Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 36 , Issue 3
Showing 1-25 articles out of 25 articles from the selected issue
Regular Articles
  • Joonwoo Lee, Yi Jae Hong, Ji Seon Yang, Soomuk Cho, Sang June Hahn, Sh ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 331-338
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Various effects of acorn extract have been reported including antioxidant activity, cytotoxicity against cancer cells, and the levels of acetylcholine and its related enzyme activities in the dementia mouse models. However, it is unclear whether acorn extract inhibits glutamate-induced calcium signaling in hippocampal neurons. This study was an investigation into the effect of acorn extract on intracellular free Ca2+ concentrations ([Ca2+]i) in cultured rat hippocampal neurons using fura-2-based digital calcium imaging and photometry. Hippocampal neurons were used between 10 and 14 d in culture from embryonic day-18 rats. Treatment with acorn extract (1 µg/mL to 1 mg/mL) for 30 min inhibited glutamate (100 µM)-induced [Ca2+]i increases in a dose-dependent manner (IC50=46.9 µg/mL). After depletion of intracellular Ca2+ stores by treatment with the inhibitor endoplasmic reticulum Ca2+-ATPase, thapsigargin (1 µM), treatment with acorn extract (50 µg/mL) for 30 min decreased the subsequent glutamate-induced [Ca2+]i increases. Acorn extract significantly inhibited (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (30 µM)-induced [Ca2+]i increases. In addition, acorn extract inhibited the AMPA-induced [Ca2+]i responses in the presence of 1 µM nimodipine. Acorn extract also significantly inhibited N-methyl-D-aspartate (100 µM)-induced [Ca2+]i increases. Acorn extract significantly inhibited 50 mM KCl -induced [Ca2+]i increases. Acorn extract significantly inhibited (S)-3,5-dihydroxyphenylglycine-induced [Ca2+]i responses. Moreover, acorn extract almost completely blocked synaptically mediated [Ca2+]i spikes induced by decreasing extracellular Mg2+ concentration to 0.1 mM. These results suggest that acorn extract inhibits synaptically induced frequent [Ca2+]i spikes through multiple pathways such as ionotropic glutamate receptors, voltage-gated Ca2+ channels and metabotropic glutamate receptors in cultured rat hippocampal neurons.
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  • Chul-Hee Kim, Taehun Lee, Ikhoon Oh, Kung-Woo Nam, Kyeong Ho Kim, Ki-B ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 339-345
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Several isolated compounds from the wood part of Thujopsis dolabrata were evaluated for their inhibitory effects against antigen-induced mast cell degranulation and interleukin-4 (IL-4) secretion, as well as IL-4 mRNA and protein expression in immunoglobulin E (IgE)-sensitized RBL-2H3 cells. Among the five isolated compounds, (−)-elema-1,3,11(13)-trien-12-ol (1) and thujopsene (2) exhibited the potent inhibitory activity against mast cell degranulation measured by β-hexosaminidase release with IC50 values of 27.4 µM and 25.1 µM, respectively. These compounds also inhibited the release of IL-4 (IC50 values of 7.0, 6.7 µM, respectively), IL-4 mRNA expression (IC50 values of 16.5, 7.2 µM, respectively) and IL-4 protein expression (IC50 values of 17.0, 9.6 µM, respectively) in antigen-induced IgE-sensitized RBL-2H3 cells. These results suggested that (−)-elema-1,3,11(13)-trien-12-ol (1) and thujopsene (2) effectively inhibits mast cell degranulation as well as IL-4 production, suggesting that these compounds from Thujopsis dolabrata can be used as candidates for IgE-mediated allergic disorders.
