Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
37 巻 , 1 号
選択された号の論文の28件中1~28を表示しています
Communication to the Editor
Regular Articles
  • Jun Lee, Jin-Mu Yi, Haejin Kim, You Jin Lee, Jong-Shik Park, Ok-Sun Ba ...
    2014 年 37 巻 1 号 p. 6-12
    発行日: 2014/01/01
    公開日: 2014/01/01
    [早期公開] 公開日: 2013/10/29
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    Angiogenesis, the process of new vessel formation from the pre-existing blood vasculature, is critical for continuous tumor growth and is considered to be a validated antitumor target. The results of our previous study demonstrate the anti-angiogenic potential of an extract of Gleditsia sinensis thorns, which has been traditionally used in Korean medicine to remedy diverse diseases, including tumors. In the present study, we attempted to identify the active anti-angiogenic constituents of the ethanol extract of G. sinensis thorns (EEGS). By virtue of in vitro activity-guided fractionation using human umbilical vein endothelial cells (HUVEC) primary endothelial cells, chromatographic separation, and NMR spectral analyses, we isolated and identified the potent active constituent, cytochalasin H, a biologically active secondary metabolite of fungi. This unexpected active constituent may have originated from the endophytic fungi, Chaetomium globosum, which naturally populate G. sinensis, the identity of which was determined by analysis of fungal community. Cytochalasin H isolated from the EEGS showed in vitro anti-angiogenic activities such as suppressed cell growth and mobility in HUVEC, and inhibited the pro-angiogenic protein-induced formation of new blood vessels in vivo. The anti-angiogenic effect of cytochalasin H was in part due to reduced expression of pro-angiogenic factor, such as endothelin-1. This is the first report regarding the isolation and identification of cytochalasin H, as an active anti-angiogenic constituent of G. sinensis thorns.
  • Wutu Fan, Xianglong Wu, Yalei Pan, Chenrui Li, Yinbo Niu, Yuankun Zhai ...
    2014 年 37 巻 1 号 p. 13-17
    発行日: 2014/01/01
    公開日: 2014/01/01
    [早期公開] 公開日: 2013/10/19
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    The current study was designed to investigate the effects of 1-(1,3-benzodioxol-5-yl-carbonyl) piperidine (1-BCP) on swimming endurance capacity which as one indicator of fatigue in the weight-loaded forced swimming mice. Mice were given either vehicle or 1-BCP (0.1, or 0.2 mmol/kg body weight daily) by intraperitoneal injection once daily for 2 weeks. The 1-BCP groups showed a significant increase in swimming time to exhaustion compared with the control group. 1-BCP increased the liver glycogen (LG) and muscle glycogen (MG) contents significantly, while decreased the lactic acid (LA) and blood urea nitrogen (BUN) levels notably compared with control group. Besides, 1-BCP treatment also significantly improved the endogenous cellular antioxidant enzymes in mice by increasing the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). Therefore, this study demonstrated for the first time that the supplementation of 1-BCP, as a positive allosteric modulator of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, could enhance the endurance capacity of mice and facilitated them recovery from fatigue. Thus, we provide a new effective therapeutic strategy for fatigue.
  • Shimako Tanaka, Shinya Uchida, Naoki Inui, Kazuhiko Takeuchi, Hiroshi ...
    2014 年 37 巻 1 号 p. 18-25
    発行日: 2014/01/01
    公開日: 2014/01/01
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    A “cocktail” approach, which involves simultaneous administration of multiple CYP-specific probes, concurrently detects the activity of multiple CYP enzymes. We developed and validated a rapid and selective LC-MS/MS method for determining the plasma concentrations of 5 CYP probe drugs and metabolites (caffeine/paraxanthine, CYP1A2 substrate; losartan/losartan carboxylic acid (E3174), CYP2C9 substrate; omeprazole/5-hydroxyomeprazole, CYP2C19 substrate; dextromethorphan/dextrorphan, CYP2D6 substrate; and midazolam/1′-hydroxymidazolam, CYP3A4 substrate) by single-step extraction, followed by a single LC-MS/MS run. An Ostro™ 96-well plate was used for extraction of CYP substrates and metabolites from human plasma and urine. Following optimization of the chromatographic conditions, all the peaks were well separated, and retention times ranged between 4.4 and 11.7 min. The total run time for a single injection was within 13 min. The accuracy and precision values suggested that the assay had high accuracy and reliability in plasma and urine samples. No significant matrix interference was observed. To demonstrate the efficacy of this method, plasma and urine concentrations of 5 CYP probe substrates and their metabolites were determined after simultaneous oral administration of 5 drugs to 4 healthy volunteers. All the substrates and metabolites were detected over an 8 h period, and the plasma concentrations of each substrate at 8 h after administration were above the lower limit of quantification. Urine concentrations of drugs and their metabolic ratio were evaluated after the administration. In conclusion, the advantage of our cocktail approach is that it enables in vivo assessment of the activity of various drug-metabolizing enzymes in a single assay.
