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Reviews
  • Takashi Suzuki
    Volume 40 (2017) Issue 12 Pages 2015-2023
    Released: December 01, 2017
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    Sialidase releases sialic acid residues from the ends of sugar chains. The sialidases are involved in many physiological processes including cell differentiation and proliferation and immune function as well as pathophysiological conditions such as various human cancers and infections. Therefore visualization of sialidase activities with high sensitivity could provide valuable insights into these isozyme’s activity. We developed novel fluorescent sialidase substrates, 2-benzothiazol-2-yl-phenol derivatives-based N-acetylneuraminic acid (Neu5Ac) (BTP-Neu5Ac) substrates, for highly sensitive and specific visualization of sialidase activity in living mammalian tissues and virus-infected cells. We found that BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in rat tissues including brain slices. BTP-Neu5Ac can also clearly detect cancer cells implanted orthotopically in mouse colons and human colon cancers. In this review, I describe imaging of sialidase activity with BTP-Neu5Ac in animal tissues, detection of colon cancer, memory formation, detection of virus-infected cells, and application to drug-resistant influenza virus detection and separation.

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  • Katsumi Iga, Akiko Kiriyama
    Volume 40 (2017) Issue 12 Pages 2024-2037
    Released: December 01, 2017
    [Advance publication] Released: October 07, 2017
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    Our study of drug–drug interaction (DDI) started with the clarification of unusually large DDI observed between ramelteon (RAM) and fluvoxamine (FLV). The main cause of this DDI was shown to be the extremely small hepatic availability of RAM (vFh). Traditional DDI prediction assuming the well-stirred hepatic extraction kinetic ignores the relative increase of vFh by DDI, while we could solve this problem by use of the tube model. Ultimately, we completed a simple and useful method for prediction of DDI. Currently, DDI prediction becomes more complex and difficult when examining issues such as dynamic changes in perpetrator level, inhibitory metabolites, etc. The regulatory agents recommend DDI prediction by use of some sophisticated methods. However, they seem problematic in requiring plural in vitro data that reduce the flexibility and accuracy of the simulation. In contrast, our method is based on the static and two-compartment models. The two-compartment model has advantages in that it uses common pharmacokinetics (PK) parameters determined from the actual clinical data, guaranteeing the simulation of the reference standard in DDI. Our studies confirmed that dynamic changes in perpetrator level do not make a difference between static and dynamic methods. DDIs perpetrated by FLV and itraconazole were successfully predicted by use of the present method where two DDI predictors [perpetrator-specific inhibitory activities toward CYP isoforms (pAi, CYPs) and victim-specific fractional CYP-isoform contributions to the clearance (vfm, CYPs)] are determined successively as shown in the graphical abstract. Accordingly, this approach will accelerate DDI prediction over the traditional methods.

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Current Topics - Recent Progress in the Study of Vasoactive Modulators in Metabolic and Cardiovascular Diseases
Current Topics : Reviews
  • Kana Maruyama, John J. McGuire, Satomi Kagota
    Volume 40 (2017) Issue 12 Pages 2039-2044
    Released: December 01, 2017
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    Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteases released from tissues or by synthetic peptide ligands administered pharmacologically. Its wide expression in the cardiovascular system, particularly within the endothelium, vasodilation activity, and link to increased expression of inflammatory cytokines positions PAR2 as a potentially important regulator of vascular pathology under conditions of tissue inflammation, and injury; and thus, a pharmaceutical target for new therapeutics. Obesity is considered a chronic low-grade systemic inflammatory condition as inflammatory cytokines released from adipocytes are closely related to development of metabolic syndrome and related disorders. Our work over the past five-years has focused on the changes in vasomotor functions of PAR2 in metabolic syndrome, using an animal model known as the SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP.ZF). In young SHRSP.ZF that had already developed impaired responses to nitric oxide, we reported that PAR2-induced endothelium-dependent vasodilation is preserved. However, this PAR2 vasodilation decreased with increasing age and further chronic exposure to the conditions of metabolism disorder. These findings raise the possibility that PAR2 regulates tissue perfusion and can protect organs from injury, which is an increasing clinical concern at later stages of metabolic syndrome. Here we present our studies on the time-dependent changes in vasoreactivity to PAR2 in metabolic syndrome and the underlying mechanisms. Furthermore, we discuss the implications of these age-related changes in PAR2 for the cardiovascular system in metabolic syndrome.