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  • Joo Hyuck Lim, So-Hyun Park, Myoung Rae Kim, Byoung-Sam Yoo, Jae Chan ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 346-351
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 17, 2012
    JOURNALS FREE ACCESS
    The role of cyclohexane diester analogues in the formation of melanin has been recently reported. In the present study, we investigated the inhibitory effect of cyclohexanediol bis-ethylhexanoate (CHEH) on melanogenesis in B16 melanoma cells and on UV-B-induced pigmentation in human skin. CHEH significantly reduced the melanin content in a dose-dependent manner, without cytotoxic effects at the effective concentrations. Moreover, CHEH dose-dependently inhibited tyrosinase activity in B16 melanoma cells, as confirmed by Western blot analysis of the tyrosinase protein levels. However, tyrosinase transcript levels remained unchanged under the same experimental conditions. These results indicate that CHEH inhibited melanogenesis in B16 melanoma cells by regulating tyrosinase activity at the post-transcriptional level. On the other hand, in a cell-free system, CHEH did not inhibit tyrosinase activity. This indicated that CHEH suppressed the pigmentation of melanocytes by indirectly regulating tyrosinase activity. Finally, in a clinical trial, a cream containing 1.0% CHEH showed good whitening effect on UV-induced pigmented human skin without adverse effects. In conclusion, we suggest that CHEH may be an effective inhibitor of melanogenesis and useful effects in the treatment of hyperpigmented disorders.
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  • Kiichiro Teruya, Yuki Myojin-Maekawa, Fumio Shimamoto, Hiromitsu Watan ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 352-359
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 08, 2012
    JOURNALS FREE ACCESS
    Gastrointestinal damage associated with radiation therapy is currently an inevitable outcome. The protective effect of Kefir was assessed for its usefulness against radiation-induced gastrointestinal damage. A Kefir supernatant was diluted by 2- or 10-fold and administered for 1 week prior to 8 Gray (Gy) X-ray irradiation at a dose rate of 2 Gy/min, with an additional 15 d of administration post-irradiation. The survival rate of control mice with normal drinking water dropped to 70% on days 4 through 9 post-irradiation. On the other hand, 100% of mice in the 10- and 2-fold-diluted Kefir groups survived up to day 9 post-irradiation (p<0.05 and p<0.01, respectively). Examinations for crypt regeneration against 8, 10 and 12 Gy irradiation at a dose rate of 4 Gy/min revealed that the crypt number was significantly increased in the mice administered both diluted Kefir solutions (p<0.01 for each). Histological and immunohistochemical examinations revealed that the diluted Kefir solutions protected the crypts from radiation, and promoted crypt regeneration. In addition, lyophilized Kefir powder was found to significantly recover the testis weights (p<0.05), but had no effects on the body and spleen weights, after 8 Gy irradiation. These findings suggest that Kefir could be a promising candidate as a radiation-protective agent.
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  • Masanobu Suwa, Shigeharu Oie, Hiroyuki Furukawa
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 360-363
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Naturally occurring bacteria, is exist in nature, and is never cultivated on conventional culture medium. We evaluated the efficacy of disinfectants against naturally occurring bacteria in in-use cotton balls soaked in 0.02% benzalkonium chloride solution which had been used to disinfect the genital area by patients undergoing self-catheterization at home and the same bacteria subcultured on nutrient broth (artificially cultivated bacteria). The colony forming units (CFU) of naturally occurring bacteria such as Serratia marcescens, Alcaligenes xylosoxidans, and Burkholderia cepacia were not decreased after 48 h exposure to 0.025–0.1% benzalkonium chloride solution, but the same strains subcultured on nutrient broth were killed within only 10 min exposure to 0.025–0.1% benzalkonium chloride solution. In addition, the CFU of these three kinds of naturally occurring bacteria were not decreased after 48 h exposure to 0.02% chlorhexidine gluconate solution, but the same strains subcultured on nutrient broth were killed within 2 h exposure to chlorhexidine gluconate solution. The result showed that disinfectant efficacy differed markedly against naturally occurring and artificially cultivated bacteria. Therefore, it is preferable to use the naturally occurring bacteria not only artificially cultivated bacteria when examining disinfectant efficacy.