  • Hisako Machida, Masafumi Seki, Nori Yoshioka, Kaori Yabuno, Koji Miyaw ...
    2014 年 37 巻 1 号 p. 26-30
    発行日: 2014/01/01
    公開日: 2014/01/01
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    An outbreak of Multi-Drug Resistance Pseudomonas aeruginosa (MDRP) infections occurred in intensive care unit (ICU) and emergency room (ER) between June and August 2007. Five patients who isolated MDRP in the outbreak of 2007 were all used bronchoscopes, thus, we suspected contamination of the bronchoscopes as the cause of outbreak. Although we did not detect MDRP from any bronchoscopes, the outbreak finally ended after all the bronchoscopes had been disinfected appropriately with the reexamination of washing process in 2008 and 2009. We retrospectively reviewed eleven patients who isolated MDRP in 2006 and 2007, and the fact was revealed that bronchoscopes were used in most patients in ICU and ER. Bronchoscopes were significantly used during 2006–2007 period, compared with 2008–2009 period in ICU and ER, and the case-control analysis among all Pseudomonas aeruginosa isolated patients identified that bronchoscopes [risk ratio (RR) 8.25, 95% confidence interval (CI) 1.328–51.26] was one of the most important risk factors for MDRP isolation. Duration from admission to MDRP isolation was significant longer in MDRP-isolated cases (19.82±12.77 d), compared with in non MDRP-isolated controls (11.76±11.69 d: p=0.0453). Our epidemiological analysis suggested the significant risk factors for an MDRP outbreak, and could contribute the estimation of the focus and prevention of future outbreaks.
  • Mikio Takeuchi, Tatsuya Niimi, Mari Masumoto, Masaya Orita, Hiroyuki Y ...
    2014 年 37 巻 1 号 p. 31-36
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC50 of 44 nM, and inhibited THP-1 and K562 cell line growth with the IC50 of 198 nM and 673 nM, respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT.
  • Chun-xiang Zhu, Lan Yan, Xiao-juan Wang, Qi Miao, Xing-xing Li, Feng Y ...
    2014 年 37 巻 1 号 p. 37-43
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Zorro2 is a member of a non-long terminal repeat (LTR) retrotransposon family in Candida albicans, but as yet no clear evidence has been provided to establish either transcription or transposition activity for Zorro2. In this study, the relative expression changes of two open reading frames in Zorro2, ORF19.7274 and ORF19.7275, were examined in response to miconazole (MCZ), and were found to be increased by this treatment. As well, the copy number and the transcripts of Zorro2 in MCZ-induced resistant daughter strains were increased compared to the parental strain, indicating that transposition of Zorro2 occurred during long-term MCZ treatment. Intriguingly, the transcription activity of Zorro2 retrotransposons was significantly inhibited when the cells were treated with MCZ together with antioxidant N-acetyl-L-cysteine (NAC). As both the level of intracellular reactive oxygen species (ROS) and the expression of genes involving DNA repair activated by MCZ were reduced when combined with the treatment of NAC, we propose that the damage caused by accumulation of ROS under MCZ stress is a major reason for the transcription and transposition activation of the Zorro2 retrotransposon.
  • Jae-Suk Choi, Min-Hee Jeon, Woi-Sook Moon, Jin-Nam Moon, Eun Jin Cheon ...
    2014 年 37 巻 1 号 p. 44-53
    発行日: 2014/01/01
    公開日: 2014/01/01
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    The potential hair growth-promoting activity of rice bran supercritical CO2 extract (RB-SCE) and major components of RB-SCE, linoleic acid, policosanol, γ-oryzanol, and γ-tocotrienol, were evaluated with the histological morphology and mRNA expression levels of cell growth factors using real-time reverse transcriptase-polymerase chain reaction (PCR) in C57BL/6 mice. RB-SCE showed hair growth-promoting potential to a similar extent as 3% minoxidil, showing that the hair follicles were induced to be in the anagen stage. The numbers of the hair follicles were significantly increased. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), and keratinocyte growth factor (KGF) were also significantly increased and that of transforming growth factor-β (TGF-β) decreased in RB-SCE-treated groups. Among the major components of RB-SCE, linoleic acid and γ-oryzanol induced the formation of hair follicles according to examination of histological morphology and mRNA expression levels of cell growth factors. In conclusion, our results demonstrate that RB-SCE, particularly linoleic acid and γ-oryzanol, promotes hair growth and suggests RB-SCE can be applied as hair loss treatment.
