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Search Results: 1 - 10 of 1484 matches for " Kazuharu Shimizu "
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Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers
Kazuya Terasawa,Kazuharu Shimizu,Gozoh Tsujimoto
Journal of Nucleic Acids , 2011, DOI: 10.4061/2011/131579
Abstract: RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future. 1. Introduction RNA interference (RNAi) is an evolutionarily conserved, gene-silencing mechanism that is triggered by double-stranded RNA (dsRNA). Two types of small RNA—namely, small interfering RNA (siRNA) and microRNA (miRNA)—are central players in RNAi. Both siRNAs and miRNAs regulate gene expression by annealing to mRNA sequence elements that are fully or partially complementary [1, 2]. Since transfected synthetic siRNAs were shown to induce RNAi in mammalian cells [3], they have been widely used to decipher gene function through suppression of gene expression, and they have also attracted attention because of their potential for therapeutic applications [4, 5]. miRNAs are a phylogenetically conserved family of endogenous small RNAs that play important roles in a wide variety of biological functions, including cell differentiation, tumor genesis, apoptosis, and metabolism [1, 2, 6, 7]. miRNAs are initially generated as long primary transcripts (pri-miRNA) that are processed in the nucleus by the enzyme complexes Drosha and DiGeorge Critical Region 8 (DGCR8) to a 70–90?nt stem-loop structure called pre-miRNA. The pre-miRNA is then exported to the cytoplasm. There, the exported pre-miRNA or exogenous dsRNA is cleaved by the enzyme Dicer into a ~22-nucleotide (nt) duplex known as miRNA or siRNA, respectively. The duplex is then incorporated into the RNA-induced silencing complex (RISC). After removing one strand called the passenger strand, the remaining strand,
Genome-wide analysis of aberrant methylation in human breast cancer cells using methyl-DNA immunoprecipitation combined with high-throughput sequencing
Yoshinao Ruike, Yukako Imanaka, Fumiaki Sato, Kazuharu Shimizu, Gozoh Tsujimoto
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-137
Abstract: Here, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation.This study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes.DNA methylation is an indispensable epigenetic modification of mammalian genomes. In mammals, it occurs predominantly at CpG dinucleotides which are sparsely distributed through the genome except at short genomic regions called CpG islands (CGIs) [1]. The state of CpG methylation regulates and stabilizes chromatin structure, and possibly regulates accessibility of these DNA regions to the transcription machinery [2]. DNA methylation is essen
Proteomic analysis of S-nitrosylation induced by 1-methyl-4-phenylpyridinium (MPP+)
Komatsubara Akira T,Asano Tomoya,Tsumoto Hiroki,Shimizu Kazuharu
Proteome Science , 2012, DOI: 10.1186/1477-5956-10-74
Abstract: Background Nitric oxide (NO) mediates its function through the direct modification of various cellular targets. S-nitrosylation is a post-translational modification of cysteine residues by NO that regulates protein function. Recently, an imbalance of S-nitrosylation has also been linked to neurodegeneration through the impairment of pro-survival proteins by S-nitrosylation. Results In the present study, we used two-dimensional gel electrophoresis in conjunction with the modified biotin switch assay for protein S-nitrosothiols using resin-assisted capture (SNO-RAC) to identify proteins that are S-nitrosylated more intensively in neuroblastoma cells treated with a mitochondrial complex I inhibitor, 1-methyl-4-phenylpyridinium (MPP+). We identified 14 proteins for which S-nitrosylation was upregulated and seven proteins for which it was downregulated in MPP+-treated neuroblastoma cells. Immunoblot analysis following SNO-RAC confirmed a large increase in the S-nitrosylation of esterase D (ESD), serine-threonine kinase receptor-associated protein (STRAP) and T-complex protein 1 subunit γ (TCP-1 γ) in MPP+-treated neuroblastoma cells, whereas S-nitrosylation of thioredoxin domain-containing protein 5 precursor (ERp46) was decreased. Conclusions These results suggest that S-nitrosylation resulting from mitochondrial dysfunction can compromise neuronal survival through altering multiple signal transduction pathways and might be a potential therapeutic target for neurodegenerative diseases.
Trastuzumab Produces Therapeutic Actions by Upregulating miR-26a and miR-30b in Breast Cancer Cells
Takehiro Ichikawa, Fumiaki Sato, Kazuya Terasawa, Soken Tsuchiya, Masakazu Toi, Gozoh Tsujimoto, Kazuharu Shimizu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031422
Abstract: Objective Trastuzumab has been used for the treatment of HER2-positive breast cancer (BC). However, a subset of BC patients exhibited resistance to trastuzumab therapy. Thus, clarifying the molecular mechanism of trastuzumab treatment will be beneficial to improve the treatment of HER2-positive BC patients. In this study, we identified trastuzumab-responsive microRNAs that are involved in the therapeutic effects of trastuzumab. Methods and Results RNA samples were obtained from HER2-positive (SKBR3 and BT474) and HER2-negetive (MCF7 and MDA-MB-231) cells with and without trastuzumab treatment for 6 days. Next, we conducted a microRNA profiling analysis using these samples to screen those microRNAs that were up- or down-regulated only in HER2-positive cells. This analysis identified miR-26a and miR-30b as trastuzumab-inducible microRNAs. Transfecting miR-26a and miR-30b induced cell growth suppression in the BC cells by 40% and 32%, respectively. A cell cycle analysis showed that these microRNAs induced G1 arrest in HER2-positive BC cells as trastuzumab did. An Annexin-V assay revealed that miR-26a but not miR-30b induced apoptosis in HER2-positive BC cells. Using the prediction algorithms for microRNA targets, we identified cyclin E2 (CCNE2) as a target gene of miR-30b. A luciferase-based reporter assay demonstrated that miR-30b post-transcriptionally reduced 27% (p = 0.005) of the gene expression by interacting with two binding sites in the 3′-UTR of CCNE2. Conclusion In BC cells, trastuzumab modulated the expression of a subset of microRNAs, including miR-26a and miR-30b. The upregulation of miR-30b by trastuzumab may play a biological role in trastuzumab-induced cell growth inhibition by targeting CCNE2.