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  • Keisuke Kobayashi, Taichi Ohshiro, Daisuke Matsuda, Wei Jiang, Bin Hon ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 364-369
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    A cardiotonic glycoside, bufalin, originally isolated from the dried white venom of Chinese toad Bufo gargarizans, was found to inhibit lipid droplet accumulation in mouse macrophages. Bufalin selectively inhibited synthesis of [14C]cholesteryl ester (CE), a main component of lipid droplet, from [14C]oleic acid and [14C]cholesterol with IC50 values of 8.6 µM and 10 µM, respectively. The postlysosomal metabolism of cholesterol to CE in macrophages was also inhibited by the compound with a similar IC50 value of 13.2 µM. However, the compound exhibited almost no effect on acyl-CoA : cholesterol acyltransferase, a key enzyme in CE synthesis localized in the endoplasmic reticulum (ER). From the fluorescent microscopic observation of cellular lipids, bufalin-treated macrophages increased the accumulation of free cholesterol in lysosomes and caused to enlarge the shape and volume of lysosomes as well as pregnenolone-treated macrophages. These findings suggest that bufalin inhibited the postlysosomal metabolism of cholesterol, leading to a reduction of lipid droplets in mouse macrophages without cytotoxicity.
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  • Katsuhiro Isoda, Eriko Tetsuka, Yoshimi Shimizu, Kanae Saitoh, Isao Is ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 370-375
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 25, 2012
    JOURNALS FREE ACCESS
    Nano-size silica material is a promising reagent for disease diagnosis, cosmetics, and the food industry. For the successful application of nanoparticle materials in bioscience, evaluation of nano-size material toxicity is important. We previously found that nano-size silica particles caused acute liver failure in mice. However, the hepatotoxicity of nanosilica particles with the diameter of 70 nm or less is unknown. Here, we investigated the relationship between particle size and toxicity using nanosilica particles with diameters of 30, 50, and 70 nm (SP30, SP50, and SP70, respectively). We observed dose-dependent increases in hepatic injury following administration of SP50 and SP30, with SP30 causing greater acute liver injury than that seen with SP50. Smaller silica nanoparticles induced liver injury even at proportionally lower dose levels. Furthermore, we investigated the combinatorial toxicity of SP30 in the presence of chemically induced liver injury (including that caused by carbon tetrachloride, paraquat, cisplatin, and acetaminophen). We observed that particles of the smallest size tested (SP30) synergized with chemical substances in causing liver injury. These data suggest that the size (diameter) of the silica nanoparticles affects the severity of nanoparticle-induced liver injury, a finding that will be useful for future investigations in nanotechnology and nanotoxicology.
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  • Yan Lin, Zhihui Liu, Yongqiang Li, Xiaoli Liao, Sina Liao, Shaofang Ce ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 376-383
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 11, 2012
    JOURNALS FREE ACCESS
    As a physical adjuvant approach in the treatment of solid tumors, regional hyperthermia plays a synergistic role in enhancing the efficacy of simultaneous chemotherapy. Paclitaxel (PTX) is an anti-mitotic taxane drug that is widely used in chemotherapy for the treatment of various human malignancies such as lung, ovarian, breast, and head and neck cancers. Since the possibility that hyperthermia can enhance the anti-tumor effects of PTX has not yet been investigated, the present study was designed to evaluate the effects of short-term hyperthermia on PTX-induced antitumor activity in the human breast cancer line MCF-7. It was found that short-term hyperthermia promoted PTX-induced suppression of cell proliferation. The IC50 for PTX was reduced from 18.2±1.0 to 15.0±0.45 nM (p<0.05). The level of PTX-induced cell apoptosis was increased from 8.5±1.2 to 16.4±2.4% (p<0.05) and from 15.2±1.4 to 34.9±2.8% (p<0.05), at the end of the first and second hyperthermia cycles, respectively; both the activity and expression of caspase-7 were enhanced. In addition, PTX-induced cell cycle arrest in the G2/M phase was further promoted by short-term hyperthermia, from 9.3±0.7 to 12.5±0.9% (p<0.05). In contrast, short-term hyperthermia affected neither tumor cell migration nor invasion in the presence or absence of PTX. The presented data thus suggest that short-term hyperthermia may serve as a feasible approach in the promotion of breast cancer cell sensitivity to PTX.
