Takaharu Negoro

Publications (19)60.75 Total impact

• Article: Neutrophil TRPM2 channels are implicated in the exacerbation of myocardial ischemia/reperfusion injury.

  Toshihito Hiroi, Teruaki Wajima, Takaharu Negoro, Masakazu Ishii, Yasuko Nakano, Yuji Kiuchi, Yasuo Mori, Shunichi Shimizu
  [show abstract] [hide abstract]
  ABSTRACT: AIMS: Transient receptor potential melastatin 2 (TRPM2) highly expressed in immunocytes is a Ca(2+)-permeable nonselective cation channel activated by oxidative stress. Myocardial ischemia/reperfusion (I/R) injury is characterized by acute inflammation associated with the augmentation of oxidative stress. We hypothesized that TRPM2 is implicated in the exacerbation of myocardial I/R injury.Methods and ResultsWildtype (Trpm2(+/+)) and Trpm2 knockout (Trpm2(-/-)) mice were subjected to ligation of the left main coronary artery followed by reperfusion. Myocardial infarction following I/R, but not ischemia alone, was more reduced in Trpm2-/- mice than in Trpm2(+/+) mice and cardiac contractile functions were also improved in Trpm2(-/-) mice. TRPM2 was highly expressed in the polymorphonuclear leukocytes (PMNs) rather than in the heart. The number of neutrophils and myeloperoxidase (MPO) activity in the reperfused area following ischemia was lowered in Trpm2(-/-) mice. When Trpm2+(/+) or Trpm2(-/-) PMNs were administered to the Trpm2(-/-) heart ex vivo through the perfusate or in vivo by intravenous injection, Trpm2(+/+) PMNs produced enlargement of the infarct size. Following in vitro regional I/R, a pharmacological inhibitor of TRPM2 reduced the infarct size. The combination of H(2)O(2) and leukotriene B(4) (LTB(4)) increased intracellular Ca(2+) concentration and their adhesion to endothelial cells in Trpm2(+/+) but not in Trpm2(-/-) PMNs. CONCLUSIONS: These findings indicate that neutrophil TRPM2 is implicated in the exacerbation of myocardial reperfusion injury. Accumulation of neutrophils in the reperfused area mediated by TRPM2 activation is likely to play a crucial role in myocardial I/R injury.
  Cardiovascular research 11/2012; · 5.80 Impact Factor
• Article: Impaired Ca²⁺ regulation of CD4⁺CD25⁺ regulatory T cells from pediatric asthma.

  Yoshiki Yamamoto, Takaharu Negoro, Akane Hoshi, Akiko Wakagi, Shunichi Shimizu, Alison H Banham, Masakazu Ishii, Haruyo Akiyama, Yuji Kiuchi, Susumu Sunaga, Takashi Tobe, Giovanna Roncador, Kazuo Itabashi, Yasuko Nakano
  [show abstract] [hide abstract]
  ABSTRACT: CD4(+)CD25(+) regulatory T (T(reg)) cells can control the allergic response to allergen, airway eosinophilia and airway hypersensitivity. We speculated that chronic inflammation persisting in asthma airways is dependent on abnormalities of these T(reg) cells. There are differences in the pathology of asthma in adults and children, and the airways of pediatric asthma are considered to be more naive than those of adults. Therefore, we analyzed the functionality of T(reg) cells in pediatric asthma and the relationship between T(reg) function and asthma symptoms. The anergic state, which is one of the defining properties of T(reg), was analyzed by measuring intracellular Ca(2+) influx following T cell receptor (TCR) stimulation. FOXP3-positive cells and FOXP3 mRNA expression were measured by flow analysis and real-time PCR with the SYBR method, respectively. CD45RO(+) cells make up approximately 99% of CD4(+)CD25(high) T cells and 89% of CD4(+)CD25(low) T cells in human adult blood. The proportion of CD45RO(+) cells in CD4(+)CD25(+) (high + low) T cells from pediatric asthma was much smaller (about 56%). Interestingly, our data indicated that CD45RO(+) T(reg) cells from pediatric asthma aberrantly increased intracellular Ca(2+) concentrations following TCR activation compared with pediatric nonasthma controls. These impaired CD45RO(+) T(reg) cell functions were correlated with asthma symptoms. The correlation was observed in the group with a highly expressed atopic phenotype and longer duration of asthma. We suggest that chronic inflammation in pediatric asthma airways may be the result of impaired regulatory functions of CD45RO(+) T(reg) cells.
  International Archives of Allergy and Immunology 05/2011; 156(2):148-58. · 2.40 Impact Factor
• Article: [Decay-accelerating factor].