MicroRNA Profile Predicts Recurrence after Resection in Patients with Hepatocellular Carcinoma within the Milan Criteria
Fumiaki Sato,Etsuro Hatano,Koji Kitamura,Akira Myomoto,Takeshi Fujiwara,Satoko Takizawa,Soken Tsuchiya,Gozoh Tsujimoto,Shinji Uemoto,Kazuharu Shimizu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016435
Abstract: Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection.
A Consideration of the Constancy of Biomass Density in Plant Populations Undergoing Self-Thinning
Kazuharu Ogawa
International Journal of Ecology , 2007, DOI: 10.1155/2007/54762
Abstract: The constancy of biomass density was considered in an entire plant population p by combining two adjacent populations p1 and p2 for which the self-thinning rule is assumed to be satisfied independently and each biomass density is also assumed to be the same constant value. Under these assumptions, the biomass density d in a population p was formulated as d=c((km−α
A codon substitution model that incorporates the effect of the GC contents, the gene density and the density of CpG islands of human chromosomes
Kazuharu Misawa
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-397
Abstract: Codon substitution rates of CpG to TpG mutations, TpG to CpG mutations, and non-CpG transitions and transversions in humans were estimated by comparing the coding regions of thousands of human and chimpanzee genes and inferring their ancestral sequences by using macaque genes as the outgroup. Since the genomic features are depending on each other, partial regression coefficients of these features were obtained.The substitution rates of codons depend on gene densities of the chromosomes. Transcription-associated mutation is one such pressure. On the basis of these results, a model of codon substitutions that incorporates the effect of genomic features on codon substitution in human chromosomes was developed.Accurate models of DNA and protein evolution are essential for studying molecular evolution. Models of DNA and protein evolution are used in homology searches [1], sequence alignments [2,3], gene finding [4], detection of natural selection [5-8], and reconstruction of phylogenetic trees [9,10].Recent studies on the mutation rates in non-coding regions have shown that the mutation rates of CpGs in the human genome are negatively correlated to the local GC content [11-14] and to the densities of functional elements [11].CpG hypermutability [15] is known to be one of the major causes of codon substitution in mammalian genes [16-19]. CpG dinucleotides are often methylated at C, and methylated C spontaneously deaminates to thymine (T) approximately 10 times [20] or more [21] rapidly than other types of point mutations. Approximately 14% of codon substitutions are caused by CpG hypermutations [22]. Statistical indications of positive selection in some sequences or individual codons may be caused by a difference in mutation rates in the synonymous and nonsynonymous sites because of CpG hypermutations [23]. Previous studies that focused on the effect of CpG hypermutation on the rate of amino acid change in the human lineage [22,24] did not incorporate the regional variati
Property of the spectrum of large-scale magnetic fields from inflation
Bamba, Kazuharu
High Energy Physics - Phenomenology , 2007, DOI: 10.1103/PhysRevD.75.083516
Abstract: The property of the spectrum of large-scale magnetic fields generated due to the breaking of the conformal invariance of the Maxwell theory through some mechanism in inflationary cosmology is studied. It is shown that the spectrum of the generated magnetic fields should not be perfectly scale-invariant but be slightly red so that the amplitude of large-scale magnetic fields can be stronger than $\sim 10^{-12}$G at the present time. This analysis is performed by assuming the absence of amplification due to the late-time action of some dynamo (or similar) mechanism.
The interrelation between the generation of large-scale electric fields and that of large-scale magnetic fields during inflation
Bamba, Kazuharu
High Energy Physics - Phenomenology , 2007, DOI: 10.1088/1475-7516/2007/10/015
Abstract: The interrelation between the generation of large-scale electric fields and that of large-scale magnetic fields due to the breaking of the conformal invariance of the electromagnetic field in inflationary cosmology is studied. It is shown that if large-scale magnetic fields with a sufficiently large amplitude are generated during inflation, the generation of large-scale electric fields is suppressed, and vice versa. Furthermore, a physical interpretation of the result and its cosmological significance are considered.
An Estimate of Growth and Maintenance Respiration in Hinoki Cypress (Chamaecyparis obtusa) Seedlings
Kazuharu Ogawa
American Journal of Plant Physiology , 2007,
Abstract: The quantitative relationship between respiration and growth was investigated for hinoki cypress (Chamaecyparis obtusa (Sieb. et Zucc) Endl.) seedlings (including roots). The relation of annual specific respiration rate to annual relative growth rate was fitted by a single straight line over all seedlings. From this relation, respiration was divided into two components, maintenance and growth respiration which were proportional to dry mass and its increment, respectively. Any seedlings consumed annually 391% of the dry mass by maintenance respiration and 24% of the dry mass increment by growth respiration. The ratio of growth respiration to total respiration, varying among seedlings, ranged from 3 to 13% with the average of 8%.
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