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  • Makiko Washiyama, Naoya Koizumi, Makiko Fujii, Masuo Kondoh, Kiyohito ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 384-389
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    The control of permeability through the paracellular route has been paid great attention to for enhanced bioavailability of macromolecular and hydrophilic drugs. The paracellular permeability is controlled by tight junctions (TJ), and claudins are the major constituents of TJ. Despite numerous studies on TJ modulation, the dynamics is not well understood, although it could be crucial for clinical applications. Here, we studied the time (t) course of electrical conductivity (Σ) in a monolayer of Madin-Darby canine kidney (MDCK) and Caco-2 cells upon treatment with modulators, the C-terminus fragments of Clostridium perfringens enterotoxin (C-CPE) and sodium caprate (C10). For C-CPE treatment, Σ remains approximately constant, then starts increasing at t=tc (percolation threshold). For C10, on the other hand, Σ increases to 1.6–2.0 fold of the initial value, stays constant, and then starts increasing again for both MDCK and Caco-2 cells at t=tc. We find that this behavior can be explained within a framework of percolation, where Σ shows a logarithmic dependence on ttc with the power of μ; μ denotes the critical exponent. We obtain μ=1.1–1.2 regardless of cell type or modulator. Notably, μ depends only on the dimensionality (d) of the system, and these values correspond to those for d=2. Percolation is thus the operative mechanism for the increase in Σ through TJ modulation. The findings provide fundamental knowledge, not only on controlled drug delivery, but also on bio-nanotechnologies including the fabrication of biological devices.
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  • Seung Hwan Park, Soojin Lee, Yoon Ki Hong, Soojin Hwang, Jang Ho Lee, ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 390-398
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 14, 2012
    JOURNALS FREE ACCESS
    SuHeXiang Wan (SHXW), a Chinese traditional medicine, has been used to treat infantile convulsions, seizures and strokes. Previously, we reported that modified SHXW, called KSOP1009, suppressed the hyper-activation of c-Jun N-terminal kinase (JNK) and Alzheimer’s disease (AD)-like phenotypes in amyloid-β42 (Aβ42)-expressing Drosophila AD models. In the present study, we, further, investigated the detailed mechanism by which KSOP1009 suppresses the AD-like phenotypes of the model flies. As seen in the brains of AD patients, pan-neuronal expression of Aβ42 in Drosophila increased activation of extracellular signal-regulated kinase (ERK), which was monitored by its phosphorylation level, and the number of glial cells in the brain. Suppression of caspase activity did not affect these phenomena, suggesting that Aβ42 induces ERK activation and glial cell proliferation independently of apoptotic processes. KSOP1009 intake significantly reduced the level of ERK activation and the number of glial cells. Moreover, KSOP1009 intake also effectively decreased the defects in the wing vein formation induced by Epidermal growth factor receptor (Egfr) overexpression in fly wings, suggesting that it may contain an inhibitory substance that inhibits the EGFR/ERK signaling pathway. In addition, the Aβ42-induced locomotive defect was partially rescued by inhibition of the elevated ERK activity through its antagonistic drug treatment. Taken together, these results suggest that KSOP1009 exerts its therapeutic effect by inhibiting the EGFR/ERK pathway and glial cell proliferation and by suppressing the JNK pathway and apoptosis.
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  • Zheng-Guang Zhu, Hong Jin, Peng-Jiu Yu, Yuan-Xin Tian, Jia-Jie Zhang, ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 399-406
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: January 11, 2013
    JOURNALS FREE ACCESS
    Mollugin, a kind of naphthohydroquinone, is a major constituent isolated from Rubia cordifolia L. and demonstrated to possess anti-inflammatory activity in recent reports. However, the effects and mechanism of action of mollugin in inflammation have not been fully defined. The present study was therefore designed to investigate whether mollugin suppresses the inflammatory response in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Mollugin attenuated the LPS-induced expression of nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin (IL)-1β and IL-6 but augmented the expression of tumor necrosis factor (TNF)-α. Mollugin did not inhibit the degradation of inhibitory kappa B (IκB)-α or the nuclear translocation of p65 nuclear factor-kappa B (NF-κB) but rather enhanced the phosphorylation of p65 subunits evoked by LPS. Mollugin did not inhibit the phosphorylation of extracellular-signal-related kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase (JNK) 1/2 either. Mollugin significantly reduced the LPS-mediated phosphorylation of Janus kinase (JAK) 2, signal transducers and activators of transcription (STAT) 1 and STAT3. Molecular docking analysis showed that mollugin binds to JAK2 in a manner similar to that of AG490, a specific JAK2 inhibitor. We conclude that mollugin may be a JAK2 inhibitor and inhibits LPS-induced inflammatory responses by blocking the activation of the JAK-STAT pathway.