  • Masaki Okawara, Yoshihiro Tokudome, Hiroaki Todo, Kenji Sugibayashi, F ...
    2014 年 37 巻 1 号 p. 54-59
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Orally administrated diosgenin, a steroidal saponin found in the roots of Dioscorea villosa, improves reduced skin thickness in ovariectomized mice, and plays an important role in the treatment of hyperlipidemia. Diosgenin has been noticed as an active element in cosmeceutical and dietary supplements. We have already elucidated that the absolute oral bioavailability of diosgenin is very low; however, a high skin distribution of diosgenin was also observed. The aim of the present study was to examine and compare the effects of β-cyclodextrin (β-CD) and 3 kinds of its derivatives such as hydroxypropyl β-CD on the diosgenin permeability using a Caco-2 model and rat jejunal perfusion. These derivatives of β-CD greatly improved the low solubility of diosgenin. No significant increase was observed in the lactate dehydrogenase leakage from Caco-2 cell, while a slight decrease was found on the transepithelial electrical resistance by diosgenin and β-CD derivatives. However, β-CD derivatives, especially hydroxyethyl β-CD and hydroxypropyl β-CD, markedly enhanced diosgenin permeability across the Caco-2 monolayer and rat jejunum. The bioavailability of diosgenin in the presence of β-CD derivatives were about 4 to 11 fold higher than diosgenin suspension. The mechanisms of these enhancement effects may be due to improvements in solubility and tight junction opening.
  • Anna Rita Togna, Omidreza Firuzi, Valentina Latina, Virinder Singh Par ...
    2014 年 37 巻 1 号 p. 60-66
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Inflammation contributes to the pathogenesis of neurodegenerative diseases and anti-inflammatory compounds may have a role in prevention or treatment of these pathologies. 4-Methylcoumarins are effective antioxidants with anti-inflammatory properties. In this study, the inhibitory effects of two 4-methylcoumarin derivatives, 7,8-dihydroxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DHEMC) and 7,8-diacetoxy-3-ethoxycarbonylmethyl-4-methylcoumarin (DAEMC) were examined on the inflammatory processes induced by lipopolysaccharide (LPS) in activated primary rat microglial cultures. LPS-induced production of nitric oxide (NO, measured by Griess method) and other pro-inflammatory mediators, thromboxane (TX) B2 and prostaglandin (PG) E2 (both determined by radioimmunoassay (RIA)), as well as tumor necrosis factor (TNF)-α (determined by enzyme-linked immunosorbent assay (ELISA)) were inhibited in the presence of 100 µM DHEMC and DAEMC. DAEMC was able to significantly inhibit NO, TXB2 and TNF-α production also at 50 µM. Both compounds at 100 µM significantly lowered cyclooxygenase-2 (COX-2) protein expression in LPS-stimulated microglial cells measured by Western blot, but only DAEMC showed an inhibitory effect on inducible nitric oxide synthase (iNOS) protein expression at 100 µM. In conclusion, our findings show that 4-methylcoumarin derivatives can modulate inflammatory pathways in microglial cells, probably by acting at the protein expression level.
  • Mika Ikehata, Mao Ogawa, Yasuki Yamada, Shota Tanaka, Kumiko Ueda, Sei ...
    2014 年 37 巻 1 号 p. 67-73
    発行日: 2014/01/01
    公開日: 2014/01/01
    [早期公開] 公開日: 2013/10/29
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    We investigated the effects of epigenetic modifiers such as DNA methyltransferase (DNMT) or histone deacetylase (HDAC) inhibitors on the cytotoxicity induced by 3 anticancer drugs (5-fluorouracil (5-FU), irinotecan (CPT-11) or its active form SN38, and oxaliplatin (L-OHP)) in human colorectal cancer (CRC) cells. Cytotoxicity in 4 CRC cell lines (HT29, SW480, SW48 and HCT116) was examined by colorimetric assay after drug treatment for 72 h. The effects of drug combinations were analyzed by an isobologram method. SW480 cells showed the lowest sensitivity to cytotoxicity induced by the anticancer drugs among the 4 CRC cell lines. In SW480 cells, DNMT inhibitors, such as decitabine (DAC), azacytidine and zebularine (Zeb), showed synergic effects on the cytotoxicity induced by anticancer drugs except for SN-38 plus Zeb, while HDAC inhibitors, trichostatin A, suberoylanilide hydroxamic acid and valproic acid, showed antagonistic effects. DAC showed the most potent synergic effects among the epigenetic modifiers studied. Thus, we examined whether the synergic effect of DAC is observed in other different CRC cell lines, HT29, SW48 and HCT116 cells. In all 4 CRC cell lines, the cytotoxicity of L-OHP was enhanced in a synergic manner by co-treatment with DAC. However, synergic effects of DAC with 5-FU or CPT-11 (SN-38) were not observed in 4 CRC cell lines.