  Takaharu Negoro, Yasuko Nakano
  Nippon rinsho. Japanese journal of clinical medicine 06/2010; 68 Suppl 6:102-4.
• Article: TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration.

  Shinichiro Yamamoto, Shunichi Shimizu, Shigeki Kiyonaka, Nobuaki Takahashi, Teruaki Wajima, Yuji Hara, Takaharu Negoro, Toshihito Hiroi, Yuji Kiuchi, Takaharu Okada, Shuji Kaneko, Ingo Lange, Andrea Fleig, Reinhold Penner, Miyuki Nishi, Hiroshi Takeshima, Yasuo Mori
  [show abstract] [hide abstract]
  ABSTRACT: Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca(2+)-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H(2)O(2)) evokes Ca(2+) influx through TRPM2 to activate Ca(2+)-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-kappaB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H(2)O(2)-induced Ca(2+) influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca(2+) influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.
  Nature medicine 08/2008; 14(7):738-47. · 27.14 Impact Factor
• Article: [Analysis of students, achievement rate and contents of assessment for objective structured clinical examination (OSCE) attempted at the faculty of pharmaceutical sciences, Showa University].

  Mari Kogo, Noriko Koyama, Takaharu Negoro, Kimiko Aoki, Isao Saito, Yasuna Kobayashi, Junichi Mashimo, Keiko Sasaki, Takashi Tobe, Toshinori Yamamoto, Yuji Kiuchi, Hitoshi Sato
  [show abstract] [hide abstract]
  ABSTRACT: The aim of this study was to analyze students, achievement rate and contents of assessment judged by instructors in objective structured clinical examination (OSCE) attempted at the Faculty of Pharmaceutical Science, Showa University. The OSCE was carried out for fourth-year students in May 28, 2005. In this trial, there were two stations, i.e., counting/measurement dispensing and subsequent audit of dispensed drugs, and 218 students and 31 instructors (as evaluators) participated. We developed a checklist to test students attitudes and skills (two stages) and overall evaluation (five stages). Each student was evaluated by two instructors. Examination time was 8 minutes for drug dispensing, and 4 minutes for the audit of dispensed drug. After the OSCE trial, we analyzed validity of examination time, contents of assessment, and differences in scores between different evaluators. More than half of the students could not finish the examination within the limit of time for dispensing the liquid and cream and audit for dispensed powder. The number of items that 60% of the students achieved was 48 (82.8%). Moreover, 20% of the assessment items did not agree among the evaluators with a disagreement rate of 20% or more. Thus, we distinguished between the items based on the extent of disagreement rates. It was suggested that most of the students achieved such a level to actually perform clinical training in pharmacies. From these results, it is necessary to set up an assignment to finish the within the time limit to extent the time limit depending upon examination contents, to standardize the evaluation to increase the agreement rate among evaluators, and to more clearly identify assessment criteria.
  Yakugaku zasshi journal of the Pharmaceutical Society of Japan 06/2007; 127(5):905-17. · 0.39 Impact Factor
• Article: Influence of SNPs in cytokine-related genes on the severity of food allergy and atopic eczema in children.

