Yuki Yamaguchi

Tokyo Institute of Technology, Edo, Tōkyō, Japan

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Publications (90)483.37 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Accurate detection and monitoring of disease-related biomarkers is important in understanding pathophysiology. We devised a rapid immunoreaction system that uses submicrometer polymer-coated fluorescent ferrite (FF) beads containing both ferrites (magnetic iron oxide) and fluorescent europium complexes.METHODS: FF beads were prepared by encapsulation of hydrophobic europium complexes into the polymer layers of affinity magnetic matrices using organic solvent. A sandwich immunoassay using magnetic collection of antibody-coated FF beads to a specific place was performed. Brain natriuretic peptide and prostate-specific antigen were selected as target detection antigens to demonstrate the feasibility of this approach. An immunohistochemical staining using magnetic collection of antibody-coated FF beads onto carcinoma cell samples was also performed.RESULTS: The sandwich immunoassays, taking advantage of the magnetic collection of antibody-coated FF beads, detected target antigens within 5 min of sample addition. Without magnetic collection, the sandwich immunoassay using antibody-coated FF beads required long times to obtain results, similar to conventional immunoassays. With the use of magnetic collection of antibody-coated FF beads, immunohistochemical staining enabled discrimination of carcinoma cells within 20 min.CONCLUSIONS: This proof of principle system demonstrates that immunoreactions involving the magnetic collection of antibody-coated FF beads allow acceleration of the antigen-antibody reaction. The simple magnetic collection of antibody-coated FF beads to a specific space enables rapid detection of disease-related biomarkers and identification of carcinoma cells.
    Clinical Chemistry 01/2014; · 7.15 Impact Factor
  • The Journal of Antibiotics 01/2014; · 2.19 Impact Factor
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    ABSTRACT: The elongation factors DSIF and NELF are responsible for promoter-proximal RNA polymerase II (Pol II) pausing. NELF is also involved in 3' processing of replication-dependent histone genes, which produce non-polyadenylated mRNAs. Here we show that DSIF and NELF contribute to the synthesis of small nuclear RNAs (snRNAs) through their association with Integrator, the large multisubunit complex responsible for 3' processing of pre-snRNAs. In HeLa cells, Pol II, Integrator, DSIF and NELF accumulate at the 3' end of the U1 snRNA gene. Knockdown of NELF results in misprocessing of U1, U2, U4 and U5 snRNAs, while DSIF is required for proper transcription of these genes. Knocking down NELF also disrupts transcription termination and induces the production of polyadenylated U1 transcripts caused by an enhanced recruitment of cleavage stimulation factor. Our results indicate that NELF plays a key role in determining the post-transcriptional fate of Pol II-transcribed genes.
    Nature Communications 01/2014; 5:4263. · 10.74 Impact Factor
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    ABSTRACT: Salicylic acid is a classic non-steroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its anti-mitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, co-crystal structure of the FECH·salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its anti-mitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.
    Molecular pharmacology 09/2013; · 4.53 Impact Factor
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    ABSTRACT: DNA methylation is a well-characterized epigenetic landmark involved in transcriptional regulation; however, mechanisms underlying its regulation remain poorly characterized. Recent studies demonstrate that activation-induced cytidine deaminase (AID) is involved in active DNA demethylation. AID is aberrantly expressed in inflammation-associated cancers and generates point mutations; however, cellular disorders attributed to its demethylation function are largely unexplored. Here we demonstrate that ectopic AID expression perturbs tumor-related gene expression. AID (with Gadd45) activated a methylated Pax5 reporter construct, and induced expression and association of endogenous Pax5 with the AID promoter, suggesting that aberrant AID expression triggers an auto-activation circuit to consolidate self-expression.
    FEBS letters 06/2013; · 3.54 Impact Factor
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    ABSTRACT: Artificial beads including magnetite and fluorescence particles are useful to visualize pathologic tissue, such as cancers, from harmless types by magnetic resonance imaging (MRI) or fluorescence imaging. Desirable properties of diagnostic materials include high dispersion in body fluids, and the ability to target specific tissues. Here we report on the development of novel magnetic nanoparticles (MNPs) intended for use as diagnosis and therapy that are coated with viral capsid protein VP1-pentamers of simian virus 40, which are monodispersive in body fluid by conjugating epidermal growth factor (EGF) to VP1. Critically, the coating of MNPs with VP1 facilitated stable dispersion of the MNPs in body fluids. In addition, EGF was conjugated to VP1 coating on MNPs (VP1-MNPs). EGF-conjugated VP1-MNPs were successfully used to target EGF receptor-expressing tumor cells in vitro. Thus, using viral capsid protein VP1 as a coating material would be useful for medical diagnosis and therapy.