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  • Tsutomu Nakahara, Akane Morita, Rina Yagasaki, Asami Mori, Kenji Sakam ...
    Volume 40 (2017) Issue 12 Pages 2045-2049
    Released: December 01, 2017
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    Pathological ocular angiogenesis is a causative factor of retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. Vascular endothelial growth factor (VEGF) plays an important role in pathological angiogenesis, and anti-VEGF agents have been used to treat the ocular diseases that are driven by pathological angiogenesis. However, adverse effects associated with the blockade of VEGF signaling, including impairments of normal retinal vascular growth and retinal function, were suggested. Therefore, the development of a safe, effective strategy to prevent pathological ocular angiogenesis is needed. Recent studies have demonstrated that inhibitors of the mammalian target of rapamycin (mTOR) target proliferating endothelial cells within the retinal vasculature. Here, we review the potential of targeting the mTOR pathway to treat pathological ocular angiogenesis.

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  • Muneyoshi Okada, Keisuke Imoto, Akira Sugiyama, Jumpei Yasuda, Hideyuk ...
    Volume 40 (2017) Issue 12 Pages 2050-2060
    Released: December 01, 2017
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    The extracellular matrix (ECM), which contributes to structural homeostasis as well as to the regulation of cellular function, is enzymatically cleaved by proteases, such as matrix metalloproteinases and cathepsins, in the normal and diseased heart. During the past two decades, matricryptins have been defined as fragments of ECM with a biologically active cryptic site, namely the ‘matricryptic site,’ and their biological activities have been initially identified and clarified, including anti-angiogenic and anti-tumor effects. Thus, matricryptins are expected to be novel anti-tumor drugs, and thus widely investigated. Although there are a smaller number of studies on the expression and function of matricryptins in fields other than cancer research, some matricryptins have been recently clarified to have biological functions beyond an anti-angiogenic effect in heart. This review particularly focuses on the expression and function of basement membrane-derived matricryptins, including arresten, canstatin, tumstatin, endostatin and endorepellin, during cardiac diseases leading to heart failure such as cardiac hypertrophy and myocardial infarction.

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Current Topics : Regular Article
  • Shota Kobayashi, Takayuki Matsumoto, Makoto Ando, Maika Iguchi, Kumiko ...
    Volume 40 (2017) Issue 12 Pages 2061-2067
    Released: December 01, 2017
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    Although vasculopathies may occur systemically, there are few reports regarding femoral arteries of type 2 diabetes. Here, we investigated whether contractile response to noradrenaline in femoral arteries would change in type 2 diabetic male Otsuka Long-Evans Tokushima Fatty (OLETF) rat at the chronic stage of disease (1 year old) versus age-matched control Long-Evans Tokushima Otsuka (LETO) rat. OLETF rat exhibited hyperglycemia, hypertension, hyperlipidemia, and hypoinsulinemia compared to age-matched LETO rat. Noradrenaline-induced contraction was increased in femoral arteries in OLETF rats compared with LETO rats whereas serotonin- or phenylephrine-induced contractions were similar between these two animals. Acetylcholine- and sodium nitroprusside-induced relaxations were similar between the two groups. Very small relaxations in femoral arteries induced by clonidine and isoprenaline were obtained in LETO but not OLETF group. Noradrenaline-induced contraction was enhanced by treatment with NG-nitro-L-arginine (L-NNA), a nitric oxide synthase (NOS) inhibitor, and the between-group difference of contraction was eliminated by such treatment. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, reduced noradrenaline-induced contraction in both groups, whereas the contraction was greater in OLETF group versus LETO. Femoral arterial protein expression of endothelial NOS, COX-1, and superoxide dismutases were similar between the two groups, whereas reduction of COX-2 expression was seen in OLETF group compared with LETO. Increased contractile responsiveness to noradrenaline is seen in OLETF rat femoral artery and this may be due to reduction of suppressive effect of NO.

