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Kajitani, Koji,Ken-Ichi, Honda,Terada, Hiroyuki,Yasui, Tomoyo,Sumi, Toshiyuki,Koyama, Masayasu,Ishiko, Osamu Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.18
The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor ${\beta}$, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non-ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower ${\beta}$-tubulin levels and comparatively higher p53/${\beta}$-tubulin or CAR/${\beta}$-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non-siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.
Dynamic Trend Analysis for Ultrasonic Irradiation in Control of Suspension Polymerization Process
Hideyuki Matsumoto,Masaya Honda,Hiroyuki Mori,Chiaki Kuroda 제어로봇시스템학회 2009 제어로봇시스템학회 국제학술대회 논문집 Vol.2009 No.8
A purpose of the present paper is to demonstrate applicability of the method of dynamic trend analysis, which is based on the linear approximation technique and the cumulative sum technique, for monitoring of the reactor temperature data in the suspension polymerization process. By executing ultrasonic irradiation at the moment when an extracted segment changes, development of the dynamic trend analysis is investigated from the viewpoint of control of characteristics of polymer particles. Then, relationships between the dynamic trend analysis and the process control are also discussed by considering control of temperature rise under ultrasonic irradiation, which would influence on produced polymer particles.
Shuhei Yamamoto,Mina Okochi,Kowichi Jimbow,Hiroyuki Honda 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3
Three-dimensional (3D) cell culture arrays of melanoma cell spheroids were assembled to evaluate the combined effect of a melanogenesis-targeting drug, Npropionyl- 4-cysteaminylphenol (NPrCAP), and heat treatment. An array-like multicellular pattern of mouse melanoma B16F1 cells in a collagen gel was established by magnetic cell labeling using a pin-holder device to exert a magnetic force. The cellular spheroids were exposed to NPrCAP and heat (42°C for 1 h) as a model of anti-cancer treatment. As a result, melanogenesis of B16F1 cells was 29-fold higher in this 3D array than in conventional two-dimensional (2D) monolayer cultures. Because the spheroid size was linearly correlated with the cell number within a spheroid, the antiproliferative effect could be evaluated in a non-destructive manner. Moreover, the half-maximal inhibitory concentration of NPrCAP coupled with heat treatment calculated from the spheroid size was 2-fold higher in the 3D array (0.30mM) than in 2D culture (0.15 mM). These results indicate that spheroid formation decreases the chemosensitivity of cancer cells, and this model would be suitable as a susceptibility assay for melanogenesis-targeting drugs. Therefore, this 3D culture model provides a better screening format to evaluate drug and physical treatments for cancer therapy than 2D formats.
Development of a Tactical Screening Method to Investigate the Characteristics of Functional Peptides
Akiko Kume,Mina Okochi,Kazunori Shimizu,Yasuko Yoshida,Hiroyuki Honda 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.1
Using spot-synthesized peptide arrays, a functional peptide can be screened as a high-binding peptide for a target molecule. We have developed a rational screening method for functional peptides by analyzing the physicochemical rules of high-binding peptide sequences. To screen the peptides simply and strategically, we prepared an exhaustive 4-mer peptide library consisting of 256 peptides (44 = 256) characterized by four physicochemical groups of 20 amino acids: Group 1, non-charged hydrophobic amino acids; Group 2, non-charged hydrophilic amino acids; Group 3, positive-charged hydrophilic amino acids; Group 4, negative-charged hydrophilic amino acids. First, our previous screening data from cell adhesion, bile acid-binding, and nanoparticle-binding peptides were applied to the fourcategory analysis, and target-specific physicochemical characteristics were obtained. We then prepared an exhaustive 4-mer peptide library using these four physicochemical groups, and screened for high-binding peptides that bind model proteins interleukin-2 and IgG. We obtained individual physicochemical rules for high-binding peptides: group 1 or 4 amino acids in position (P) 1, group 1 in P2 and P4 for IL-2, and group 2 and 3 amino acids at all position for IgG. Therefore, this system, which employs the use of a simple and strategic peptide library, will be useful in the development of functional peptides.
Nao Yamaoka,Kazunori Shimizu,Yu Imaizumi,Yohei Okada,Hiroyuki Honda 한국바이오칩학회 2019 BioChip Journal Vol.13 No.2
Degeneration of motor neurons and skeletal muscles or the collapse of neuromuscular junctions (NMJs) causes progressive motility disturbances in many neuromuscular diseases. Although various microdevices for the co-culture of skeletal muscle myotubes and motor neurons have been developed to investigate neuromuscular diseases in vitro, it remains difficult to isolate single myotubes and motor neurons from the device for single-cell analyses, such as gene expression analysis. Here, we developed open chamber-coculture microdevices that contain cell culture chambers with narrow widths. Given the small chamber width (0.2 mm), the device significantly prevented the overlap among myotubes within the chamber. The percentage of non-overlapping was 95.6 ± 7.7% for the 0.2-mmwidth chamber and 11.8 ± 6.4% for the 7-mm-width chamber as a control. In addition, the device with the 0.2-mm chamber promoted myotube maturation, as indicated by the longer widths and lengths of the myotubes relative to those in the control chamber. Single C2C12 myotubes and human induced pluripotent stem cell (hiPSC)-derived motor neurons were successfully collected from the device with the 0.2-mm chamber using a micromanipulator equipped with a glass capillary. Furthermore, myotubes and hiPSC-derived motor neurons were co-cultured in the device with the 0.2- mm chamber, and the formation of NMJs were observed. Thus, the developed device is a useful tool for performing single-cell analysis for studying neuromuscular diseases in vitro.
Imanishi, Toshiaki,Hanai, Taizo,Aoyagi, Ichiro,Uemura, Jun,Araki, Katsuhiro,Yoshimoto, Hiroshi,Harima, Takeshi,Honda , Hiroyuki,Kobayashi, Takeshi The Korean Society for Biotechnology and Bioengine 2002 Biotechnology and Bioprocess Engineering Vol.7 No.5
In order to control glucose concentration during fed-batch culture for antibiotic production, we applied so called “software sensor” which estimates unmeasured variable of interest from measured process variables using software. All data for analysis were collected from industrial scale cultures in a pharmaceutical company. First, we constructed an estimation model for glucose feed rate to keep glucose concentration at target value. In actual fed-batch culture, glucose concentration was kept at relatively high and measured once a day, and the glucose feed rate until the next measurement time was determined by an expert worker based on the actual consumption rate. Fuzzy neural network (FNN) was applied to construct the estimation model. From the simulation results using this model, the average error for glucose concentration was 0.88 g/L. The FNN model was also applied for a special culture to keep glucose concentration at low level. Selecting the optimal input variables, it was possible to simulate the culture with a low glucose concentration from the data sets of relatively high glucose concentration. Next, a simulation model to estimate time course of glucose concentration during one day was constructed using the on-line measurable process variables, since glucose concentration was only measured off-line once a day. Here, the recursive fuzzy neural network (RFNN) was applied for the simulation model. As the result of the simulation, average error of RFNN model was 0.91 g/L and this model was found to be useful to supervise the fed-batch culture.