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Takashi Ito

Publications and source records attributed to Takashi Ito.

At least 19 recordsLinked to original sources
PubMed2024

Strategic targeting of Cas9 nickase induces large segmental duplications.

Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.

Saccharomyces cerevisiae↗
PubMed2007

Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation.

We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.

Adenosine Diphosphate↗
PubMed2006

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress.

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.

Adenoviridae↗
PubMed2006

Regulation of N-cadherin-based cell-cell interaction by JSAP1 scaffold in PC12h cells.

We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell-cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell-cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Stereocontrolled route to vicinal diamines by [3.3] sigmatropic rearrangement of allyl cyanate: asymmetric synthesis of anti-(2R,3R)- and syn-(2R,3S)-2,3-diaminobutanoic acids.

A stereocontrolled route via allyl 1,2-diols to vicinal diamines based on the [3.3] sigmatropic rearrangement of allyl cyanate has been developed. Our approach consists of two consecutive steps: stereoselective construction of allyl anti- and syn-1,2-diols followed by [1,3]-chirality transfer by sigmatropic rearrangement, which allow an access to anti-(2R,3R)- and syn-(2R,3S)-2,3-diaminobutanoic acids. [reaction: see text]

Acetaldehyde↗
PubMed2006

Dorfin-CHIP chimeric proteins potently ubiquitylate and degrade familial ALS-related mutant SOD1 proteins and reduce their cellular toxicity.

The ubiquitin-proteasome system (UPS) is involved in the pathogenetic mechanisms of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Dorfin is a ubiquitin ligase (E3) that degrades mutant SOD1 proteins, which are responsible for familial ALS. Although Dorfin has potential as an anti-ALS molecule, its life in cells is short. To improve its stability and enhance its E3 activity, we developed chimeric proteins containing the substrate-binding hydrophobic portion of Dorfin and the U-box domain of the carboxyl terminus of Hsc70-interacting protein (CHIP), which has strong E3 activity through the U-box domain. All the Dorfin-CHIP chimeric proteins were more stable in cells than was wild-type Dorfin (Dorfin(WT)). One of the Dorfin-CHIP chimeric proteins, Dorfin-CHIP(L), ubiquitylated mutant SOD1 more effectively than did Dorfin(WT) and CHIP in vivo, and degraded mutant SOD1 protein more rapidly than Dorfin(WT) does. Furthermore, Dorfin-CHIP(L) rescued neuronal cells from mutant SOD1-associated toxicity and reduced the aggresome formation induced by mutant SOD1 more effectively than did Dorfin(WT).

Amyotrophic Lateral Sclerosis↗
PubMed2006

High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system.

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.

Carbocyanines↗
PubMed2006

Mechanism of constipation in familial amyloid polyneuropathy: a case report.

Previously no functional study has been available for the mechanism of constipation in familial amyloid polyneuropathy (FAP). We performed a gut function test in a 78-year-old woman with transthyretin-type FAP who had severe constipation. The gut function test showed a prolonged colonic transit time, a low anal pressure at rest and upon squeezing, loss of the spontaneous phasic rectal contractions, and weak abdominal strain. All these abnormalities suggested a peripheral autonomic dysfunction, corresponding to the enteric neuropathy that was confirmed upon autopsy.

Aged↗
PubMed2006

A large-scale full-length cDNA analysis to explore the budding yeast transcriptome.

We performed a large-scale cDNA analysis to explore the transcriptome of the budding yeast Saccharomyces cerevisiae. We sequenced two cDNA libraries, one from the cells exponentially growing in a minimal medium and the other from meiotic cells. Both libraries were generated by using a vector-capping method that allows the accurate mapping of transcription start sites (TSSs). Consequently, we identified 11,575 TSSs associated with 3,638 annotated genomic features, including 3,599 ORFs, to suggest that most yeast genes have two or more TSSs. In addition, we identified 45 previously undescribed introns, including those affecting current ORF annotations and those spliced alternatively. Furthermore, the analysis revealed 667 transcription units in the intergenic regions and transcripts derived from antisense strands of 367 known features. We also found that 348 ORFs carry TSSs in their 3'-halves to generate sense transcripts starting from inside the ORFs. These results indicate that the budding yeast transcriptome is considerably more complex than previously thought, and it shares many recently revealed characteristics with the transcriptomes of mammals and other higher eukaryotes. Thus, the genome-wide active transcription that generates novel classes of transcripts appears to be an intrinsic feature of the eukaryotic cells. The budding yeast will serve as a versatile model for the studies on these aspects of transcriptome, and the full-length cDNA clones can function as an invaluable resource in such studies.