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Regular Articles
  • Dae-Sung Kim, Su-Bin Cha, Min-Cheol Park, Seol-A Park, Hye-Soo Kim, Wo ...
    Volume 40 (2017) Issue 12 Pages 2068-2074
    Released: December 01, 2017
    [Advance publication] Released: September 22, 2017
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    Scopoletin was recently shown to stimulate melanogenesis through cAMP-response element-binding protein (CREB) phosphorylation. In this study, we investigated the molecular events of melanogenesis-induced by scopoletin. After exposure to scopoletin, the protein levels of tyrosinase and tyrosianse related protein-1 (TRP-1) were significantly increased in B16F10 cells. The mRNA levels of tyrosinase and microphthalmia-associated transcription factor (MITF) were also enhanced by scopoletin. cAMP production and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were increased by scopoletin treatment. Scopoletin-mediated increase of intracellular melanin and tyrosinase expression were significantly attenuated by protein kinase A (PKA) inhibitors (H-89 and KT5720), while a protein kinase C (PKC) inhibitor (Ro-32-0432) had no effect and a p38 MAPK inhibitor (SB203580) partially blocked the scopoletin-induced intracellular melanin and tyrosinase expression. Moreover, scopoletin synergistically with cell-permeable cAMP analog (dibutyryl cAMP) significantly induced tyrosinase activity and melanin content in B16F10 cells. The silencing of p38 MAPK by small interfering RNA (siRNA) decreased the scopoletin-induced tyrosinase expression in B16F10 cells. These results suggest that scopoletin could induce melanin synthesis through the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

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  • Toshiya Kudo, Sachie Nakatani, Misato Kakizaki, Ai Arai, Keisuke Ishid ...
    Volume 40 (2017) Issue 12 Pages 2075-2080
    Released: December 01, 2017
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    Chondroitin sulfate (CS) is a sulfated polysaccharide produced by chondrocytes. Alkaline phosphatase (ALP) is an important enzyme involved in the mineralization of chondrocytes. In recent years, it has been reported that CS regulates the differentiation of various cells. In this study, we investigated the effect of supplemented CS on ALP activity and mineralization of the chondrogenic cell line, ATDC5. In addition, hyaluronic acid (HA), a non-sulfated and acidic polysaccharide, was used in comparison to CS. CS and HA significantly suppressed ALP activity without affecting ATDC5 cell proliferation. In addition, although the inhibition of ALP activity was observed at every time point, Alp mRNA expression level was not affected by CS. The suppressive effect of CS on ALP activity was abrogated by pre-treatment with chondroitinase ABC (CSase). CS and L-homoarginine (hArg), an inhibitor of ALP, significantly suppressed mineralization in ATDC5 cells. In conclusion, supplemented CS directly inhibits ALP to prevent the progression of chondrocytes from differentiation to mineralization.

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  • Yanqing Song, Huan Gao, Sixi Zhang, Yue Zhang, Xiangqun Jin, Jingmeng ...
    Volume 40 (2017) Issue 12 Pages 2081-2087
    Released: December 01, 2017
    [Advance publication] Released: September 23, 2017
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    The purpose of this study is to develop a new method of preparing salvianolic acid extracts (SAE) water-in-oil-in-water (W/O/W) multiple emulsion (ME). SAE injection is used in the treatment of brain infarct and promotion of blood circulation in China. However, the injection is not convenient, and the oral preparation has poor bioavailability. Hence, a new preparation that is convenient and stable with good biological availability is required. SAE ME was prepared by two-step emulsification method. Combined with single-factor investigation and orthogonal test, the embedding rate and centrifugal retention rate were taken as the comprehensive indexes to optimize the formulation of SAE ME. The ME size was tested by laser particle size analyzer. The pharmacokinetic studies were conducted in Sprague-Dawley rats with HPLC-MS/MS method. The blood coagulation and hemorheology tests were conducted to assess the effect of preparation in rats. The best preparation technique for SAE ME is by the use of trospium chloride; SAE represent 12% of water in the phase, lipophilic emulsifier hydrophilic lipophilic balance value=4.3, lipophilic emulsifier is 20% of the oil phase. The median diameter of particle is (0.608±0.05) µm and the Cmax of ME is 3-fold higher compared to Cmax of free drug. The oral biavailability of ME is 26.71-fold higher than that of free drug with good effect on blood circulation. SAE ME is stable hence, improves the biological availability and slows down drug release.