  • Takaaki Kitajima, Masashi Muroi, Naomi Yamashita, Ken-ichi Tanamoto
    2014 年 37 巻 1 号 p. 74-80
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Body and excrement extracts from Dermatophagoides farinae were used to study stimulation of Toll-like receptors (TLRs). The excrement extract stimulated nuclear factor (NF)-κB-dependent reporter activity to an extent similar to lipopolysaccharide (LPS) in a mouse macrophage cell line, J774A.1, but the activity of the body extract was negligible. The excrement extract also activated NF-κB in HEK293 cells expressing TLR1/TLR2, TLR2/TLR6 and CD14/TLR4/MD-2, whereas no activation was observed in cells expressing TLR3, TLR5, TLR7, TLR8 or TLR9. Although the excrement extract required co-expression of CD14, TLR4 and MD-2 in HEK293 cells to activate NF-κB, efficient activation was still observed in I-13.35 cells, a bone-marrow macrophage cell line established from LPS-hypo-responsive C3H/HeJ mice. The excrement extract activated NF-κB in HEK293 cells expressing TLR2 alone, but the activation was significantly increased by co-expression of CD14. Polymyxin B inhibited CD14/TLR4/MD-2- and CD14/TLR2-mediated activation of NF-κB but not the activation in I-13.35 cells. These results indicate that CD14/TLR4/MD-2-dependent and CD14/TLR2-dependent mechanisms are involved in the activation of NF-κB by the excrement extract of D. farinae and suggest that the extract also contains substances that activate NF-κB through non-TLR-mediated mechanisms.
  • Azusa Taura, Kazuyuki Furuta, Tomoko Yamaguchi, Kenji Kawabata, Satosh ...
    2014 年 37 巻 1 号 p. 81-86
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Mast cells are involved in various immunological responses, although it remains unknown how their terminal differentiation is regulated. We previously established a culture model that mimics the process of mast cell maturation in the cutaneous tissue and found that growth factor independent 1 (Gfi1) was up-regulated whereas its paralogue Gfi1b down-regulated. Here we investigated the roles of Gfi1 and Gfi1b in the process of mast cell maturation using a murine mast cell line, MC9. Gfi1 and Gfi1b cDNAs were stably expressed in MC9 cells using the recombinant lentivirus. Histamine synthesis was significantly induced by stem cell factor (SCF) alone, whereas tryptase expression was significantly augmented in the presence of both SCF and Swiss 3T3 cells. Since exogenously expressed Gfi1 and Gfi1b might affect their expression levels in MC9 cells, we investigated the relationship between the expression profiles of Gfi1/Gfi1b proteins and maturation indices, such as histamine synthesis and tryptase expression. The comparison suggested that histamine synthesis during the co-culture period was positively regulated by Gfi1b while augmented expression of tryptase was abolished by one-sided expression of Gfi1/Gfi1b. Our findings indicated the involvement of Gfi1 and Gfi1b in the process of murine mast cell maturation.
  • Shinji Tanaka, Tadashi Toki, Mika Yokoyama, Hiroki Shimizu, Tomonori Y ...
    2014 年 37 巻 1 号 p. 87-95
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Inhibitor of kappaB (IκB) kinase beta (IKKβ) plays a critical role in nuclear factor-kappaB (NF-κB) activation and production of proinflammatory cytokines in various inflammatory diseases including rheumatoid arthritis. We previously reported a novel IKKβ inhibitor Compound D, 4-[6-(cyclobutylamino)imidazo[1,2-b]pyridazin-3-yl]-2-fluoro-N-{[(2S,4R)-4-fluoropyrrolidin-2-yl]methyl}benzamide, which is efficacious in experimental arthritis models. In the present study, we characterized the pharmacological properties of Compound D and investigated the mechanisms of the anti-arthritic effect. Compound D inhibited IKKβ kinase activity with 160-fold selectivity against IKKα. The cellular analyses revealed that Compound D selectively blocked NF-κB promoter activity among major cellular signaling pathways, such as the activator protein-1 pathway, consistent with inhibition of the NF-κB signaling pathway including phosphorylation of IκBα. In addition, Compound D inhibited NF-κB-driven production of tumor necrosis factor alpha (TNFα) and interleukin-6 comparably. The correlation between inhibitory effect on TNFα production and plasma concentration of the compound was observed in vivo. Consecutive administration of Compound D decreased gene expression of proinflammatory cytokines and inflammatory mediators in the paws of arthritic mice with attenuation of paw swelling. Notably, Compound D was rapidly distributed to the arthritic paws, rather than healthy paws, and where it decreased the gene expression of proinflammatory cytokines by a single oral administration. Furthermore, Compound D completely inhibited arthritis progression even when treatment occurred after disease development. These data suggest that the downregulation of proinflammatory cytokines in local inflamed joints is one of the mechanisms underlying the anti-arthritic effect of the IKKβ inhibitor, Compound D.