5' Untranslated Regions↗
PubMed2006

Degradation of NFAT5, a transcriptional regulator of osmotic stress-related genes, is a critical event for doxorubicin-induced cytotoxicity in cardiac myocytes.

Nuclear factor-activated T cell 5 (NFAT5), a novel member of the NFAT family of proteins, was originally identified as a transcriptional factor responsible for adaptation to hyperosmotic stress. Though NFAT5 is ubiquitously expressed, the biological functions of NFAT5 remain to be clarified, especially in the tissues that are not exposed to hypertonicity, including hearts. In the present study, we focused on the cardioprotective roles of NFAT5 against the cardiotoxic anti-tumor agent doxorubicin (Dox). In cultured cardiomyocytes, transcripts of the hypertonicity-inducible genes, such as taurine transporter (TauT) and sodium/myo-inositol transporter, were down-regulated by Dox. Interestingly, NFAT5 protein, but not mRNA, was decreased in cardiomyocytes exposed to Dox. Treatment of proteasome inhibitors, MG-132 or proteasome-specific inhibitor 1, prevented the Dox-mediated decrease of NFAT5 protein. Further, ubiquitin-conjugated NFAT5 was not detected in cultured cardiomyocytes treated with MG-132 and/or Dox, as assessed by immunoprecipitation assay, suggesting Dox-induced degradation through ubiquitin-independent proteasome pathway. Importantly, inhibition of NFAT5 with overexpression of dominant-negative NFAT5 decreased cell viability and increased creatine kinase leakage into culture medium. Consistently, small interfering RNA targeting NFAT5 gene enhanced myocyte death. These findings suggest that Dox promoted the degradation of NFAT5 protein, reducing cell viability in cardiomyocytes. This is the first demonstration that NFAT5 is a positive regulator of cardiomyocyte survival.

Animals↗
PubMed2006

A novel Cdc42-interacting domain of the yeast polarity establishment protein Bem1. Implications for modulation of mating pheromone signaling.

In Saccharomyces cerevisiae, the Rho-type small GTPase Cdc42 is activated by its guanine-nucleotide exchange factor Cdc24 to polarize the cell for budding and mating. A multidomain protein Bem1 interacts not only with Cdc42 but also with Cdc24 and the effectors of Cdc42, including the p21-activated kinase Ste20, to function as a scaffold for cell polarity establishment. Although Bem1 interacts with Cdc24 and Ste20 via its PB1 and the second SH3 domains (SH3b), respectively, it is unclear how Bem1 binds Cdc42. Here we show that a region comprising the SH3b and its C-terminal flanking segment termed CI (SH3b-CI) directly interacts with Cdc42. A dual-bait reverse two-hybrid approach revealed that the CI is critical to the interaction: N253D substitution in the CI abolishes the binding of the SH3b-CI to Cdc42 but not to the proline-rich region of Ste20, whereas W192K substitution in the SH3b has the opposite effect. Nevertheless, the SH3b-CI interacts with Ste20 proline-rich region and Cdc42 in a mutually exclusive manner. The N253D substitution renders cellular growth temperature-sensitive and suppresses mating. The W192K-induced mating defect is exacerbated by the N253D substitution and suppressed by increasing the dosage of Ste20 provided that the CI is intact. Intriguingly, Cdc42 can mediate an indirect interaction of the SH3b-CI to the CRIB domain of Ste20. These results suggest that the SH3b and the CI collaborate in tethering of Ste20 to Bem1 to ensure efficient mating pheromone signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Diverse DNA methylation statuses at alternative promoters of human genes in various tissues.

