Hidenori Ohnishi

Publications (31)64.02 Total impact

• Article: Hypothermia augments NF-kappaB activity and the production of IL-12 and IFN-gamma.

  Takahiro Arai, Hideo Kaneko, Hidenori Ohnishi, Eiko Matsui, Toshiyuki Fukao, Norio Kawamoto, Kimiko Kasahara, Naomi Kondo
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  ABSTRACT: The differentiation of Th1 and Th2 is strictly regulated by humoral and cellular factors. The imbalance between Th1 and Th2 is considered to be the pathogenesis of allergic and autoimmune disorders. It is important to elucidate the effect of environmental factors, such as temperature, on the expression of cytokines of Th1 and Th2. We investigated the expression of IFN-gamma, IL-4, IL-5, IL-10 and IL-12 from LPS- or PHA-stimulated PBMCs at 30 degrees C or 37 degrees C using ELISA and Real-time PCR. We measured the change of NF-kappaB activity at 30 degrees C or 37 degrees C with LPS stimulation using the reporter gene assay. IFN-gamma production from LPS-stimulated PBMCs at 30 degrees C was up-regulated compared with 37 degrees C. IL-5 and IL-10 production from PHA-stimulated PBMCs at 30 degrees C were down-regulated compared with 37 degrees C. This augmented IFN-gamma production was caused by the up-regulation of IL-12 production from CD14+ blood monocytes. Both IL-12 mRNA and IL12 protein at 30 degrees C were up-regulated compared with 37 degrees C. NF-kappaB, the key molecule for the expression of IL-12, was also augmented at 30 degrees C compared with 37 degrees C. Hypothermia up-regulated the expression of IL-12 and IFN-gamma due to the augmented NF-kappaB activity. It is suggested that hypothermia modifies the pattern of cytokine gene expression.
  Allergology International 10/2008; 57(4):331-8.
• Article: [QOL questionnaire for pediatric patients with bronchial asthma and their parents or caregivers. Preparation and evaluation of the short form version 2008 (Gifu)].

  Naomi Kondo, Koichiro Hirayama, Eiko Matsui, Takahide Teramoto, Hideo Kaneko, Toshiyuki Fukao, Kenji Orii, Minako Kawamoto, Michinori Funato, Hidenori Ohnishi, Norio Kawamoto, Hideyuki Morita, Takeshi Kimura, Masatoshi Nada, Tetsuji Tokumi, Tomohiro Hori, Rinko Watanabe
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  ABSTRACT: The QOL questionnaire version 2001 for pediatric patients with bronchial asthma and their parents or caregivers includes 15 questions for patients under the age of 4 years and 20 questions for patients over the age of 4 years. We have already reported that the QOL questionnaire version 2001 reflects reliability (including reproducibility), factorial validity, and changes in paroxysmal attacks of asthma. In this study, we revised the questionnaire for use in routine medical practice. In this study, based on the data of a previous report, the number of questions was reduced further and it was revised to the questionnaire the short form by integrated data. The revised version 2008 (Gifu) consisted of emotional burden, asthma attack, instability of symptoms and proper acceptance of asthma as a common factor, moreover 4 or more years old added load of exercise factor which consisted of two questions in each factor. This QOL short form questionnaire version 2008 (Gifu) is a disease specific questionnaire in comparison with health control, bronchial asthma and non-asthmatic patients, such as atopic dermatitis and allergic rhinitis. Although Cronbach's alpha fell with reduction of the number of questions, we conclude that it was acceptable in the clinical practice.
  Arerugī = [Allergy] 09/2008; 57(8):1022-33.
• Article: A novel polymorphism, E254K, in the 5-lipoxygenase gene associated with bronchial asthma.