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  • Yi-Jia Yan, Lei-Lei Bao, Li-Jun Zhang, Jun Bian, Tai-shan Hu, Mei-zhen ...
    Volume 40 (2017) Issue 12 Pages 2088-2095
    Released: December 01, 2017
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    This study aimed to investigate the effect of hematoporphyrin dimethylether (HDME)-mediated photodynamic therapy for laser-induced choroidal neovascularization (CNV) in adult Brown Norway rats. HDME was administered via tail vein at 14 d after the laser photocoagulation, and the rats received irradiance with a laser light at 570 nm at 15 min after injection. CNV was evaluated by fundus photography, fundus fluorescein angiography, optical coherence tomography, and hematoxylin and eosin staining. We found that CNV was occurred at 7 d after photocoagulation and reaching peak activity at 14 d after photocoagulation. There is a significant reduction in the total area of the fluorescein leakage and the number of strong fluorescein leakage spots on 7 d after HDME-mediated photodynamic therapy (PDT). The results suggest that HDME-mediated PDT inhibits laser-induced CNV in rats, representing a promising therapy for wet age-related macular degeneration.

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  • Huiyoung Kwon, In Ho Jung, Ji Hyun Yi, Jae Hoon Kim, Jung Hoon Park, S ...
    Volume 40 (2017) Issue 12 Pages 2096-2104
    Released: December 01, 2017
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    Ziziphus jujuba is a plant, which bears fruits and seeds that are used for medicinal purposes in Traditional oriental medicine. The seed of Zizyphus jujuba var. spinosa (EZJ) has been also traditionally used for psychiatric disorders in Chinese and Korean medicines. Recent findings have indicated that EZJ improves memory impairment, a common symptom of various neurological diseases. However, the effects of EZJ on amyloid β (Aβ) toxicity, which is a main cause of Alzheimer’s disease (AD), remain to be elucidated. To illuminate the potential anti-AD effect and mechanism in the mouse hippocampal tissue, we examined the effect of standardized EZJ on Aβ-induced synaptic long-term potentiation (LTP) deficit in the hippocampal tissue. EZJ blocked Aβ-induced LTP deficits in a concentration-dependent manner. Moreover, EZJ increased brain-derived neurotrophic factor (BDNF) level in naïve hippocampal slices. The finding that the blockade of BDNF receptor reduced the effect of EZJ suggests that EZJ ameliorates the Aβ-induced LTP deficit through BDNF/topomyosin receptor kinase B (TrkB) signaling. However, transcription or translation inhibitors failed to block the effect of EZJ, suggesting that BDNF synthesis is not required for the action of EZJ on LTP. Finally, we found that EZJ stimulates plasmin activity. In contrast, plasmin inhibitor blocked the effect of EZJ on the Aβ-induced LTP deficit. Our findings indicate that EZJ ameliorates Aβ-induced LTP deficits through BDNF/TrkB signaling. This phenomenon is induced by a regulatory effect of EZJ on the post-translation modification of BDNF.