We characterized the DNA methylation status at 144 tissue-biased and 37 non-tissue-biased alternative promoters of 61 human genes in five normal tissues. Analysis of the collected data revealed that (i) DNA methylation status differed greatly among alternative promoters belonging to the same gene; (ii) DNA methylation status differed between tissues for the majority of the individual promoters, and (iii) 80-90% of CpG-island-containing promoters were not methylated on either allele throughout the five tissues examined. Furthermore, although the statistical significance was not as clear as for the above features, we also found that (iv) the DNA methylation patterns of tissue-biased promoters changed more drastically than those of non-tissue-biased promoters; (v) tissue-biased promoters tended to be less methylated than their respective alternative promoters in the tissues where they were preferentially expressed, and (vi) the 'null' methylation pattern of a given promoter was enriched in the tissues where the transcription was most active. These findings together indicate that there are dynamic physiological changes of DNA methylation. DNA methylation appears to play a significant role in differential usage of alternative promoters and may be related to functional diversification between CpG-island-containing promoters and CpG-island-less promoters.

Brain↗
PubMed2006

Marked strain differences in the pharmacokinetics of an alpha4beta1 integrin antagonist, 4-[1-[3-Chloro-4-[N-(2-methylphenyl)-ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]-methoxybenzoic Acid (D01-4582), in Sprague-Dawley rats are associated with albumin genetic polymorphism.

Strain differences in pharmacokinetics of an alpha4beta1 integrin antagonist, 4-[1-[3-chloro-4-[N-(2-methylphenyl)-ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid (D01-4582), in Sprague-Dawley rat strains (SD rat and CD rat) and their mechanism were investigated. Total plasma clearances of D01-4582 were 31.5 and 5.23 ml/min/kg in SD and CD rats, respectively. From in vivo studies, hepatic uptake process was thought to be involved in the strain differences. Differences in the uptake of D01-4582 by isolated hepatocytes prepared from the both strains were not observed when hepatocytes were incubated with simple buffer, but marked differences were observed when hepatocytes were incubated with plasma. When the dissociation constants (Kd) for the plasma protein binding of D01-4582 were examined in six rat strains, each strain was classified into two groups: a high-Kd group, which included SD rats, Brown Norway rats, and Wistar rats; and a low-Kd group, which included CD rats, Lewis rats, and Eisai hyperbilirubinemic rats. Since all rat strains in the low-Kd group showed higher area under the concentration-time curve for D01-4582 than rats in the high-Kd group, it was considered that the strain differences in the pharmacokinetics of D01-4582 were due to differences in the binding affinity. Purified albumin also showed strain differences in Kd. The cDNA sequence of the albumin was analyzed, and 11 substitutions were observed. V238L and T293I were found only in the high-Kd group, suggesting that these amino acid changes reduced the binding affinity of albumin for D01-4582. In conclusion, the strain differences in D01-4582 pharmacokinetics were suggested to be caused by an alteration in Kd, associated with albumin genetic polymorphism.

Albumins↗
PubMed2006

Electrochemical characterization of recessed nanodisk-array electrodes prepared from track-etched membranes.

Recessed nanodisk-array electrodes (RNEs) fabricated from track-etched polycarbonate membranes (TEPCMs) having cylindrical nanopores (50 nm in diameter) were characterized using cyclic voltammetry (CV). Such electrodes were prepared by depositing a gold thin film onto a TEPCM via sputtering or thermal evaporation. CV of the RNEs showed the transition from linear to radial diffusion modes of redox-active molecules with decreasing scan rate. The resulting change in maximum faradic current, which is the peak current in a peak-shaped CV and the plateau current in a sigmoidal CV, provides a simple means for calculating the pore length and effective pore density within a RNE. This method permits us to assess the completeness of the seal between a TEPCM and gold film as well as the extent to which air bubbles block the nanopores.

Journal Article↗