    Journal of Biotechnology 06/2013; · 3.18 Impact Factor
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    ABSTRACT: Vesnarinone is a synthetic quinolinone derivative used in the treatment of cardiac failure and cancer. It is also known to cause agranulocytosis as a side effect, which restricts its use, although the mechanism underlying agranulocytosis is not well understood. Here, we show that vesnarinone binds to valosin-containing protein (VCP), which interacts with poly-ubiquitinated proteins and is essential for the degradation of IκBα to activate NFκB. We show that vesnarinone binding to VCP impairs the degradation of IκBα and that the impairment of the degradation of IκBα is the result of the inhibition of the interaction between VCP and the 26S proteasome. These results suggest that vesnarinone suppresses NFκB activation by inhibiting the VCP-dependent degradation of poly-ubiquitinated IκBα, resulting in the suppression of TNFα mRNA expression.
    Molecular pharmacology 02/2013; · 4.53 Impact Factor
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    ABSTRACT: As the oscillating gradient spin-echo sequence has shown promise as a means to probe tissue microstructure, it was applied here to diffusion-tensor imaging of in vivo rat brain. The apparent diffusion tensor (ADT) was estimated for motion-probing gradient (MPG) frequencies in the range 33.3-133.3 Hz, and regions-of-interest (ROIs) in the corpus callosum (CC), visual cortex (VC), cerebellar white matter (CBWM) and cerebellar grey matter (CBGM) were selected for detailed analysis. There were substantial, approximately linear changes to the ADT with increasing MPG frequency for all four ROIs. All ROIs showed clear increases in mean diffusivity. CBWM had a substantial decrease in fractional anisotropy, whereas the CC and VC had minor increases of the same parameter. All eigenvalues of the ADT tended to increase with frequency for the CBWM, CBGM and VC, but only the principal eigenvalue increased strongly for the CC. On the other hand, there was no evidence that the orientation of the principal eigenvector varied systematically with MPG frequency for any of the ROIs. The relationship between the behaviour of the eigenvalues and the behaviours of the mean diffusivity and fractional anisotropy is investigated in detail. Pixelwise linear fits to the MD from individual animals found elevated changes across the cerebellum. The data acquired for this work encompassed a range of effective diffusion-times from 7.5 ms down to 1.875 ms, and some ideas on how the results might be used to extract quantitative information about brain tissue microstructure are discussed.
    NeuroImage 12/2012; · 6.25 Impact Factor
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    ABSTRACT: Vitamin K2 (VK2, menaquinone) is known to have anticancer activity in vitro and in vivo. Although its effect is thought to be mediated, at least in part, by the induction of apoptosis, the underlying molecular mechanism remains elusive. Here, we identified Bcl-2 antagonist killer 1 (Bak) as a molecular target of VK2-induced apoptosis. VK2 directly interacts with Bak and induces mitochondrial-mediated apoptosis. Although Bak and Bax, another member of the Bcl-2 family, are generally thought to be functionally redundant, only Bak is necessary and sufficient for VK2-induced cytochrome c release and cell death. Moreover, VK2-2,3 epoxide, an intracellular metabolite of VK2, was shown to covalently bind to the cysteine-166 residue of Bak. Several lines of evidence suggested that the covalent attachment of VK2 is critical for apoptosis induction. Thus, this study reveals a specific role for Bak in mitochondria-mediated apoptosis. This study also provides insight into the anticancer effects of VK2 and suggests that Bak may be a potential target of cancer therapy.
    Molecular pharmacology 12/2012; · 4.53 Impact Factor
  • Yuki Yamaguchi, Hirotaka Shibata, Hiroshi Handa
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    ABSTRACT: DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) were originally identified as factors responsible for transcriptional inhibition by 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) and were later found to control transcription elongation, together with P-TEFb, at the promoter-proximal region. Although there is ample evidence that these factors play roles throughout the genome, other data also suggest gene- or tissue-specific roles for these factors. In this review, we discuss how these apparently conflicting data can be reconciled. In light of recent findings, we also discuss the detailed mechanism by which these factors control the elongation process at the molecular level.
    Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
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    ABSTRACT: NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB.
