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Search Results: 1 - 10 of 1062 matches for " Hodaka Fujii "
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Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain  [PDF]
Toshitsugu Fujita, Hodaka Fujii
Advances in Bioscience and Biotechnology (ABB) , 2012, DOI: 10.4236/abb.2012.35081
Abstract: Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
Direct Identification of Insulator Components by Insertional Chromatin Immunoprecipitation
Toshitsugu Fujita, Hodaka Fujii
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026109
Abstract: Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. However, non-biased methods to identify molecules bound to specific genomic loci in vivo are limited. Here, we applied insertional chromatin immunoprecipitation (iChIP) to direct identification of components of insulator complexes, which function as boundaries of chromatin domain. We found that the chicken β-globin HS4 (cHS4) insulator complex contains an RNA helicase protein, p68/DDX5; an RNA species, steroid receptor RNA activator 1; and a nuclear matrix protein, Matrin-3, in vivo. Binding of p68 and Matrin-3 to the cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). Thus, our results showed that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP.
Locus-Specific Biochemical Epigenetics/Chromatin Biochemistry by Insertional Chromatin Immunoprecipitation
Toshitsugu Fujita,Hodaka Fujii
ISRN Biochemistry , 2013, DOI: 10.1155/2013/913273
Abstract:
Identification of Proteins Associated with an IFNγ-Responsive Promoter by a Retroviral Expression System for enChIP Using CRISPR
Toshitsugu Fujita, Hodaka Fujii
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0103084
Abstract: Isolation of specific genomic regions retaining molecular interactions is essential for comprehensive identification of molecules associated with the genomic regions. Recently, we developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology for purification of specific genomic regions. Here, we developed a retroviral expression system for enChIP using CRISPR. We showed that the target genomic locus can be purified with high efficiency by using this system. We also showed that contamination of potential off-target sites is negligible by using this system if the guide RNA (gRNA) for the target site has a sufficiently long unique sequence in its seed sequence. enChIP combined with stable isotope labeling using amino acids in cell culture (SILAC) analysis identified proteins whose association with the interferon (IFN) regulatory factor-1 (IRF-1) promoter region increases in response to IFNγ stimulation. The list of the associated proteins contained many novel proteins in the context of IFNγ-induced gene expression as well as proteins related to histone deacetylase complexes whose involvement has been suggested in IFNγ-mediated gene expression. Finally, we confirmed IFNγ-induced increased association of the identified proteins with the IRF-1 promoter by ChIP. Thus, our results showed that the retroviral enChIP system using CRISPR would be useful for biochemical analysis of genome functions including transcription and epigenetic regulation.
Locus-Specific Biochemical Epigenetics/Chromatin Biochemistry by Insertional Chromatin Immunoprecipitation
Toshitsugu Fujita,Hodaka Fujii
ISRN Biochemistry , 2013, DOI: 10.1155/2013/913273
Abstract: Comprehensive understanding of regulation mechanisms of biological phenomena mediated by functions of genomic DNA requires identification of molecules bound to genomic regions of interest in vivo. However, nonbiased methods to identify molecules bound to specific genomic loci in vivo are limited. To perform biochemical and molecular biological analysis of specific genomic regions, we developed the insertional chromatin immunoprecipitation (iChIP) technology to purify the genomic regions of interest. We applied iChIP to direct identification of components of insulator complexes, which function as boundaries of chromatin domain, showing that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP. In addition, recently, we succeeded in identifying proteins and genomic regions interacting with a single copy endogenous locus. In this paper, we will discuss the application of iChIP to epigenetics and chromatin research. 1. Introduction Detailed biochemical and molecular biological analysis of chromatin domains is critical for understanding mechanisms of genetic and epigenetic regulation of gene expression, hetero- and euchromatinization, X-chromosome inactivation, genomic imprinting, and other important biological phenomena [1]. However, biochemical nature of chromatin domains is poorly understood. This is mainly because methods for performing biochemical and molecular biological analysis of chromatin structure are limited [2–8]. Identification of regulatory regions of gene expression has been extensively attempted in the last several decades. Conventionally, these analyses have been performed by using artificial methods such as reporter assay [9] and in silico identification of genomic regions conserved among species [10]. More recently, enhancer-specific modifications are being used to identify enhancer regions in the genome (see review [11]). However, although these approaches have been successful for relatively easy targets such as immediate early genes, it has been shown that they could produce artifactual results in many circumstances. In fact, deletion studies of candidate regulatory endogenous genomic regions have shown that the candidate regions identified by using these conventional methods could often be dispensable for expression of the genes of interest. Furthermore, these approaches cannot be used when regulatory genomic regions are far from regulated loci, for example, on other chromosomes. In fact, long-range interaction including interchromosomal interaction has been suggested to play
Identification of a Regulatory T Cell Specific Cell Surface Molecule that Mediates Suppressive Signals and Induces Foxp3 Expression
Rui Wang, Qi Wan, Lina Kozhaya, Hodaka Fujii, Derya Unutmaz
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002705
Abstract: Regulatory T (Treg) cells control immune activation and maintain tolerance. How Tregs mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in Tregs activated through the T cell receptor (TCR). Ectopic expression of GARP in human na?ve T (TN) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in TN cells induced expression of Treg master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human Treg cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.