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  • Masayuki Nishihara, Kyoko Nishikura, Norimichi Morikawa, Shota Yokoyam ...
    Volume 40 (2017) Issue 12 Pages 2105-2109
    Released: December 01, 2017
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    Several studies reported that the administration of oxaliplatin often induced allergy, but few studies have analyzed the pathogenesis. In this study, we examined the relationship between the incidence of allergy and status of oxaliplatin administration, patient background, laboratory data, or combined drugs. The subjects were 144 patients with colorectal or gastric cancer in whom oxaliplatin administration was started and completed between 2010 and 2016. They were divided into 2 groups: allergy and non-allergy groups. We extracted important factors influencing its appearance using multivariate analysis, and analyzed items of which the influence was suggested, using receiver operating characteristic (ROC) analysis. In 11 patients (7.6%), allergy appeared. The median frequency of appearance was 9 times (range: 5–13), being similar to that previously reported. On multivariate analysis, albumin (Alb) was extracted as an important factor. The cut-off value of Alb for the risk of allergy was 4.1 g/dL. An increase in the number of protein conjugates may have increased the risk of functioning as a hapten. Furthermore, the results suggested that the more frequency of oxaliplatin administration might increase the incidence of allergy, although it was not extracted as an important factor. In addition to young and female patients, as previously indicated, careful follow-up may be necessary for those with an Alb level of ≥4.1 g/dL especially after the 6th course.

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  • Yuta Ohno, Yusuke Otsuka, Masakatsu Nohara, Tomomi Furihata, Yoshiki K ...
    Volume 40 (2017) Issue 12 Pages 2110-2116
    Released: December 01, 2017
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    While it is well known that L-carnitine [3-hydroxy-4-(trimethylazaniumyl)-butanoate] is an essential molecule for β-oxidation, it provides anti-oxidative effects as well. Since these effects have been observed in photoreceptor cells, the carnitine’s intracellular concentration is considered to play a protective role against oxidative damage to those cells. However, even though its high hydrophilicity makes it likely that carnitine import is accomplished via a dedicated host transport system, the specific uptake process into those cells is currently unknown. Therefore, in this study, we sought to identify and characterize photoreceptor cell carnitine uptake transporter(s) utilizing 661W cells as a photoreceptor cell model. The results of our uptake assays showed that carnitine was transported into 661W cells in a saturable manner (Km=5.5 mM), and that the activity was susceptible to extracellular pH and Na+. While these data suggest the involvement of a transporter in 661W cell carnitine uptake, the observed transport profile did not correspond to any of the currently known carnitine transporters such as organic cation/carnitine transporter 1 (Octn1), Octn2, Octn3, B0,+ and Ct2. In fact, in our experiments, the mRNA expressions for such carnitine transporters in 661W cells were consistently very low and the carnitine transporter substrates did not inhibit the uptake activities. Taken as a whole, our results indicate that carnitine is transported into 661W cells in a carrier-mediated manner. However, since its transport modes cannot be fully explained by known carnitine transporters, it is highly likely that photoreceptor cells utilize a unique molecularly-based carnitine uptake system.

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  • Guanqun Ge, Yan Yan, Hui Cai
    Volume 40 (2017) Issue 12 Pages 2117-2124
    Released: December 01, 2017
    [Advance publication] Released: September 30, 2017
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    Ginsenoside Rh2 (G-Rh2), a component extracted from roots of ginseng, exhibited anti-cancer pharmacological activities by inhibiting proliferation and inducing apoptosis in lung cancer cells. However, the mechanisms of G-Rh2 suppressing lung cancer development remained elusive. This study tried to investigate the possible mechanism involved in anti-proliferative effect of G-Rh2 in lung cancer cells. As results, G-Rh2 inhibited the proliferation of H1299 cells in a dose-dependent manner by inducing cell apoptosis. Activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein homologous protein (CHOP), and caspase-4 were involved in G-Rh2-induced apoptosis of H1299 cells. It was also found that G-Rh2 could up-regulate expressions of ATF4, CHOP and caspase-4 in H1299 cells in a dose-dependent manner. In addition, NAC (N-acetylcysteine, a reactive oxygen species (ROS) scavenger) treatment dramatically decreased ROS generation in H1299 cells; both of NAC and 4-PBA (4-phenylbutyrate, a specific endoplasmic reticulum (ER) stress inhibitor) administration impaired apoptosis and expression levels of ATF4, CHOP and caspase-4 in G-Rh2 incubated H1299 cells. In vivo assays extended the significance of these results, showing that G-Rh2 inhibited lung cancer growth and the inhibition effects of G-Rh2 in tumor growth were significantly reduced by inhibition of ER stress. In conclusion, G-Rh2 inhibited proliferation of H1299 cells by inducing ROS mediated ER stress dependent cell apoptosis.

