Toyama-shi, Japan
Toyama-shi, Japan

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Furukawa M.,Fukushima Medical University | Ohkawara H.,Fukushima Medical University | Ogawa K.,Fukushima Medical University | Ikeda K.,Fukushima Medical University | And 10 more authors.
Journal of Biological Chemistry | Year: 2017

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant up-regulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 were significantly increased in the MM patients when compared with healthy controls. Gas6 was overexpressed in the human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditional medium from the human BM stromal cell line HS-5 induced cell proliferation and anti-apoptosis of the MM cells with extracellular signal-regulated kinase, Akt, and nuclear factor-κB phosphorylation, which were reversed by the neutralizing antibody to Gas6 or IL-6. The TAM family receptor Mer, which has been identified as a Gas6 receptor, was overexpressed in BM cells of MM patients. The knockdown of Mer by siRNA inhibited cell proliferation, anti-apoptosis, and up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) in MM cells stimulated by an HS-5 cell-conditioned medium. Furthermore, the Gas6-neutralizing antibody reduced the up-regulation of IL-6 and ICAM-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathogenesis of MM, suggesting that Gas6-Mer-related signaling pathways may be a promising novel target for treating MM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.


Takabatake R.,Japan National Agriculture and Food Research Organization | Akiyama H.,Japan National Institute of Health Sciences | Sakata K.,Japan National Institute of Health Sciences | Onishi M.,FASMAC Co. | And 7 more authors.
Journal of the Food Hygienic Society of Japan | Year: 2011

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (Cf), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined Cf values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD R), and the determined bias and (RSDR) values for themethod were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.


Takabatake R.,Japan National Agriculture and Food Research Organization | Onishi M.,Fasmac Co. | Koiwa T.,Food and Agricultural Materials Inspection Center | Futo S.,Fasmac Co. | And 5 more authors.
Journal of the Food Hygienic Society of Japan | Year: 2010

A novel real-time PCR-based analytical method was established for the event-specific quantification of a GM soybean event MON89788. The conversion factor (C f) which is required to calculate the GMO amount was experimentally determined. The quantitative method was evaluated by a single-laboratory analysis and a blind test in a multi-laboratory trial. The limit of quantitation for the method was estimated to be 0.1% or lower. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSDR), and the determined bias and RSDR values for the method were both less than 20%. These results suggest that the established method would be suitable for practical detection and quantification of MON 89788.


Takabatake R.,Japan National Agriculture and Food Research Organization | Futo S.,FASMAC Co. | Minegishi Y.,NIPPONGENE CO. | Watai M.,Japan Food Research Laboratories | And 7 more authors.
Food Science and Technology Research | Year: 2010

Novel real-time PCR-based quantitative methods were developed for three GM maize events; MON863, NK603 and TC1507. The quantitative methods were designed to amplify an event-specific segment for MON863 and NK603, and a construct-specific segment for TC 1507. We also developed an eventspecific quantitative method for T25. The conversion factor (Cf), which is required for calculating the GMO amount, was determined using three types of real-time PCR equipment; the ABI PRISM 7700, 7900HT and 7500. The quantitative methods were evaluated by blind testing in an interlaboratory study using the ABI PRISM 7700 and 7900HT, and in a multilaboratory trial using the ABI PRISM 7500. The trueness, precision, and limit of quantitation were determined. Although the biases expressing the trueness for MON863, TC 1507, and T25 were slightly high, all the data suggested that the developed methods were suitable for identification and quantification of these GM maize events.


Takabatake R.,Japan National Agriculture and Food Research Organization | Masubuchi T.,Japan National Agriculture and Food Research Organization | Futo S.,FASMAC Co. | Minegishi Y.,NIPPON GENE Co. | And 6 more authors.
Journal of the Food Hygienic Society of Japan | Year: 2014

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize event, MIR162. We first prepared a standard plasmid for MIR162 quantification. The conversion factor (Cf) required to calculate the genetically modified organism (GMO) amount was empirically determined for two real-time PCR instruments, the Applied Biosystems 7900HT (ABI7900) and the Applied Biosystems 7500 (ABI7500) for which the determined Cf values were 0.697 and 0.635, respectively. To validate the developed method, a blind test was carried out in an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDR). The determined biases were less than 25% and the RSDR values were less than 20% at all evaluated concentrations. These results suggested that the limit of quantitation of the method was 0.5%, and that the developed method would thus be suitable for practical analyses for the detection and quantification of MIR162.