A simple and efficient method for the preparation of live leukocytes from peripheral blood using the LeukoCatchTM system  [PDF]
Ayumi Okamoto, Kosuke Torigata, Minami A. Sakurai, Daisuke Okuzaki, Hodaka Fujii, Toshinari Ohmine, Daisaku Miura, Shoichi Kimura, Norikazu Yabuta, Hiroshi Nojima
Advances in Bioscience and Biotechnology (ABB) , 2012, DOI: 10.4236/abb.2012.35082
Abstract: Leukocytes from peripheral blood (PB) are of great value for diagnosis as well as basic and clinical research. However, no easy, centrifugation-free method is available for the isolation of live leukocytes from blood. We here develop a simple and quick method for the purification of viable leukocytes from whole blood using novel tools, named tLeukoCatch (tip-type) or sLeukoCatch (syringe-type), which is equipped with three Pall filter layers and captures leukocytes but not red blood cells (RBCs) in whole blood. Indeed, we showed that several million leukocytes per mL (~35% of the recovery rate) were captured and eluted from whole blood. The number of contaminant RBCs decreased from several million to several thousand. When mouse blood was hemolysed, almost all of the lysed RBC fragments were removed by passage through sLeukoCatch. Optical microscopic observation confirmed that the recovered leukocytes were sufficiently healthy to respond to growth stimuli. Efficient leukocyte recovery was also confirmed for hemolysed human blood. These results suggest that the LeukoCatchTM system is useful for bedside diagnosis and basic research with blood samples.
Differences in Susceptibility to Okadaic Acid, a Diarrhetic Shellfish Poisoning Toxin, between Male and Female Mice
Hodaka Suzuki
Toxins , 2013, DOI: 10.3390/toxins5010009
Abstract: The mouse bioassay (MBA) for diarrhetic shellfish poisoning (DSP) toxins has been widely used in many countries of the world. In the Japanese and EU methods, male mice are designated to be used for MBA. Female mice were described to be less susceptible than male mice. To the best of our knowledge, however, there have been no reports on the details of sex differences in susceptibility to DSP toxins. In this study, we investigated whether, and to what extent, female mice are less sensitive to DSP toxins. A lethal dose of okadaic acid (OA), one of the representative DSP toxins, was injected intraperitoneally into mice. The mice were observed until 24 hours after injection. Both male and female mice of ICR and ddY strains, which are designated in the Japanese official method, were compared. All the mice were four weeks old and weighed 18–20 g. The experiments were repeated twice. The lethality was 70%–100%. Survival analysis showed no sex differences in susceptibility to OA, but ICR female mice showed significant resistance compared with other groups in one out of two trials. These results indicate that sex differences were not clear but, nonetheless, male mice showed more stable?results.
Sensation of Balance Dysregulation Caused/Aggravated by a Collection of Electromagnetic Waves in a Dental Implant  [PDF]
Yoshiro Fujii
Open Journal of Antennas and Propagation (OJAPr) , 2014, DOI: 10.4236/ojapr.2014.23004
Abstract: Cell phone and personal computer users have increased considerably in recent years, particularly in more developed countries. These devices have facilitated communication on a global scale. However, there have been a number of reports of abnormalities occurring in the body due to the electromagnetic waves emitted by such electronic devices. The long lists of both general and severe symptoms, including headaches, fatigue, tinnitus, dizziness, memory loss, irregular heartbeat, and whole-body skin symptoms, have been reported that are apparently associated with the condition of electromagnetic hypersensitivity. In dentistry, titanium dental implants may be commonly associated with antenna-like activity, but the underlying mechanism remains unknown. In the current case studies, balance difficulties were found to occur when the patients had titanium dental implants. These implants seemed to be acting as antennae and collecting harmful electromagnetic waves. Further studies are required to confirm this hypothesis.
Gold Alloy Dental Inlay for Preventing Involuntary Body Movements Caused by Electromagnetic Waves Emitted by a Cell Phone  [PDF]
Yoshiro Fujii
Open Journal of Antennas and Propagation (OJAPr) , 2014, DOI: 10.4236/ojapr.2014.24005
Abstract: Cell phone and personal computer use has increased considerably in recent years, particularly in developed countries. These devices have facilitated communication on a global scale. However, there have been a number of reports of health problems related to the electromagnetic waves emitted by such electronic devices. A long list of both general and severe symptoms, including headaches, fatigue, tinnitus, dizziness, memory loss, irregular heartbeat and whole-body skin le-sions, have been reported. These are reportedly associated with the condition known as electro-magnetic hypersensitivity (EHS). This report shows how a subject’s abnormal involuntary body movements, caused by electromagnetic waves emitted by a cell phone, are prevented by placing a gold alloy inlay in the subject’s mouth. It appears that the subject’s involuntary movements are the result of balance dysregulation resulting from EHS. The subject’s various symptoms improve after the specific dental treatment. However, the underlying mechanism of the symptoms and the rea-sons why this treatment is so successful remain unknown. Further research is required to clarify these issues.
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