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  • Koichi Takao, Akira Shirahata, Keijiro Samejima, Robert Anthony Casero ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 407-411
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO.
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  • Tatsuya Hisada, Miki Katoh, Kotaro Hitoshi, Yuya Kondo, Miho Fujioka, ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 412-416
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 15, 2012
    JOURNALS FREE ACCESS
    A fentanyl patch is widely used for the treatment of cancer pain. Its few adverse effects include constipation and drowsiness. The absorption volume of transdermally applied fentanyl may differ according to its site of application and variability in patch adhesion. Since fentanyl is predominantly metabolized by the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 in the liver, its concentration may vary in cases of physiologically reduced CYP3A4 activity in the liver (liver disease and aging) or on co-administration of drugs. The clinical significance of measuring plasma concentration of fentanyl is high, but conventional methods require complicated processes such as solid-phase extraction and liquid–liquid extraction before the sample is injected into an HPLC system. In this study, a simple liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was developed for determining plasma fentanyl concentrations by deproteinization with acetonitrile. A recovery test was conducted using an absolute calibration curve to confirm the method’s linearity and inter- and intra-day reproducibility. The required plasma volume for detection was reduced from 1 mL in the conventional method to 20 µL in the present study, and a good calibration curve was obtained in the concentration range from 0.05 to 5 ng/mL. These findings suggest that the method for sample preparation and quantification developed in this study are appropriate for measuring fentanyl concentration in human plasma in clinical settings.
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  • Megumi Tsukamoto, Asuka Hatabu, Yoshitake Takahashi, Hiroshi Matsuda, ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 417-424
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: January 10, 2013
    JOURNALS FREE ACCESS
    Many of the neurodegenerative diseases associated with a decrease in regional cerebral blood flow (rCBF) are untreatable, and the appropriate therapeutic strategy is to slow the progression of the disease. Therefore, it is important that a definitive diagnosis is made as soon as possible when such diseases are suspected. Diagnostic imaging methods, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), play an important role in such a definitive diagnosis. Since several problems arise when evaluating these images visually, a procedure to evaluate them objectively is necessary, and studies of image analyses using statistical evaluations have been suggested. However, the assumed data distribution in a statistical procedure may occasionally be inappropriate. Therefore, to evaluate the decrease of rCBF, it is important to use a statistical procedure without assumptions about the data distribution. In this study, we propose a new procedure that uses nonparametric or smoothed bootstrap methods to calculate a standardized distribution of the Z-score without assumptions about the data distribution. To test whether the judgment of the proposed procedure is equivalent to that of an evaluation based on the Z-score with a fixed threshold, the procedure was applied to a sample data set whose size was large enough to be appropriate for the assumption of the Z-score. As a result, the evaluations of the proposed procedure were equivalent to that of an evaluation based on the Z-score.
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  • Jong Hoon Won, Jung Eun Ji, Kyong Hoon Ahn, Seok Kyun Kim, Jong Min Ch ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 425-431
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.
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  • Tsutomu Yamaki, Kazuo Ohtake, Keiko Ichikawa, Masaki Uchida, Hiroyuki ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 432-441
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 15, 2012
    JOURNALS FREE ACCESS
    We investigated whether poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 monolayer to hydrophilic macromolecules and clarified the disposition of tight junction (TJ) proteins. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran (FD-4) permeation were determined after treatment with PLA. TJ proteins were visualized using immunofluorescence microscopy after PLA exposure and depletion, and their expression levels were determined. The barrier function of TJs was also evaluated by measuring the alterations in the TEER and in the localization of TJ proteins. PLA induced an increase in hydrophilic macromolecule, FD-4, permeation through Caco-2 cell monolayers and a decrease in the TEER in a concentration-dependent manner, without any significant impact on the cell viability. This increased paracellular permeability induced by PLA was found to be internalized of claudin-4, ZO-1, tricellulin and mainly occludin from cell–cell junction to the subcellular space. ZO-1 appeared to play an important role in the reconstitution of TJ strand structures following PLA depletion. These results indicate that the PLA led to the internalization of TJ proteins to the subcellular space, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.