  Takaharu Negoro, Kanami Orihara, Tomoko Irahara, Hiroshi Nishiyama, Kanae Hagiwara, Risa Nishida, Hiroki Takagi, Kazue Satoh, Yoshiki Yamamoto, Shunichi Shimizu, Tamio Hagiwara, Masakazu Ishii, Toshihiro Tanioka, Yasuko Nakano, Ken Takeda, Isao Yoshimura, Yoji Iikura, Takashi Tobe
  [show abstract] [hide abstract]
  ABSTRACT: Although many single nucleotide polymorphism (SNP) studies have reported an association of atopy, allergic diseases and total serum immunoglobulin E (IgE) levels, almost all of these studies sought risk factors for the onset of these allergic diseases. Furthermore, many studies have analyzed a single gene and hardly any have analyzed environmental factors. In these analyses, the results could be masked and the effects of other genes and environmental factors may be decreased. Here, we described the correlation between four genes [interleukin (IL)-4 (C-590T), IL-4 receptor (A1652G), FCER1B (G6842A) and STAT6 (G2964A)] in connection with IgE production; the role of IL-10 (C-627A) as a regulatory cytokine of allergy; and the severity of food allergy (FA) and atopic eczema (AE) in 220 Japanese allergic children. In addition to these SNPs, environmental factors, i.e., patient's attitude, indoor environment, and so on, were also investigated in this study. Our study was retrospective, and the correlation was analyzed by our defined clinical scores divided into three terms: worst symptoms, recent symptoms and general amelioration at the most recent examination during the disease course. Our results indicated that IL-10 AA, the genotype with lower IL-10 production, is associated with higher IgE levels in the serum (p < 0.0001, estimate; 0.912). Marginal liver abnormalities were observed in the subject group with both FA and AE (p < 0.1191, estimate; 0.1490). Our defined clinical scores enabled evaluation of various aspects of disease severity. Based on the scores, while no single SNP selected in this study determined severity, the combination of the SNP with laboratory data and environmental factors appeared to determine severity.
  Pediatric Allergy and Immunology 12/2006; 17(8):583-90. · 2.46 Impact Factor
• Article: A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin.

  Yasuko Nakano, Sachiko Tajima, Ai Yoshimi, Haruyo Akiyama, Motoo Tsushima, Toshihiro Tanioka, Takaharu Negoro, Motowo Tomita, Takashi Tobe
  [show abstract] [hide abstract]
  ABSTRACT: Human plasma contains at least three forms of adiponectin: a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We purified HMW adiponectin from human plasma using its affinity to gelatin and obtained monoclonal antibodies against it. On Western blot analysis, the reactivity of these monoclonal antibodies was shown to be restricted to a non-heat-denatured form of adiponectin molecules. On heating, the collagen-like domain of adiponectin molecules became denatured, and thus the trimer form could not be maintained. From these, monoclonal antibodies against HMW adiponectin were suggested to react with the intact trimer of adiponectin. With these monoclonal antibodies, we developed a sandwich ELISA system for quantifying adiponectin in human serum. Its specificity was verified by analysis of serum fractions separated by gel-filtration chromatography, and our ELISA system was found to be HMW adiponectin-specific. With this novel ELISA, the HMW adiponectin concentrations were 8.4 +/- 5.5 microg/ml (mean +/- SD) in healthy women and 6.2 +/- 3.6 microg/ml in healthy men. Also, serum with a lower HMW adiponectin concentration was shown to have a lower HMW ratio (i.e., HMW adiponectin/total adiponectin).
  The Journal of Lipid Research 08/2006; 47(7):1572-82. · 5.56 Impact Factor
• Article: [Immunological tests: Decay-accelerating factor].

  Takaharu Negoro, Takashi Tobe
  Nippon rinsho. Japanese journal of clinical medicine 08/2005; 63 Suppl 7:98-100.
• Article: Possible involvement of hydroxyl radical on the stimulation of tetrahydrobiopterin synthesis by hydrogen peroxide and peroxynitrite in vascular endothelial cells.