  Chunying Bai, Eiko Matsui, Hidenori Ohnishi, Kaori Kimata, Kimiko Kasahara, Hideo Kaneko, Zenichiro Kato, Toshiyuki Fukao, Naomi Kondo
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  ABSTRACT: Cysteinyl-leukotrienes are important pro-inflammatory mediators in bronchial asthma (BA) and are derived from arachidonic acid by the action of 5-lipoxygenase. We identified a novel polymorphism, c.760 G>A (E254K), in exon 6 of the 5-lipoxygenase gene (5-LO). This substitution was detected in 11 out of 180 patients with BA, but not in any of the 150 non-allergic subjects. The frequency of c.760 G>A showed a significant difference between BA and non-allergic subjects (P=0.0007). The c.760 G>A polymorphism existed at the surface edge of the C-terminal catalytic domain, and the E-to-K substitution changed the charge of the side chain from negative to positive. Thus, our results suggest that E254K in the 5-LO might be associated with BA.
  International Journal of Molecular Medicine 03/2008; 21(2):139-44. · 1.98 Impact Factor
• Article: Development of fluorescence-linked immunosorbent assay for high throughput screening of interferon-gamma.

  Eiji Matsukuma, Zenichiro Kato, Kentaro Omoya, Kazuyuki Hashimoto, Ailian Li, Yutaka Yamamoto, Hidenori Ohnishi, Hidenori Hiranuma, Hisakazu Komine, Naomi Kondo
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  ABSTRACT: Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples. We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA. We developed a new system for measuring hIFN-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1% and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA. The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.
  Allergology International 04/2006; 55(1):49-54.
• Article: Molecular mechanism of a temperature-sensitive phenotype in peroxisomal biogenesis disorder.

  Kazuyuki Hashimoto, Zenichiro Kato, Tomoko Nagase, Nobuyuki Shimozawa, Kazuo Kuwata, Kentaro Omoya, Ailian Li, Eiji Matsukuma, Yutaka Yamamoto, Hidenori Ohnishi, Hidehito Tochio, Masahiro Shirakawa, Yasuyuki Suzuki, Ronald J A Wanders, Naomi Kondo
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  ABSTRACT: Peroxisomal biogenesis disorders include Zellweger syndrome and milder phenotypes, such as neonatal adrenoleukodystrophy (NALD). Our previous study of a NALD patient with a marked deterioration by a fever revealed a mutation (Ile326Thr) within a SH3 domain of PEX13 protein (Pex13p), showing a temperature-sensitive (TS) phenotype in peroxisomal biogenesis. Clinical TS phenotypes also have been reported in several genetic diseases, but the molecular mechanisms still remain to be clarified. The immunofluorescent staining with anti-Pex13p antibody also revealed TS phenotype of the I326T mutant protein itself in the patient cells. Protease digestion of the recombinant Pex13p-SH3 domain showed an increase of protease susceptibility, suggesting a problem of mutant protein fold. Conformational analyses against urea denaturation using urea gradient gel electrophoresis or fluorescence emission from tryptophan residue revealed that the mutant protein should be easily unfolded. Far-UV circular dichroism (CD) spectra demonstrated that both wild-type and the mutant protein have antiparallel beta-sheets as their secondary structure with slightly different extent. The thermal unfolding profiles measured by CD showed a marked lower melting temperature for I326T protein compared with that of wild-type protein. Analysis of the protein 3D-structure indicated that the Ile326 should be a core residue for folding kinetics and the substitution of Ile326 by threonine should directly alter the kinetic equilibrium, suggesting a marked increase of the unfolded molecules when the patient had a high fever. Structural analyses of the protein in the other genetic diseases could provide an avenue for better understanding of genotype-phenotype correlations.
  Pediatric Research 09/2005; 58(2):263-9. · 2.70 Impact Factor
• Article: Systematic optimization of active protein expression using GFP as a folding reporter.