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  • Kazuyuki Furuya, Noriko Yamamoto, Yuki Ohyabu, Teruyuki Morikyu, Hiroh ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 442-451
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Selective androgen receptor modulators (SARMs) comprise a new class of molecules that induce anabolic effects with fewer side effects than those of other anabolic agents. We previously reported that the novel SARM S-101479 had a tissue-selective bone anabolic effect with diminished side effects in female animals. However, the mechanism of its tissue selectivity is not well known. In this report, we show that S-101479 increased alkaline phosphatase activity and androgen receptor (AR) transcriptional activity in osteoblastic cell lines in the same manner as the natural androgen ligand dihydrotestosterone (DHT); conversely, stimulation of AR dimerization was very low compared with that of DHT (34.4%). S-101479 increased bone mineral content in ovariectomized rats without promoting endometrial proliferation. Yeast two-hybrid interaction assays revealed that DHT promoted recruitment of numerous cofactors to AR such as TIF2, SRC1, β-catenin, NCoA3, gelsolin and PROX1 in a dose-dependent manner. SARMs induced recruitment of fewer cofactors than DHT; in particular, S-101479 failed to induce recruitment of canonical p160 coactivators such as SRC1, TIF2 and notably NCoA3 but only stimulated binding of AR to gelsolin and PROX1. The results suggest that a full capability of the AR to dimerize and to effectively and unselectively recruit all canonical cofactors is not a prerequisite for transcriptional activity in osteoblastic cells and resulting anabolic effects in bone tissues. Instead, few relevant cofactors might be sufficient to promote AR activity in these tissues.
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  • Yuuka Miyake, Kei Mayumi, Hideto Jinno, Toshiko Tanaka-Kagawa, Shizuo ...
    Type: Regular Article
    2013 Volume 36 Issue 3 Pages 452-461
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) plays important roles in the glucuronidation of various drugs and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies, because many of their physiological characteristics are similar to those of humans. In this study, the similarities and differences in enzymatic properties of UGT1A1 between humans and minipigs were precisely identified. Minipig UGT1A1 (mpUGT1A1) cDNA was firstly cloned by the rapid amplification of cDNA ends (RACE) method, and the corresponding protein as well as human UGT1A1 (hUGT1A1) enzyme was expressed in insect cells. Then the kinetics of estradiol at 3-hydroxy position (E-3OH) and 7-ethyl-10-hydroxycamptothecin (SN-38) glucuronidation by recombinant UGT1A1s as well as human and minipig liver microsomes were analyzed. The homology between mpUGT1A1 and hUGT1A1 at the amino acid level was 80.9%. E-3OH and SN-38 glucuronidation by recombinant hUGT1A1 and mpUGT1A1 showed allosteric sigmoidal kinetics. The CLmax value (29.1 µL/min/mg protein) for E-3OH glucuronidation of mpUGT1A1 was significantly higher (1.4-fold) than that of hUGT1A1, whereas the CLmax value (0.83 µL/min/mg protein) for SN-38 glucuronidation was significantly lower (27%) than that of hUGT1A1; however, the kinetic models and parameter levels for E-3OH and SN-38 glucuronidation by human and minipig liver microsomes did not parallel those in the respective species. These findings suggest that the enzymatic properties of UGT1A1 are considerably different between humans and minipigs. The information on species differences in UGT1A1 function gained in this study should help with in vivo extrapolation of xenobiotic metabolism and toxicity.
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Notes
  • Yasuhisa Okajima, Tomoko Yoshida, Hiroaki Fujimori, Junhui Wang, Hirom ...