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  • Charith UB Wijerathne, Hee-Seon Park, Hye-Yun Jeong, Ji-Won Song, Og-S ...
    Volume 40 (2017) Issue 12 Pages 2125-2133
    Released: December 01, 2017
    [Advance publication] Released: September 22, 2017
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    Quisqualis indica (QI) has been used for treating disorders such as stomach pain, constipation, and digestion problem. This study was aimed to evaluate the therapeutic efficacy of QI extract on treating benign prostatic hyperplasia (BPH) in LNCaP human prostate cancer cell line and a testosterone-induced BPH rat model. LNCaP cells were treated with QI plus testosterone propionate (TP), and androgen receptor (AR) and prostate specific antigen (PSA) expression levels were assessed by Western blotting. To induce BPH, the rats were subjected to a daily subcutaneous injection of TP (3 mg/kg) for 4 weeks. The rats in treatment group were orally gavaged with QI (150 mg/kg) together with the TP injection. In-vitro studies showed that TP-induced increases in AR and PSA expression in LNCaP cells were reduced by QI treatment. In BPH-model rats, the prostate weight, testosterone in serum, dihydrotestosterone (DHT) concentration and 5α-reductase type 2 mRNA expression in prostate tissue were significantly reduced following the treatment with QI. TP-induced prostatic hyperplasia and the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 were significantly attenuated in QI-treated rats. In addition, QI induced apoptosis by up-regulating caspase-3 and -9 activity and decreasing the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio in prostate tissues of BPH rats. Further investigation showed that TP-induced activation of AKT and glycogen synthase kinase 3β (GSK3β) was reduced by QI administration. Therefore, our findings suggest that QI attenuates the BPH state in rats through anti-proliferative and pro-apoptotic activities and might be useful in the clinical treatment of BPH.

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  • Hiroyasu Sakai, Shoko Tabata, Minami Kimura, Saori Yabe, Yosuke Isa, Y ...
    Volume 40 (2017) Issue 12 Pages 2134-2139
    Released: December 01, 2017
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    5-Fluorouracil (5-FU) is widely used as an anti cancer drug and is known to cause severe diarrhea. Recently we suggested that levels of chemokine (C-X-C motif) ligand 1 (CXCL1) and neutrophil recruitment in the colonic mucosa were drastically increased by the 5-FU administration in mice. Hange-shashin-to (HST) is prescribed in Japan for treat gastritis, stomatitis, and inflammatory diarrhea. We therefore examined the effects of HST and its active ingredients on 5-FU-induced CXCL1 upregulation in cultured colon tissue, and also examined the effects of HST on 5-FU-induced diarrhea development in the mouse. The distal colon isolated from the mouse was incubated with 5-FU and HST. Mice were given 5-FU (50 mg/kg, intraperitoneally (i.p.)) daily for four days. HST (300 mg/kg, per os (p.o.)) was administered 30 min before mice received 5-FU. mRNA levels of CXCL1 in the colon were examined using quantitative RT-PCR. 5-FU enhanced CXCL1 mRNA in the colon but the effect by 5-FU was markedly suppressed by application of HST and its active ingredients, baicalein and 6-gingerol. Nuclear factor kappa B (NF-κB) was activated by 5-FU treatment in cultured colon tissue, which was also suppressed by HST and the combination of baicalein and 6-gingerol. Furthermore, HST reduced 5-FU-induced diarrhea development. Under such experimental condition, CXCL1 gene, protein levels of neutrophil elastase and myeloperoxidase upregulation induced by 5-FU in the colon was attenuated by HST. These findings suggest that HST, especially baicalein and 6-gingerol, prevent the development of neutrophil recruitment and diarrhea by the inhibition of NF-κB activity.