  • Noriaki Nagai, Yoshimasa Ito
    2014 年 37 巻 1 号 p. 96-104
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Tranilast (TL), an antiallergic agent, has been clinically used in the treatment of bronchial asthma, although its clinical use has been limited by its poor solubility in water, photodegradation and systemic side effects. In this study, we prepared a gel ointment containing TL nanoparticles (TLnano gel ointment), and investigated its usefulness. In addition, we demonstrated the preventive effects of the TLnano gel ointment on inflammation in adjuvant-induced arthritis (AA) rats. The TLnano gel ointment was prepared using Bead Smash 12 (a bead mill) and additives including sodium docusate, 2-hydroxypropyl-β-cyclodextrin, methylcellulose and Carbopol 934; the mean particle diameter of the TL nanoparticles was 71.0±25.4 nm. In in vitro skin penetration experiments, the amount of penetrated TL, the penetration rate (Jc) and the penetration coefficient through the skin (Kp) of the TLnano gel ointment were significantly higher than those of a gel ointment containing TL microparticles (TLmicro gel ointment; particle diameter 50.5±26.3 µm). The TL concentrations in the skin tissue and plasma of rats receiving the TLnano gel ointment were also higher than in rats receiving the TLmicro gel ointment. In addition, the application of the TLnano gel ointment attenuated the increase in paw edema of the hind feet of AA rats in comparison with AA rats treated with the TLmicro gel ointment. These results suggest that TL nanoparticles can be applied to the formulation of a transdermal system, and that a transdermal formulation using TL nanoparticles might be a delivery option for the clinical treatment of RA.
  • Minako Karahashi, Hiroto Fukuhara, Miki Hoshina, Takeshi Sakamoto, Toh ...
    2014 年 37 巻 1 号 p. 105-112
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Fibrates are used in biochemical and pharmacological studies as bioactive tools. Nevertheless, most studies have lacked information concerning the concentrations of fibric acids working inside tissues because a simple and sensitive method is not available for their quantitation. This study aimed to develop a simple and sensitive bioanalytical method for the quantitation of clofibric, bezafibric and fenofibric acids in samples of very small portions of tissues. Fibric acids were extracted into n-hexane–ethyl acetate from tissue homogenates (10 mg of liver, kidney or muscle) or serum (100 µL) and were derivatized with 4-bromomethyl-6,7-dimethoxycoumarin, followed by HPLC with fluorescence detection. These compounds were separated isocratically on a reversed phase with acetonitrile–water. Standard analytical curves were linear over the concentration range of 0.2–20 nmol/10 mg of liver. Precision and accuracy were within acceptable limits. Recovery from liver homogenates ranged from 93.03 to 112.29%. This method enabled the quantitation of fibric acids in 10 mg of liver from rats treated with clofibric acid, bezafibric acid or fenofibrate. From these analytical data, it became clear that there was no large difference in ratio of acyl-CoA oxidase 1 (Acox1) mRNA level to fibric acid content in the liver among the three fibric acids, suggesting that these three fibric acids have similar potency to increase expression of the Acox1 gene, which is a target of peroxisome proliferator-activated receptor α. Thus, the proposed method is a simple, sensitive and reliable tool for the quantitation of fibric acids working in vivo inside livers.
  • Zhao-Bo Li, Chang-Fu Li, Jia Li, Yan-Sheng Zhang
    2014 年 37 巻 1 号 p. 113-122
    発行日: 2014/01/01
    公開日: 2014/01/01
    [早期公開] 公開日: 2013/10/19
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    As part of the efforts to understand isoflavonoid metabolism in Pueraria lobata at the molecular level, the cDNAs encoding two divergent 4-coumarate : coenzyme A ligases (4CLs, designated Pl4CL1 and Pl4CL2, respectively) were isolated from P. lobata roots. Sequence analysis revealed that Pl4CL1 had an N-terminal extension of twenty-one amino acid residues compared to Pl4CL2. Phylogenetic analysis showed that Pl4CL1 and Pl4CL2 fell into angiosperm Class II and Class I, respectively. Through in vitro biochemical assays, both Pl4CLs were found to have the capacity to utilize 4-coumarate and trans-cinnamate as substrates, while neither of them could convert sinapate. Pl4CL2 had a broader substrate specificity than Pl4CL1. The affinity of Pl4CL1 for 4-coumarate was 2.6-fold higher than that of Pl4CL2 (with the Km values of 3.5 µM and 9.1 µM, respectively). Combining the dataset including gene expression profiles, metabolites measurements, and biochemical properties, our results indicated that Pl4CL1, just as other angiosperm Class II 4CLs, might play a role in isoflavone biosynthesis in P. lobata, while Pl4CL2 belongs to angiosperm Class I, and may function as a housekeeping enzyme concerning lignification.