    Type: Note
    2013 Volume 36 Issue 3 Pages 462-466
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Extensive DNA damage leads to the activation of poly(ADP-ribose) polymerase and subsequently to the formation of poly(ADP-ribose). When the damage is severe or leads to cell death, poly(ADP-ribose) may leak into the blood circulation. The metabolism of poly(ADP-ribose) in the bloodstream is not well understood. Thus, in the present study, the metabolism of 32P-labeled poly(ADP-ribose) was followed in mice after injection of this labeled compound into the tail vein. The results showed that 5 min after injection more than half of the radioactivity was concentrated in acid-soluble fractions, namely in low molecular weight compounds in the blood, liver, and kidneys. Most of this radioactivity was in the form of inorganic phosphate, detected 5 min post-injection in the blood, kidneys, and urine. By contrast, the metabolites ADP-ribose and phosphoribosyl-AMP were not detected in any of the tissues nor in blood or urine. Taken together, these findings suggest that once poly(ADP-ribose) enters the bloodstream it is rapidly degraded, thereby preventing its accumulation in the blood.
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  • Suran Ryu, Ji-Sun Shin, Young-Wuk Cho, Hyoung Kook Kim, Soo Heui Paik, ...
    Type: Note
    2013 Volume 36 Issue 3 Pages 467-474
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    Since inhibition of angiotensin II type 1 (AT1) receptor reduces chronic inflammation associated with hypertension, we evaluated the anti-inflammatory potential and the underlying mechanism of fimasartan, a Korean Food and Drug Administration approved anti-hypertension drug, in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Fimasartan suppressed the expressions of inducible nitric oxide synthase (iNOS) by down-regulating its transcription, and subsequently inhibited the productions of nitric oxide (NO). In addition, fimasartan attenuated LPS-induced transcriptional and DNA-binding activities of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). These reductions were accompanied by parallel reductions in the nuclear translocation of NF-κB and AP-1. Taken together, our data suggest that fimasartan down-regulates the expression of the iNOS in macrophages via NF-κB and AP-1 inactivation.
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  • Ken Shirato, Shogo Sato, Madoka Sato, Yoko Hashizume, Kaoru Tachiyashi ...
    Type: Note
    2013 Volume 36 Issue 3 Pages 475-480
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: January 15, 2013
    JOURNALS FREE ACCESS
    Splenic marginal zone macrophages expressing macrophage receptor with collagenous structure (MARCO) contribute to the clearance of blood-borne pathogens. We determined a splenic adherent cell fraction abundantly containing cells expressing a higher level of MARCO by flow cytometry, and examined the effects of daily administration of an anabolic dose of β2-agonist clenbuterol on the phagocytic capacity of the cells in mice. After 6 weeks of clenbuterol (1.0 mg/kg body weight/d) or vehicle administration to the mice, splenic adherent cells were isolated. These cells were separated into three cell-size subpopulations. Among them, the small-cell subpopulation contained abundantly the cells with markedly higher levels of MARCO and exhibited more intense phagocytic capacity against Escherichia coli, as compared with the other subpopulations. The phagocytic capacity of the small cells was significantly reduced after clenbuterol administration. These results suggest that the utilization of clenbuterol as doping drug impairs bacterial clearance in the spleen.
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  • Hiroyuki Fujita, Eiichi Hinoi, Takumi Watanabe, Takashi Iezaki, Misa T ...
    Type: Note
    2013 Volume 36 Issue 3 Pages 481-484
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    We have recently shown that the transcription factor Paired box-5 (Pax5) promotes bone formation in vivo and osteoblastogenesis in vitro. Here, we demonstrated the involvement of Pax5 in bone remodeling after ovariectomy (OVX). A significant increase was seen in vertebrae bone volume in transgenic mice preferentially overexpressing Pax5 in osteoblasts by using the mouse α1(I)Collagen promoter, whereas OVX significantly reduced vertebrae bone volume in wild-type (WT) mice without significantly affecting that in Pax5 transgenic mice. Preferential osteoblastic Pax5 overexpression invariably led to significant increases in osteoblastic and osteoclastic parameters in mice with sham operation. However, OVX significantly increased osteoclastic parameters in WT mice, without additionally increasing osteoblastic and osteoclastic parameters in Pax5 transgenic mice. These results suggest that osteoblastic Pax5 would play a role in OVX-induced bone loss through a mechanism relevant to the promotion of both osteoblastic bone formation and osteoclastic bone resorption in vivo.