  Shunichi Shimizu, Masakazu Ishii, Yoshiyuki Miyasaka, Teruaki Wajima, Takaharu Negoro, Tamio Hagiwara, Yuji Kiuchi
  [show abstract] [hide abstract]
  ABSTRACT: We recently described that hydrogen peroxide (H2O2) stimulates the synthesis of tetrahydrobiopterin (BH4) through the induction of the rate-limiting enzyme GTP-cyclohydrolase I (GTPCH), and increases tetrahydrobiopterin content in vascular endothelial cells. Tetrahydrobiopterin is easily oxidized by peroxynitrite (ONOO-), but not by hydrogen peroxide. The aim of this study was to determine the effect of hydroxyl radical and peroxynitrite, which are both toxic biological oxidants, on tetrahydrobiopterin synthesis and the regulation of its content in vascular endothelial cells. In the cell-free assay system, tetrahydrobiopterin was rapidly oxidized by the hydroxyl radical and peroxynitrite, but not by hydrogen peroxide. However, the addition of not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite to vascular endothelial cells transiently decreased tetrahydrobiopterin content, and then markedly increased its content. Interestingly, total biopterin content was also decreased by early treatment with oxidants. Moreover, oxidants induced the expression of GTP-cyclohydrolase I, and the increase of the tetrahydrobiopterin content was blocked by the treatment with GTP-cyclohydrolase I inhibitor. Both the hydrogen peroxide- and peroxynitrite-induced increases in tetrahydrobiopterin content and findings suggest that not only hydrogen peroxide but also the hydroxyl radical and peroxynitrite stimulates tetrahydrobiopterin synthesis through GTP-cyclohydrolase I expression, and that the hydroxyl radical plays a central role in the stimulation of tetrahydrobiopterin synthesis. Moreover, the transient decrease in BH4 to tetrahydrobiopterin.
  The International Journal of Biochemistry & Cell Biology 05/2005; 37(4):864-75. · 4.63 Impact Factor
• Article: Reduction of GTP cyclohydrolase I feedback regulating protein expression by hydrogen peroxide in vascular endothelial cells.

  Masakazu Ishii, Shunichi Shimizu, Teruaki Wajima, Tamio Hagiwara, Takaharu Negoro, Akira Miyazaki, Takashi Tobe, Yuji Kiuchi
  [show abstract] [hide abstract]
  ABSTRACT: We examined the effect of H(2)O(2) on the expression of GTP cyclohydrolase I (GTPCH) feedback regulating protein (GFRP). Addition of H(2)O(2) to endothelial cells decreased GFRP mRNA levels, in contrast to the increase of tetrahydrobiopterin (BH(4)) content and GTPCH mRNA levels. The inhibitors of nitric oxide (NO) synthase and GTPCH had no influence on the decrease of GFRP mRNA levels in H(2)O(2)-treated cells. It is suggested that H(2)O(2) induces BH(4) synthesis through not only induction of GTPCH but also reduction of GFRP. The decrease of GFRP mRNA level appears to be independent of the produced NO and BH(4).
  Journal of Pharmacological Sciences 03/2005; 97(2):299-302. · 2.08 Impact Factor
• Article: Induction of CD4+ regulatory T cells by TPA in mice: contra-suppression by CD8+ T cells.