  • Naoki Sato, Genichiro Seo, Yoshimi Benno
    2014 年 37 巻 1 号 p. 123-129
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×108 colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 104.8 and 105.8 cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.
  • Yoichi Nishiya, Tsuyoshi Yamagata, Ayumu Fukuda, Sachiko Yokokawa, Tak ...
    2014 年 37 巻 1 号 p. 130-136
    発行日: 2014/01/01
    公開日: 2014/01/01
    [早期公開] 公開日: 2013/10/26
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    KW-7158 is a novel therapeutic candidate for treating overactive bladder (OAB) with a unique mode of action: suppression of sensory afferent nerves. However, the molecular target of this compound remains unknown. We herein report the identification of the KW-7158 target to be equilibrative nucleoside transporter-1 (ENT1). A membrane protein expression library of ca. 7000 genes was expressed in a dorsal root ganglion cell line, which we had previously generated, and subjected to screening for binding with a fluorescent derivative that retains high binding activity to the target. The screening revealed that only cells transfected with an ENT1 expression vector exhibited significant binding. We next performed [3H]KW-7158 binding experiments and an adenosine influx assay and found that KW-7158 binds to and inhibits ENT1. To further demonstrate the pharmacological relevance, we evaluated other known ENT1 inhibitors (nitrobenzylthioinosine, dipyridamole, draflazine) in an in vitro bladder strip contraction assay and the rat spinal cord injury OAB model. We found that all of the inhibitors exhibited anti-OAB activities, of which the potencies were comparable to that of adenosine influx inhibition in vitro. These studies demonstrated that the pharmacological target of KW-7158 is ENT1, at least in the rat OAB model. Our results will aid understanding of the precise mechanism of action of this drug and may also shed new light on the use of the adenosine pathway for the treatment of OAB.
  • Yusuke Kono, Shigeru Kawakami, Yuriko Higuchi, Fumiyoshi Yamashita, Mi ...
    2014 年 37 巻 1 号 p. 137-144
    発行日: 2014/01/01
    公開日: 2014/01/01
    [早期公開] 公開日: 2013/10/19
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    Tumor-associated macrophages (TAMs) have an alternatively activated macrophage phenotype (M2) and promote cancer cell proliferation, angiogenesis and metastasis. Nuclear factor-kappaB (NF-κB) is one of the master regulators of macrophage polarization. Here, we investigated the effect of inhibition of NF-κB activity by small interfering RNA (siRNA) on the pro-tumor response of macrophages located in the tumor microenvironment in vitro. We used mouse peritoneal macrophages cultured in conditioned medium from colon-26 cancer cells as an in vitro TAM model (M2-like macrophages). Transfection of NF-κB (p50) siRNA into M2-like macrophages resulted in a significant decrease in the secretion of interleukin (IL)-10 (a T helper 2 (Th2) cytokine) and a significant increase of T helper 1 (Th1) cytokine production (IL-12, tumor necrosis factor-α, and IL-6). Furthermore, vascular endothelial growth factor production and matrix metalloproteinase-9 mRNA expression in M2-like macrophages were suppressed by inhibition of NF-κB expression with NF-κB (p50) siRNA. In addition, there was a reduction of arginase mRNA expression and increase in nitric oxide production. The cytokine secretion profiles of macrophages cultured in conditioned medium from either B16BL6 or PAN-02 cancer cells were also converted from M2 to classically activated (M1) macrophages by transfection of NF-κB (p50) siRNA. These results suggest that inhibition of NF-κB activity in M2-like macrophages alters their phenotype toward M1.