    Cell Reports 10/2012; · 7.21 Impact Factor
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    ABSTRACT: Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100-300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.
    PLoS ONE 01/2012; 7(12):e50481. · 3.53 Impact Factor
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    ABSTRACT: SV40 is comprised of the viral minichromosome and the capsid proteins VP1, VP2, and VP3. Complete reconstitution of SV40 virions in vitro remains a challenge. Here we describe in vitro reconstitution of SV40 particles that contain ~5-kb circular nucleosomal DNA with hyperacetylated histones and are encapsidated in a coat composed of VP1, VP2, and VP3, closely mimicking the characteristics of authentic SV40 virions. When inoculated into mammalian cells, VP1/2/3 particles containing nucleosomal DNA carrying a reporter gene yielded a significantly higher level of gene expression than VP1-only particles containing the corresponding naked DNA. The elevated gene expression resulted mainly from enhanced association of the particles with the cell surface and from facilitation of subsequent uptake into cells. Thus, the in vitro reconstitution system reported here should be useful for the elucidation of Polyomaviridae assembly mechanisms and for the development of novel carriers for gene delivery.
    Virology 09/2011; 420(1):1-9. · 3.35 Impact Factor
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    ABSTRACT: Histone variants perform unique functions and are deposited onto DNA by mechanisms distinct from those of canonical histones. The H2A variant, H2A.Z, also known as Htz1 in Saccharomyces cerevisiae, is not uniformly distributed across the genome but facilitates transcriptional activation at target gene promoters and anti-silencing at heterochromatin loci. Htz1 is also involved in DNA replication, DNA repair, chromosome segregation and cell cycle control. Its sequence identity to canonical H2A is only ∼60%, and it is likely that the nonconserved residues are responsible for Htz1-specific functions. However, precise roles of these variant-specific residues are not well understood. To gain insights into the molecular basis underlying the functional differences between canonical and variant histones, 117 alanine-scanning point mutants of Htz1 were constructed for this study, and chemical genetic screens were carried out. Consequently, seven Htz1 residues that conferred one or more abnormal phenotypes when mutated were identified. Based on primary sequence and functional conservation between H2A and Htz1, two of these residues (F32 and I109) appear to have an Htz1-specific role, whereas the rest seem to have functions shared between H2A and Htz1. This study provides a useful resource for future investigations into functional convergence and divergence between canonical and variant histones.
    Genes to Cells 04/2011; 16(5):590-607. · 2.73 Impact Factor
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    ABSTRACT: Recent studies have showed that transcription elongation factors regulate early development. Foggy/Spt5 is a subunit of DRB sensitivity-inducing factor, which negatively and positively regulates transcription elongation. Here, we report that the positive function of Foggy/Spt5 is required for gata1 expression during zebrafish embryonic hematopoiesis. Antisense morpholino oligonucleotide (MO)-mediated knockdown of foggy/spt5 has led to a reduction in the expression of gata1 and the gata1 target genes alas2 and hbae3 and inhibited proper hemoglobin production. By contrast, expression of hematopoietic stem cell and endothelial markers, including scl, lmo2, gata2, fli-1, and flk-1, and expression of biklf, whose product directs gata1 expression via its direct binding to the gata1 promoter, were unaltered, suggesting that gata1 is a functionally important target gene of Foggy/Spt5. The MO-mediated gata1 repression was relieved by forced expression of wild-type foggy/spt5, but not by a mutant lacking the positive function. Therefore, this study provides evidence that Foggy/Spt5 plays an important role in gata1 gene expression and erythropoiesis through its transcriptional activation domain.
    Genes to Cells 02/2011; 16(2):231-42. · 2.73 Impact Factor
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    ABSTRACT: For a long time, not much attention had been paid to post-initiation steps in transcription, because it was widely believed that transcriptional control was brought about almost entirely through the regulation of transcription initiation. However, it has become clear that the process of elongation is also tightly controlled by a collection of regulatory factors called transcription elongation factors and contributes, for example, to rapid induction of immediate-early genes and to the control over the viral life cycle. Transcription elongation has attracted attention also because this process is coupled with various RNA processing events. In this review, we discuss biochemical and physiological aspects of elongation control, particularly focusing on the role of the negative elongation factor NELF.