  Kentaro Omoya, Zenichiro Kato, Eiji Matsukuma, Ailian Li, Kazuyuki Hashimoto, Yutaka Yamamoto, Hidenori Ohnishi, Naomi Kondo
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  ABSTRACT: Many recombinant proteins have been used as drugs; however, human proteins expressed using heterologous hosts are often insoluble. To obtain correctly folded active proteins, many optimizations of expression have been attempted but usually are found to be applicable only for specific targets. Interleukin-18 (IL-18) has a key role in many severe disorders including autoimmune diseases, and therapeutic approaches using IL-18 have been reported. However, production of IL-18 in Escherichia coli resulted in extensive inclusion body formation and previous conventional screenings of expression conditions could obtain only a condition with a low yield. To address the problem, we applied a folding reporter system using green fluorescent protein (GFP) for screening of the expression conditions for hIL-18. The established system efficiently screened many conditions, and optimized conditions for the expression of hIL-18 significantly enhanced the final yield of the active protein. Systematic screening using a GFP reporter system could be applied for the production of other proteins and in other organisms.
  Protein Expression and Purification 09/2004; 36(2):327-32. · 1.59 Impact Factor
• Article: Generation of highly stable IL-18 based on a ligand-receptor complex structure.

  Yutaka Yamamoto, Zenichiro Kato, Eiji Matsukuma, Ailian Li, Kentaro Omoya, Kazuyuki Hashimoto, Hidenori Ohnishi, Naomi Kondo
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  ABSTRACT: Human interleukin-18 (hIL-18), initially cloned as an IFN-gamma-inducing factor, has a key role in many inflammatory diseases. We have previously developed a high production system for correctly folded active hIL-18 protein, leading to the revelation of the 3D-structure and the receptor binding mode. These findings can strongly indicate the experimental and medical applications of IL-18; however, the recombinant protein is prone to be inactivated forming multimers. Recently, therapeutic approaches using recombinant IL-18 have shown the effectiveness for treatment of cancer; indicating the necessity of a more stable protein for therapy with intertrial reliability. Here we have generated a highly stable hIL-18 with replacement of cysteine by serine based on the tertiary structure and the binding mechanism, retaining the biological activity. Similar rational designs can be applied to develop new therapeutic molecules of other cytokines.
  Biochemical and Biophysical Research Communications 05/2004; 317(1):181-6. · 2.48 Impact Factor
• Article: Optimized gene synthesis and high expression of human interleukin-18.

  Ailian Li, Zenichiro Kato, Hidenori Ohnishi, Kazuyuki Hashimoto, Eiji Matsukuma, Kentaro Omoya, Yutaka Yamamoto, Naomi Kondo
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  ABSTRACT: Human interleukin-18 (hIL-18), originally known as an IFN-gamma-inducing factor, is a recently cloned cytokine that is secreted by Kupffer cells of the liver and by stimulated macrophages. We have previously established a method of expression and purification of IL-18. The yield however remains low and the insufficient expression of a heterologous protein could be due to skewed codon usage between the expression host and the cDNA donor. The sequence of mature hIL-18 has 37 a.a. rare codons for Escherichia coli in a total of 157 a.a. To overcome this problem, gene synthesis was performed with optimized codons for the expression host E. coli. The final yield of the hIL-18 protein with optimized codons was about five times higher than the yield with the native sequence. Using a minimal medium, this system produces large quantities of labeled proteins that can be used in NMR analysis. Our simple and efficient production system can be applied to the production of other cytokines for new structural and therapeutic use.
  Protein Expression and Purification 12/2003; 32(1):110-8. · 1.59 Impact Factor
• Article: The structure and binding mode of interleukin-18.

  Zenichiro Kato, JunGoo Jee, Hiroaki Shikano, Masaki Mishima, Izuru Ohki, Hidenori Ohnishi, Ailian Li, Kazuyuki Hashimoto, Eiji Matsukuma, Kentaro Omoya, Yutaka Yamamoto, Teruyo Yoneda, Takane Hara, Naomi Kondo, Masahiro Shirakawa
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  ABSTRACT: Interleukin-18 (IL-18), a cytokine formerly known as interferon-gamma- (IFN-gamma-) inducing factor, has pleiotropic immunoregulatory functions, including augmentation of IFN-gamma production, Fas-mediated cytotoxicity and developmental regulation of T-lymphocyte helper type I. We determined the solution structure of IL-18 as a first step toward understanding its receptor activation mechanism. It folds into a beta-trefoil structure that resembles that of IL-1. Extensive mutagenesis revealed the presence of three sites that are important for receptor activation: two serve as binding sites for IL-18 receptor alpha (IL-18Ralpha), located at positions similar to those of IL-1 for IL-1 receptor type I (IL-1RI), whereas the third site may be involved in IL-18 receptor beta (IL-18Rbeta) binding. The structure and mutagenesis data provide a basis for understanding the IL-18-induced heterodimerization of receptor subunits, which is necessary for receptor activation.
  Natural Structural Biology 12/2003; 10(11):966-71.
• Article: Interleukin‐18 is associated with the severity of atopic dermatitis