  • Dae-Hyun Roh, Seo-Yeon Yoon
    2014 年 37 巻 1 号 p. 145-151
    発行日: 2014/01/01
    公開日: 2014/01/01
    [早期公開] 公開日: 2013/10/24
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    Sigma-1 receptors (Sig-1Rs) play a role in different types of pain and in central sensitization mechanism in spinal cord. However, it is currently unexplored whether Sig-1Rs are involved in orofacial pain processing. Here we show whether a selective Sig-1R antagonist, BD1047 reduces nociceptive responses in the mouse orofacial formalin model and the number of Fos-immunoreactive (ir) cells in the trigeminal nucleus caudalis (TNC). In addition, it was examined whether the phosphorylation of extracellular signal-regulated kinase (pERK) or p38 (pp38) mitogen-activated protein kinases (MAPK), which are closely linked to pain signaling and sensitization, in TNC was modified by BD1047. The 5% formalin (10 µL) was subcutaneously injected into the right upper lip, and the rubbing responses with ipsilateral fore- or hind paw were counted for 45 min. BD1047 (1, 3 or 10 mg/kg) were intraperitoneally treated 30 min before formalin injection. High dose of BD1047 (10 mg/kg) produced significant anti-nociceptive effects in the first and the second phase. The number of Fos-ir cells in ipsilateral side of TNC was also reduced by BD1047 as compared to that in saline-treated animals. In addition, the number of pp38-ir cells in ipsilateral TNC was decreased in BD1047-treated animals, whereas the number of pERK-ir cells was not modified. Collectively, these results demonstrate that Sig-1Rs play a pivotal role in the orofacial pain processing, and the pp38 signaling pathway can be associated with Sig-1R’s action in TNC.
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  • Mina Lee, Seung Hyun Kim, Hee Kyoung Lee, Yekyung Cho, Jimmy Kang, San ...
    2014 年 37 巻 1 号 p. 152-157
    発行日: 2014/01/01
    公開日: 2014/01/01
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    The extract of Siegesbeckia pubescens herb and its chemical constituents were tested for the ability to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia. The methanol extract and the 90% MeOH fraction of S. pubescens effectively attenuated lipopolysaccharide-induced nitric oxide production. Several steps of chromatography yielded eight ent-kaurane diterpenes (18) and one ent-pimarane diterpene (9) from the 90% MeOH fraction. Among these compounds, compounds 29 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglia. Compounds 3 and 9 concentration-dependently decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), supported by quantitative real time polymerase chain reaction (PCR) and Western blot analysis. These results suggest that ent-kaurane and ent-pimarane diterpenes isolated from S. pubescens are expected to be potential candidates against neuroinflammation-related disease.
  • Jin-Ju Kim, Sovannarith Korm, Won-Seok Kim, Ok-Seon Kim, Ji-Seon Lee, ...
    2014 年 37 巻 1 号 p. 158-163
    発行日: 2014/01/01
    公開日: 2014/01/01
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    We aimed to identify a novel flavonoid from the in-house natural products to suppress matrix metalloproteases (MMPs), which is responsible for degradation of collagen and other extracellular matrix proteins. Total eight natural products were screened for identification of a novel MMP-9 suppressor using MMP-9 reporter system, where the prompt initial screening with multiple samples is readily examined. Among the extracts used in the present study, one extract (Citrus unshiu) was found active in this assay system. Furthermore, three representative flavonoids in this active extract of Citrus unshiu peel were tested in MMP-9 reporter system. Nobiletin (NB) of the tested flavonoids suppressed MMP-9 expression without cytotoxicity, which was validated by both real-time polymerase chain reaction (PCR) and zymography analyses. Sustained p38 mitogen activated protein kinase (MAPK) activity, closely associated with induction of MMP-9 under stress condition, was markedly reduced by NB treatment, which implies that modulation of p38MAPK by nobiletin is responsible for reduction of MMP9 expression. Hence, nobiletin, identified from MMP-9 reporter system based screening, may be further applied for the purpose of delaying collagen degradation in skin fibroblasts.
  • Kimie Sai, Kouichi Kurose, Tomoko Koizumi, Noriko Katori, Jun-ichi Saw ...
    2014 年 37 巻 1 号 p. 164-168
    発行日: 2014/01/01
    公開日: 2014/01/01
    ジャーナル フリー HTML
    Human orosomucoid (ORM) is a major acute-phase plasma protein, encoded by 2 highly homologous genes, ORM1 and ORM2. Human ORM induction is assumed to be regulated by each proximal promoter region, where putative glucocorticoid responsive elements and CCAAT/enhancer binding protein (C/EBP)β binding sites are located. However, the details of the differential regulation of these genes remain unknown. To explore this, we assessed the role of the distal promoter region of each ORM in HeLa cells. Luciferase-reporter activities of full constructs, containing approximately 1.1 kbp (FULL), and those of deletion constructs, containing up to 188 bp region (DEL) upstream of the transcription start sites of ORM1 and ORM2 were compared under both basal and inducer-treated conditions. For ORM1 and ORM2 DEL constructs, significantly increased activities after dexamethasone (DEX) treatments (alone and combined with interleukin (IL)-1β) were observed. Significantly higher FULL construct activities than DEL construct activities were observed for ORM1 after IL-1β treatment, while those for ORM2 were significantly lower at basal level and after DEX treatments. Upon C/EBPβ overexpression, FULL construct activities were significantly higher than those of DEL constructs at basal level and after IL-1β treatment for ORM1, and at basal level and after inducer-treatments for ORM2. Higher transcription-induction activity in the distal promoter region was evident for ORM1 in the absence of C/EBPβ overexpression, and for ORM2 under C/EBPβ overexpression conditions. These findings suggest that the ORM distal promoter region differentially regulates expression of ORM genes at basal level and in acute phase responses.