  Takaharu Negoro, Kazue Satoh, Fumio Iinuma, Takashi Tobe, Mitsuo Watanabe
  [show abstract] [hide abstract]
  ABSTRACT: Among the eight inbred mouse strains employed in our preceding report, 12-O-tetradecanoylphorbol 13-acetate (TPA) painting alone induced CD4+ regulatory T (Tr) cells in four strains (e.g., C3H/He) at 6-8 weeks of age, but not in the remaining strains (e.g., C57BL/6, BALB/c). In the present study, the effect of growth from 4-14 weeks on delayed-type hypersensitivity (DTH) response was investigated in three inbred murine strains, C3H/He (H-2k), C57BL/6 (H-2b) and BALB/c (H-2d) mice. In all strains older than 10 weeks, DTH response was suppressed exclusively by TPA painting. The defect of suppressive activity for DTH in several of the strains at 6-8 weeks of age was dependent on the presence of cells, which blocked regulatory cell activity at 6-8 weeks of age, but not at 10 weeks of age. The age-dependent difference in regulatory activity was caused by the presence of CD8+ contra-regulatory T (Tcr) cells. CD8+ contra-regulatory T cells are required to contact regulatory cells in order to block DTH suppressive activity. Adhesion molecules were of great importance in contra-suppression, as antibody treatment to LFA-1 or ICAM-1 blocked this activity. ICAM-1 expression on CD4 T cells greatly increased following growth in 10 week-BALB/c mice receiving TPA than 6-week-old mice, however, a slight increase in growth occurred in 6-to-10-week-old animals in which TPA was absent. The degree of increment in body weight was very similar in these inbred strains. Thymus involution in C3H/He mice was the earliest signal among these mice. This result may suggest that the period of differentiation and maturation of T cells in a first lymphoid tissue for the growth process differs in these three inbred strains. This study provides an interesting example of genetic control of maturation or proliferation of peripheral T cells.
  Biological & Pharmaceutical Bulletin 03/2002; 25(2):172-8. · 1.66 Impact Factor
• Source

  Available from: pharm.or.jp

  Article: … and histamine on the expression of thymus-and activation-regulated chemokine mRNA in peripheral blood mononuclear cells and human bronchial epithelial cells

  Takaharu Negoro, Keiko Takekawa, Kazue Satoh, Yasuko Nakano, Keita Kasahara, Tetsuji Ozawa, Yoji Iikura, Mitsuru Adachi, Takashi Tobe
  [show abstract] [hide abstract]
  ABSTRACT: We hypothesized that participation of sensory neuropeptides, substance P and neurokinin A in the airway in-flammation caused to resistance against the glucocorticoids (GCs) treatment. We have measured the expression level of cytokines and chemokines mRNAs in bronchoalveolar lavage cells (BAL), transbronchial lung biopsies (TBLB) and peripheral blood mononuclear cells (PBMC) by reverse transcriptase polymerase chain reaction (RT-PCR). The mRNAs of interleukin-4 (IL-4) and -5, which are typical Thelper 2 (Th2)-type cytokines, were scarcely detected in all of bronchoalvelar lavage fluid (BALF) and TBLB samples. But the mRNA of Th1-type cytokines, such as IL-12, IL-18, and interferon-γ (IFN-γ), tended to be expressed higher than that of Th2-type cytokines at the early stage of asthma before intervention with GCs. The mRNAs of IL-18, IFN-γ, macrophage-derived chemokine (MDC) and thymus-and activation-regulated chemokine (TARC) tended to be expressed higher than that of non-asthmatic subjects. In vitro study demonstrated that the expression of TARC mRNA induced by the combination of IL-4, IFN-γ, and neurokinin A in human bronchial epithelial cells, BEAS-2B was not inhibited by the treatment of the cells with dexamethasone even at 10 –6 M. We proposed that the part of resistance to GCs in the asthmatic patient without GC receptor β expression might be associated with the induction of TARC expression in airway epithelial cells by tachykinines and histamine.
  Journal of Health Science. 01/2002; 48:534-544.
• Article: Partial purification of cytotoxic substances from moxa extract.