    Experimental Cell Research 10/2010; 316(17):2723-30. · 3.56 Impact Factor
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    ABSTRACT: Recent estimates indicate that approximately 60% of human genes include alternative polyadenylation sites. Hence, control of alternative polyadenylation can have a great impact on gene expression and cellular function. Cleavage factor (CF) Im is a 3'-end processing factor that is essential for in vitro processing. CFIm purified from HeLa cells is associated with three polypeptides of 25, 59 and 68 kD, and it is generally thought to be a heterodimer composed of the 25-kD subunit and one of the larger subunits. Previously, we serendipitously discovered that knockdown of CFIm25 causes an upstream shift in the utilization of alternative polyadenylation sites. Here, we investigated whether this is because of an inherent property of the CFIm complex and, if so, what structural elements are important for its function. The major conclusions of this study are that (i) contrary to previous assumptions, CFIm forms stable heterotetramers through dimerization of CFIm25 and (ii) the CFIm complex per se is responsible for the control of alternative polyadenylation. (iii) However, the structurally related CFIm68 and CFIm59 are functionally redundant and (iv) CFIm68 appears to have a higher specific activity. Thus, this study establishes that CFIm not only plays a general role in 3'-end processing but also plays a regulatory role in poly(A) site selection.
    Genes to Cells 09/2010; 15(9):1003-13. · 2.73 Impact Factor
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    ABSTRACT: Of 20 natural amino acids, leucine is particularly important for promoting cellular protein synthesis. The effect of leucine involves mammalian target of rapamycin (mTOR), a key protein kinase controlling cell growth. Leucine enhances mTOR-mediated phosphorylation of S6K1 and 4E-BP, thereby promoting protein synthesis. However, how the presence of leucine is sensed and transmitted to mTOR is poorly understood. Here, we show evidence that UBR1 and UBR2 might be cellular targets of leucine. UBR1 and UBR2 are E3 ubiquitin ligases that recognize the identity of N-terminal residues and contribute to selective destabilization of target proteins according to the N-end rule. Using leucine-immobilized affinity beads, we identified UBR1 and UBR2 as leucine-binding proteins from leucine-responsive rat hepatoma H4IIE cells. Over-expression of UBR1 or UBR2 resulted in a reduction in mTOR-dependent S6K1 phosphorylation, whereas knockdown of UBR1 or UBR2 increased S6K1 phosphorylation in amino acid-starved human 293T cells. We also found that leucine binds to the substrate-recognition domain of UBR2 and inhibits degradation of N-end rule substrates in vitro. These findings suggest that UBR1 and UBR2 are negative regulators of the leucine-mTOR signaling pathway. Leucine might activate this pathway in part through inhibition of their ubiquitin ligase activity.
    Genes to Cells 03/2010; 15(4):339-49. · 2.73 Impact Factor
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    ABSTRACT: Half a century ago, thalidomide was widely prescribed to pregnant women as a sedative but was found to be teratogenic, causing multiple birth defects. Today, thalidomide is still used in the treatment of leprosy and multiple myeloma, although how it causes limb malformation and other developmental defects is unknown. Here, we identified cereblon (CRBN) as a thalidomide-binding protein. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1) and Cul4A that is important for limb outgrowth and expression of the fibroblast growth factor Fgf8 in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity. This study reveals a basis for thalidomide teratogenicity and may contribute to the development of new thalidomide derivatives without teratogenic activity.
    Science 03/2010; 327(5971):1345-50. · 31.20 Impact Factor
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    ABSTRACT: Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.
    Genes & development 12/2009; 23(23):2765-77. · 12.08 Impact Factor

Publication Stats

3k Citations
483.37 Total Impact Points


  • 1998–2013
    • Tokyo Institute of Technology
      • • Graduate School of Bioscience and Biotechnology
      • • Integrated Research Institute
      Edo, Tōkyō, Japan
  • 2012
    • National Institute of Radiological Sciences
      • Molecular Imagining Center
      Chiba-shi, Chiba-ken, Japan
  • 2004–2012
    • Weizmann Institute of Science
      • Department of Biological Chemistry
      Tel Aviv, Tel Aviv, Israel
  • 2007
    • University of Yamanashi
      Kōhu, Yamanashi, Japan
    • University of Southern California
      Los Angeles, California, United States
    • Fox Chase Cancer Center
      Philadelphia, Pennsylvania, United States
    • Hippokration General Hospital, Athens
      Athínai, Attica, Greece
  • 2006
    • Osaka City University
      • Graduate School of Medicine
      Ōsaka, Ōsaka, Japan
    • Kyoto University
      • Graduate School of Biostudies
      Kyoto, Kyoto-fu, Japan