Ohnishi T.,Japan National Institute of Health Sciences | Lim B.,Jeju National University | Nojima N.,Center for Inspection of Imported Foods and Infectious Deseases | Kunitoshi O.,Center for Inspection of Imported Foods and Infectious Deseases | And 10 more authors.
Biocontrol Science | Year: 2016

Kudoa septempunctata is the causative agent of a food-borne disease associated with the ingestion of raw olive flounder. As the current qRT-PCR method for its detection is time-consuming, a rapid and simple method is required. Recently, a new real-time loop-mediated isothermal amplification (LAMP) method and an immunochromatography method, whose sensitivities are intended to be compatible with that designated in the official analytical method (105 spores/g olive flounder), have been developed. To validate these new methods, we performed an inter-laboratory study across seven laboratories. Both methods could not detect less than 104 spores/g; however, these methods were able to detect more than 105 spores/g in olive flounder samples. These results demonstrated that the sensitivities of these methods were compatible with the designated level in the official analytical method. We concluded that these new methods were acceptable as the screening methods for K. septempunctata.


Akiyama H.,Japan National Institute of Health Sciences | Makiyama D.,Japan National Institute of Health Sciences | Nakamura K.,Japan National Institute of Health Sciences | Sasaki N.,Tokyo University of Agriculture and Technology | And 5 more authors.
Analytical Chemistry | Year: 2010

The herbicide-tolerant genetically modified Roundup Ready canola (Brassica napus) line RT73 has been approved worldwide for use in animal feed and human food. However, RT73 Brassica rapa lines derived from interspecific crosses with RT73 B. napus have not been approved in Japan. Here, we report on a novel system using individual kernel analyses for the qualitative detection of RT73 B. rapa in canola grain samples. We developed a duplex real-time polymerase chain reaction (PCR) method to discriminate B. napus and B. rapa DNA using scatter plots of the end-point analyses; this method was able to discriminate a group comprising B. rapa and Brassica juncea from a group comprising B. napus, Brassica carinata, and Brassica oleracea. We also developed a duplex real-time PCR method for the simultaneous detection of an RT73-specific sequence and an endogenous FatA gene. Additionally, a DNA-extraction method using 96-well silica-membrane plates was developed and optimized for use with individual canola kernels. Our detection system could identify RT73 B. rapa kernels in canola grain samples enabling the accurate and reliable monitoring of RT73 B. rapa contamination in canola, thus playing a role in its governmental regulation in Japan. © 2010 American Chemical Society.


By establishing a simple method for extracting a nucleic acid from a human specimen, and using the LAMP method, which is an isothermal gene amplification method showing superior quickness and convenience, with a concept of measurement different from the conventional ones, there is established a test method including steps up to detection with a measurement apparatus. There is provided a method for detecting a target site, which is an LAMP method using four kinds of primers, FIP, F3 primer, BIP, and B3 primer, which are designed on the basis of six regions of a template polynucleotide, F1 region, F2 region, F3 region, B1 region, B2 region, and B3 region, wherein: the template polynucleotide contains the target site, the four kinds of primers are designed so that the target site exists between the F1 region and the F2 region, or between the B1 region and the B2 region; a loop primer designed on the basis of a region between the F1 region and the F2 region or between the B1 region and the B2 region on the side on which the target site exists, and an oligonucleotide probe that can associate with a region including the target site are used, the loop primer and the probe are designed so that the 5 end of the loop primer and the 3 end of the probe associate with the template polynucleotide at positions close to each other, one of the loop primer and the probe is modified with a fluorescent molecule around the 5 end in the case of the loop primer or the 3 end in the case of the probe, and the other is modified with a quenching molecule.


By establishing a simple method for extracting a nucleic acid from a human specimen, and using the LAMP method, which is an isothermal gene amplification method showing superior quickness and convenience, with a concept of measurement different from the conventional ones, there is established a test method including steps up to detection with a measurement apparatus. There is provided a method for detecting a target site, which is an LAMP method using four kinds of primers, FIP, F3 primer, BIP, and B3 primer, which are designed on the basis of six regions of a template polynucleotide, F1 region, F2 region, F3 region, B1 region, B2 region, and B3 region, wherein: the template polynucleotide contains the target site, the four kinds of primers are designed so that the target site exists between the F1 region and the F2 region, or between the B1 region and the B2 region; a loop primer designed on the basis of a region between the F 1 region and the F2 region or between the B 1 region and the B2 region on the side on which the target site exists, and an oligonucleotide probe that can associate with a region including the target site are used, the loop primer and the probe are designed so that the 5 end of the loop primer and the 3 end of the probe associate with the template polynucleotide at positions close to each other, one of the loop primer and the probe is modified with a fluorescent molecule around the 5 end in the case of the loop primer or the 3 end in the case of the probe, and the other is modified with a quenching molecule.


PubMed | NIPPON GENE Co. and University of Toyama
Type: Journal Article | Journal: Experimental cell research | Year: 2015

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1 and HIF-2 seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1 and HIF-2. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment.

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