  Ikusuke Hatsukari, Naoko Hitosugi, Rie Ohno, Shinichi Nakamura, Satoshi Mizukami, Hiroshi Nagasaka, Isao Matsumoto, Hirotaka Kikuchi, Hirofumi Nishikawa, Masaru Niitsu, Masami Kawase, Takaharu Negoro, Kazue Satoh, Hideki Nakashima, Hiroshi Sakagami
  [show abstract] [hide abstract]
  ABSTRACT: The major cytotoxic activity of Moxa was extracted with CH2Cl2 and partially purified by three cycles of silica gel column chromatography. The active fractions showed higher cytotoxicity against six human tumor cell lines (two oral squamous cell carcinoma, one salivary gland tumor, one melanoma, two leukemia) than three normal oral human cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell). All fractions failed to protect the cells from the cytopathic effect induced by HIV infection. ESR spectroscopy showed that all fractions produced little or no radical under alkaline conditions, while showing much lower O2- scavenging activity, generated by hypoxanthine-xanthine oxidase reaction, than antioxidants and polyphenols. Active fractions induced DNA fragmentation in HL-60 cells, but failed to modify the mobility and activity of mitochondrial Mn-containing superoxide dismutase (MnSOD), in contrast to Moxa smoke. These data suggest that the active principles in the Moxa extract might be different from that in Moxa smoke, which produced carbon radical and modified MnSOD mobility and activity.
  Anticancer research 22(5):2777-82. · 1.73 Impact Factor
• Article: Inhibition of NO production by activated macrophages by phenolcarboxylic acid monomers and polymers with radical scavenging activity.

  Takako Ogiwara, Kazue Satoh, Takaharu Negoro, Haruo Okayasu, Hiroshi Sakagami, Seiichiro Fujisawa
  [show abstract] [hide abstract]
  ABSTRACT: Phenolcarboxylic acids (caffeic acid, p-coumaric acid, ferulic acid) and their dehydrogenation polymer (DHP) were compared for their ability to inhibit the nitric oxide (NO) production by lipopolysaccharide (LPS)-activated mouse macrophage-like cells Raw 264.7 and to scavenge superoxide (O2-) (generated by hypoxanthine and xanthine oxidase reaction), hydroxyl radical (generated by Fenton reaction) and NO radical (generated by NOC-7), using ESR spectroscopy in vitro. All phenolcarboxylic acids effectively inhibited the NO production by activated Raw 264.7 cells. Among them, caffeic acid showed the highest cytotoxic activity, radical intensity and O2- scavenging activity, but the least NO scavenging activity. Caffeic acid also inhibited the NO production most effectively. Polymers of caffeic acid (DHP-CA) and p-coumaric acid (DHP-pCA) showed higher cytotoxicity, radical intensity and radical scavenging activity and more efficiently inhibited the NO production, as compared with the corresponding monomers. DHP-CA showed higher radical generation and O2- scavenging activity than DHP-pCA. The potent O2- scavenging activity of caffeic acid was probably due to the chemical reaction of O2- to the cathecol groups. Caffeic acid, DHP-CA and DHP-pCA induced the cytotoxicity, possibly due to autogenerating radicals, because these compounds efficiently produced radicals under alkaline conditions. In summary, caffeic acid acted as a polyphenolics in phenylcarboxylic acids. A possible link between cytotoxicity and radical generation of phenylcarboxylic acids is proposed.
  Anticancer research 23(2B):1317-23. · 1.73 Impact Factor
• Article: Diverse biological activities of fermented pine seed shell extract.

  Saori Mihara, Senwa Unten, Hanzo Kakuta, Kazue Satoh, Takaharu Negoro, Hideki Nakashima, Nobuhiko Komatsu, Masato Fujimaki, Hirotaka Kikuchi, Hirofumi Nishikawa, Kiminobu Minagawa, Fumika Suzuki, Takao Satoh, Hiroshi Sakagami
  [show abstract] [hide abstract]
  ABSTRACT: Intraperitoneal administration of fermented pine seed shell extract (PSSE) (up to 2 g/kg) induced no apparent acute toxicity to mice. Pretreatment of mice with PSSE protected them from the lethality of Escherichia coli infection. PSSE showed a very weak cytotoxic activity against both normal and tumor cells and no anti-HIV activity, but stimulated the mouse macrophage-like Raw 264.7 cells to produce nitric oxide (NO) and citrulline. ESR spectroscopy showed that PSSE produced no detectable radicals, but effectively scavenged O2- (generated by the hypoxanthine-xanthine oxidase reaction), hydroxyl radical (generated by the Fenton reaction) and NO (generated by NOC-7). Comparison of PSSE with other natural products, such as polyphenols and vitamins, further confirmed the close association between radical intensity and radical scavenging activity, suggesting the bimodal action of natural products. Although the biological activities of PSSE were relatively lower than those of other natural products, the present study suggests the possible medicinal efficacy of PSSE.
  Anticancer research 22(3):1569-74. · 1.73 Impact Factor
• Article: Induction of cell death by pro-oxidant action of Moxa smoke.