  • Shinya Masuda, Tomohiro Tanaka, Hiroaki Masuzaki, Kazuwa Nakao, Sadayo ...
    2014 年 37 巻 1 号 p. 169-173
    発行日: 2014/01/01
    公開日: 2014/01/01
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    Increased spontaneous locomotive activity and oxygen consumption have been reported in transgenic mice overexpressing leptin in the liver. In the present study, we examined whether the overexpression of leptin altered glycolytic and oxidative metabolic enzymatic activities as well as the composition of myosin heavy chain (MHC) isoforms in skeletal muscle. Enzymatic activities of lactate dehydrogenase (LDH) and citrate synthase (CS) were quantified in gastrocnemius muscle (GAS) and the red portion of tibialis anterior muscle (TA) from leptin transgenic (Tg) mice and non-Tg mice. The composition of MHC isoforms was measured in soleus muscle (SOL) and extensor digitorum longus muscle (EDL) from the two groups. In red TA, LDH-to-CS ratio was significantly lower in Tg than in non-Tg (p=0.014), whereas no significant change was observed in GAS. The composition of MHC isoforms was not significantly different in SOL or EDL between Tg and non-Tg groups. Our data indicate that chronic overexpression of leptin reduces the ratio of glycolytic to oxidative capacity without changing muscle fiber types particularly in red muscles. This metabolic change may contribute to the increased spontaneous locomotive activity and oxygen consumption in Tg mice reported previously.
  • Nobuhito Hamano, Yoichi Negishi, Kyohei Takatori, Yoko Endo-Takahashi, ...
    2014 年 37 巻 1 号 p. 174-177
    発行日: 2014/01/01
    公開日: 2014/01/01
    ジャーナル フリー HTML
    Ultrasound (US) is used in the clinical setting not only for diagnosis but also for therapy. As a therapeutic US technique, high-intensity focused ultrasound (HIFU) can be applied to treat cancer in a clinical setting. Microbubbles increased temperature and improved the low therapeutic efficiency under HIFU; however, microbubbles have room for improvement in size, stability, and targeting ability. To solve these issues, we reported that “Bubble liposomes” (BLs) containing the US imaging gas (perfluoropropane gas) liposomes were suitable for ultrasound imaging and gene delivery. In this study, we examined whether BLs and HIFU could enhance the ablation area of the tumor and the antitumor effect. First, we histologically analyzed the tumor after BLs and HIFU. The ablation area of the treatment of BLs and HIFU was broader than that of HIFU alone. Next, we monitored the temperature of the tumor, and examined the antitumor effect. The temperature increase with BLs and HIFU treatment was faster and higher than that with HIFU alone. Moreover, treatment with BLs and HIFU enhanced the antitumor effect, which was better than with HIFU alone. Thus, the combination of BLs and HIFU could be efficacious for cancer therapy.
  • Kwan Hee Park, Mi Sook Jeong, Kwang Jun Park, Young Wook Choi, Seong J ...
    2014 年 37 巻 1 号 p. 178-183
    発行日: 2014/01/01
    公開日: 2014/01/01
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    The roots of Rosa multiflora THUNB. (RM) has been used in oriental traditional medicines as remedies for scabies, rheumatic arthralgia and stomatitis which were practicably related with today’s inflammatory and allergic diseases. In the present study, we evaluated whether RM root extract (RME) and its major constituent, 2-(3,4-dihydroxyphenyl)-6-(4-hydroxyphenyl)-8-(2,4-dihydroxyphenyl)-2,3-trans-6,7-cis-7,8-trans-3,4,7,8-tetrahydro-2H,6H-pyrano[2,3-f] chromene-3,7,9-triol (RM-3) belongs to condensed tannins, improve atopic dermatitis (AD)-like skin lesions in NC/Nga mice induced by mite antigen. Topical application of RME as well as RM-3 improved skin severity and suppressed mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) on skin tissues, in addition, significantly reduced T helper 2 (Th2) immune responses via interleukin 10 (IL-10) up-regulation. Thus, RME, contains lots of condensed tannins such as RM-3 which possesses potent anti-inflammtory and immune-modulatory effects, may be useful for treatment of skin allergies and can be developed as new alternative herbal therapy against AD.
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