe, Masakatsu   Yoshihara, Kazuo   Derguini, Fadila   Nakanishi, Koji  

    The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtii is negatively phototactic in the presence of the native chromophore all-trans retinal. In contrast, analog chromophores such as a ring-acyclic retinal and those in which trans/cis isomerization about the C11 dbd C12 double bond was blocked induced predominantly positive phototaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted hypothesis (1980, Microbiol. Rev. 44, 572-630) on phototaxis mechanisms.
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  • Correct Disulfide Pairing Is Required for the Biological Activity of Crustacean Androgenic Gland Hormone (AGH): Synthetic Studies of AGH

    Ohira, Tsuyoshi   Ishii, Akira   Nozaki, Takamichi   Goto, Kiyomi   Nakahara, Yuko   Takahashi, Tetsuo   Hasegawa, Yuriko   Nagasawa, Hiromichi   Nakahara, Yoshiaki  

    Androgenic gland hormone (AGH) Of file woodlouse, Armadillidium vulgare, is heterodimeric glycopeptide. In this study we synthesized AGH with it homogeneous N-linked glycan using the expressed protein ligation method. Unexpectedly, disulfide bridge arrangement of it semisynthetic peptide differed from that of a recombinant peptide prepared in it baculovirus expression system, and the semisynthetic peptide showed no biological activity in vivo. To confirm that the loss of biological activity resulted from disulfide bond isomerization, AGH with it GlcNAc moiety was chemically synthesized by file selective disulfide formation. This synthetic AGH showed biological activity in vivo. These results indicate that the native conformation of AGH is not the most thermodynamically stable form, and correct disulfide linkages are important for conferring AGH activity.
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  • Dielectric hysteresis and rotation of dipoles in polyvinylidene fluoride

    Takahashi, Tetsuo   Date, Munehiro   Fukada, Eiichi  

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  • Negative Phototaxis from Blue Light and the Role of Third Rhodopsinlike Pigment in Halobacterium Cutirubrum

    Takahashi, Tetsuo   Watanabe, Masahiko   Kamo, Naoki   Kobatake, Yonosuke  

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  • Protein Phosphatase Type 1-Interacting Protein Ysw1 Is Involved in Proper Septin Organization and Prospore Membrane Formation during Sporulation RID E-8806-2010

    Ishihara, Makoto   Suda, Yasuyuki   Inoue, Ichiro   Tanaka, Takayuki   Takahashi, Tetsuo   Gao, Xiao-Dong   Fukui, Yasuhisa   Ihara, Sayoko   Neiman, Aaron M.   Tachikawa, Hiroyuki  

    Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1 Delta mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1 Delta cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.
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  • Differentiation of photocycle characteristics of flavin-binding BLUF domains of alpha- and beta-subunits of photoactivated adenylyl cyclase of Euglena gracilis

    Ito, Shinji   Murakami, Akio   Iseki, Mineo   Takahashi, Tetsuo   Higashi, Shoichi   Watanabe, Masakatsu  

    Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PAC alpha and PAC beta), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PAC alpha F2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PAC beta F2 domain, which like PAC alpha F2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PAC beta F2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PAC alpha F2 were 0.28 0.32 and 34 44 s. The remarkable differences between PAC alpha F2 and PAC beta F2 may be related to the sensitivity of the photoactivation. In PAC alpha F2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PAC beta F2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PAC beta F2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PAC beta F2 plays a main role in suppressing the quantum efficiency.
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  • 5356776 DNA measuring method : Hidek Kambara, Kazunor Okano, Satoshi Takahashi, Keiichi Nagai, Tetsuo Nishikawa, Hachiouji, Japan assigned to Hitachi Ltd

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  • 5356776 DNA measuring method : Hidek Kambara, Kazunor Okano, Satoshi Takahashi, Keiichi Nagai, Tetsuo Nishikawa, Hachiouji, Japan assigned to Hitachi Ltd

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  • 5356776 DNA measuring method : Hidek Kambara, Kazunor Okano, Satoshi Takahashi, Keiichi Nagai, Tetsuo Nishikawa, Hachiouji, Japan assigned to Hitachi Ltd

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  • 5356776 DNA measuring method : Hidek Kambara, Kazunor Okano, Satoshi Takahashi, Keiichi Nagai, Tetsuo Nishikawa, Hachiouji, Japan assigned to Hitachi Ltd

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  • 5356776 DNA measuring method : Kambara Hidek; Okano Kazunor; Takahashi Satoshi; Nagai Keiichi; Nishikawa Tetsuo Hachiouji, Japan Assigned to Hitachi Ltd

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  • 5356776 DNA measuring method : Kambara Hidek; Okano Kazunor; Takahashi Satoshi; Nagai Keiichi; Nishikawa Tetsuo Hachiouji, Japan Assigned to Hitachi Ltd

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