  Naoko Hitosugi, Rie Ohno, Ikusuke Hatsukari, Shinichi Nakamura, Satoshi Mizukami, Hiroshi Nagasaka, Isao Matsumoto, Kazue Satoh, Takaharu Negoro, Ken Hashimoto, Hiroshi Sakagami
  [show abstract] [hide abstract]
  ABSTRACT: Moxa smoke induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells, but not in other cell lines. The cytotoxic activity of Moxa smoke was significantly reduced by a popular antioxidant, N-acetyl-L-cysteine (NAC). Moxa smoke showed oxidation potential (measured by NO monitor) and produced carbon radical (measured by ESR spectroscopy). The addition of NAC significantly reduced both the oxidation potential and carbon radical intensity of Moxa smoke. Activity staining of polyacryamide gel electrophoresis of MnSOD revealed the possible modification of the conformation and/or activity of this enzyme at an early stage of HL-60 cell death. These data suggest that Moxa smoke induces cytotoxicity by its pro-oxidant action.
  Anticancer research 22(1A):159-63. · 1.73 Impact Factor
• Article: Interaction between dental metals and antioxidants, assessed by cytotoxicity assay and ESR spectroscopy.

  Naoto Kinoshita, Takahiko Yamamura, Hiroshi Teranuma, Tadashi Katayama, Mikako Tamanyu, Takaharu Negoro, Kazue Satoh, Hiroshi Sakagami
  [show abstract] [hide abstract]
  ABSTRACT: Among dental metals, copper showed the highest cytotoxicity against human oral squamous cell carcinoma and human submandibular gland carcinoma cells, followed by palladium-alloy, gold and silver. Normal human cells (gingival fibroblast, pulp cells, periodontal ligament fibroblast) were relatively resistant to these metals. The palladium-alloy failed to induce internucleosomal DNA fragmentation, a biochemical hallmark of apoptosis, in human promyelocytic leukemic HL-60 cells. The cytotoxic activity of the palladium-alloy was significantly reduced by a non-cytotoxic concentration of N-acetyl-L-cysteine, or more efficiently by sodium ascorbate. However, higher concentrations of sodium ascorbate enhanced the cytotoxic activity of palladium-alloy. ESR spectroscopy showed that the palladium-alloy enhanced the intensity of ascorbate radical, suggesting the possible interaction between metals and antioxidants. All metals, except copper, did not significantly affect the generation of superoxide anion (by hypoxanthine-xanthine oxidase reaction), hydroxyl radical (by Fenton reaction) and nitric oxide (from NOC-7 in the presence of C-PTIO). These data demonstrate for the first time that antioxidants modify the biological activity of dental metals.
  Anticancer research 22(6C):4017-22. · 1.73 Impact Factor
• Article: Deficit of TRPM2 channels reduces myocardial ischemia-reperfusion injury in mice

  Toshihito Hiroi, Teruaki Wajima, Yuji Kaneko, Takaharu Negoro, Yuji Kiuchi, Yasuo Mori, Shunichi Shimizu
• Article: Involvement of TRPM2 channels in the early vascular permeability induced by acetic acid and zymosan

  Yuji Kaneko, Yuko Kakihana, Toshihito Hiroi, Takaharu Negoro, Disuke Kamei, Masakazu Ishii, Yuji Kiuchi, Yasuo Mori, Shunichi Shimizu