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  • Yoko Idota, Hitomi Harada, Takumi Tomono, Kaori Morimoto, Shoko Kobaya ...
    Type: Note
    2013 Volume 36 Issue 3 Pages 485-491
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: January 15, 2013
    JOURNALS FREE ACCESS
    Alginate (ALA), which is an intercellular polysaccharide associated with brown algae, is used as a food additive, a health food and a medicine. Here, we first examined the adsorption of strontium (Sr) and cesium (Cs) by ALA in vitro, and then evaluated the effects of ALA on absorption and excretion of Sr and Cs in rats, in order to evaluate its potential usefulness for minimizing radiation damage from materials released after a nuclear accident. Both Sr and Cs were concentration-dependently adsorbed by sodium alginate (ALA-Na) in vitro. In rats given diet containing either ALA-Na or calcium alginate (ALA-Ca) for two weeks, the plasma concentration of Sr gradually decreased compared with the controls (normal diet); however, in the case of Cs, the plasma concentration was decreased only in the ALA-Ca group, but not the ALA-Na group. Moreover, we examined the effect of preadministration of diet containing either ALA-Na or ALA-Ca on absorption of Sr and Cs administered orally as the chloride salts to rats. Absorption of both Sr and Cs was reduced in the ALA-Ca group, while absorption of only Sr was reduced in the ALA-Na group. Safety assessments indicated that ALA-Ca is safer than ALA-Na. These results indicate that ALA-Ca reduces absorption and promotes excretion of both Sr and Cs, while ALA-Na does so only for Sr.
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  • Takuya Watanabe, Shinya Dohgu, Fuyuko Takata, Tsuyoshi Nishioku, Akio ...
    Type: Note
    2013 Volume 36 Issue 3 Pages 492-495
    Published: March 01, 2013
    Released: March 01, 2013
    JOURNALS FREE ACCESS
    The blood–brain barrier (BBB) is formed by brain endothelial cells. Many immortalized brain endothelial cell lines have been established; these have been used as in vitro BBB models. The aim of the present study was to assess the paracellular barrier properties of the immortalized mouse brain endothelial cell lines bEND.3, bEND.5 cells, and mouse brain endothelial cell 4 (MBEC4), and those of the primary mouse brain endothelial cells pMBECs. bEND.3 cells showed low permeability to sodium fluorescein and obvious staining of tight junction proteins (claudin-5, occludin and ZO-1) similar to pMBECs; these barrier properties of MBEC4 and bEND.5 cells were low. In addition, bEND.3 cells expressed the highest level of claudin-5 among all cells. These results suggest that bEND.3 cells are a convenient and useful model for evaluating BBB function, especially the paracellular barrier.
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  • Tsutomu Yamaki, Masaki Uchida, Yusuke Kuwahara, Yohei Shimazaki, Kazuo ...
    Type: Note
    2013 Volume 36 Issue 3 Pages 496-500
    Published: March 01, 2013
    Released: March 01, 2013
    [Advance publication] Released: December 15, 2012
    JOURNALS FREE ACCESS
    We have already reported that poly-L-arginine (PLA) remarkably enhanced the in vivo nasal absorption of hydrophilic macromolecules without producing any significant epithelial damage in rats. In the present study, we examined whether PLA could enhance the absorption of a model hydrophilic macromolecule, fluorescein isothiocyanate-dextran (FD-4), across the intestinal mucosa, as well as the nasal mucosa, by an in situ closed-loop method using the rat intestine. PLA was found to enhance the intestinal absorption of FD-4 in a concentration-dependent manner within the concentrations investigated in this study, but segment-specific differences were found to be associated with this effect (ileum>jejunum>duodenum≧colon). The factors responsible for the segment-specific differences were also investigated by intestinal absorption studies using aprotinin, a trypsin inhibitor, and an analysis of the expression of occludin, a tight junction protein. In the small intestine, the differences in the effect of PLA on the absorption of FD-4 may be related to the enzymatic degradation of PLA. In the colon, the reduced effect of PLA on the absorption of FD-4 may be related to the smaller surface area for absorption and the higher expression of occludin compared with other segments.
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