  Hidenori Ohnishi, Zenichiro Kato, Mizuho Watanabe, Osamu Fukutomi, Ryosuke Inoue, Takahide Teramoto, Naomi Kondo
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  ABSTRACT: Background: Interleukin (IL)-18 acts as both a Th1 and Th2 cytokine, but its association with allergic diseases remains unclear. The aim of the present study was to measured plasma IL-18 and serum IgE levels in atopic children to evaluate how IL-18 is associated with allergic diseases.Methods: The plasma IL-18 and serum IgE levels in 51 atopic children, 28 healthy control children and 14 healthy control adults were measured by enzyme-linked immunosorbent assay (ELISA). The 5′ end of the IL-18 gene of 48 atopic children and 20 healthy control children was sequenced.Results: The plasma IL-18 level was significantly elevated in children with bronchial asthma and/or atopic dermatitis. Plasma IL-18 levels in the moderate or severe atopic dermatitis group were significantly higher than those in either the control group or the mild atopic dermatitis group. There was a positive correlation between plasma IL-18 and serum IgE levels. Three allelic combinations of polymorphisms in the IL-18 gene promoter region were observed. There was no significant difference in the plasma IL-18 levels between groups carrying these genotypes. However, bronchial asthma patients had significantly higher frequencies of the −137 G/G genotype than did control children.Conclusions: The plasma IL-18 level was elevated, particularly in patients with atopic dermatitis. As the clinical severity of atopic dermatitis increased, the plasma IL-18 level also tended to increase. These findings suggest that IL-18 may be associated with the severity of atopic dermatitis.
  Allergology International 08/2003; 52(3):123 - 130.
• Article: [Evaluation before and after pranlukast administration with the QOL questionnaire (revised version 2001) for pediatric patients with bronchial asthma and their parents or caregivers].

  Naomi Kondo, Takahide Teramoto, Ryosuke Inoue, Osamu Fukutomi, Eiko Matsui, Shinji Shinoda, Mizuho Watanabe, Heima Sakaguchi, Minako Aoki, Wataru Morimoto, [......], Yasuhiko Takemoto, Yoko Tanaka, Tsutomu Asano, Kaori Yoshikawa, Tomoko Nagase, Kazuyuki Hashimoto, Yukiko Fujita, Yutaka Yamamoto, Airen Ri, Khoichirou Hirayama
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  ABSTRACT: We conducted a longitudinal investigation with the QOL questionnaire (revised version 2001) before and after the 4-week-administration of a leukotriene receptor antagonist pranlukast. A significant improvement in the < 4 yrs group was observed at week 1, and that in > or = 4 yrs group at week 2. Under these conditions, the overall QOL score, physical domains and mental domains, significantly improved in both the < 4 yrs group and the > or = 4 yrs group. Overall, a slight correlation was observed between ratio changes in QOL scores and differences in symptom scores. However, no correlation was found in part of patients, suggesting that the QOL questionnaire allows measurement of mental changes in the patients themselves and their parents or caregivers for therapeutic effects which cannot be determined with ordinary physical findings only. In "event present" group, a significant difference in physical and mental domains was revealed by the comparison of QOL scores before and after administration. And furthermore in "event absence" group, the p-value for physical domain and mental domain was 0.0505 and 0.0912 in the < 4 yrs group, respectively, 0.0101 and 0.0446 in the > or = 4 yrs group, respectively. The above results led us to consider the QOL questionnaire (revised version 2001) useful for routine medical care. Furthermore, pranlukast was considered useful for improvement not only of physical symptoms of bronchial asthma but also of the patient's QOL, although the placebo effects in this open trial must be considered.
  Arerugī = [Allergy] 06/2002; 51(5):421-9.