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  • Takuma Yamashita, Haruka Shinotsuka, Yuki Takahashi, Kana Kato, Makiya ...
    Volume 40 (2017) Issue 12 Pages 2140-2145
    Released: December 01, 2017
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    Cell-derived nanosized vesicles or exosomes are expected to become delivery carriers for functional RNAs, such as small interfering RNA (siRNA). A method to efficiently load functional RNAs into exosomes is required for the development of exosome-based delivery carriers of functional RNAs. However, there is no method to find exosome-tropic exogenous RNA sequences. In this study, we used a systematic evolution of ligands by exponential enrichment (SELEX) method to screen exosome-tropic RNAs that can be used to load functional RNAs into exosomes by conjugation. Pooled single stranded 80-base RNAs, each of which contains a randomized 40-base sequence, were transfected into B16-BL6 murine melanoma cells and exosomes were collected from the cells. RNAs extracted from the exosomes were subjected to next round of SELEX. Cloning and sequencing of RNAs in SELEX-screened RNA pools showed that 29 of 56 clones had a typical RNA sequence. The sequence found by SELEX was enriched in exosomes after transfection to B16-BL6 cells. The results show that the SELEX-based method can be used for screening of exosome-tropic RNAs.

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  • Yan Fang, Lisheng Chu, Lin Li, Jun Wang, Yan Yang, Jingjing Gu, Jianpi ...
    Volume 40 (2017) Issue 12 Pages 2146-2152
    Released: December 01, 2017
    [Advance publication] Released: October 04, 2017
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    Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation is one of the new therapeutic strategies for treating ischemic stroke. However, the poor survival rate of transplanted BMSCs in ischemic tissue limits the therapeutic efficacy of this approach. Oxidative stress is a major mechanism underlying the pathogenesis of brain ischemia and has a negative impact on the survival of transplanted BMSCs. Tetramethylpyrazine (TMP) has been reported to possess potent antioxidant activity. In the present study, we aimed to investigate the protective effects of TMP pretreatment on BMSCs survival of hydrogen peroxide (H2O2)-induced apoptosis in vitro and to elucidate the potential antiapoptotic mechanisms of TMP pretreatment on BMSCs. BMSCs were pretreated with TMP (10, 25, 50, 100, and 200 µmol/L) for 24 h and then exposed to 500 µmol/L of H2O2 for 24 h. We found that TMP pretreatment significantly increased cell viability and decreased cell apoptosis and intracellular reactive oxygen species (ROS) generation. Furthermore, the protective effects of TMP were related to increased Bcl-2 expression, attenuated Bax expression, and enhanced levels of phosphorylated Akt (p-Akt) and extracellular regulated protein kinases1/2 (p-ERK1/2). Further studies found that these beneficial effects of TMP were significantly blocked by wortmannin (an inhibitor of phosphoinositide-3 kinase (PI3K)) or PD98059 (an inhibitor of ERK1/2). In conclusion, our results confirm that TMP protects BMSCs against H2O2-induced apoptosis by regulating the PI3K/Akt and ERK1/2 signaling pathways, suggesting that TMP may be used in combination with BMSCs to improve cell survival for the treatment of ischemic stroke.

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  • Hiroshi Ashigai, Emiko Ikeshima, Kumiko Koizumi, Keiko Nakashima, Mai ...
    Volume 40 (2017) Issue 12 Pages 2153-2157
    Released: December 01, 2017
    [Advance publication] Released: September 29, 2017
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    Oxygen transportation and regulation of some physiological processes are facilitated by blood flow. Furthermore, blood flow is regulated by various factors such as nitric oxide (NO) and the autonomic nerve system. In modern life, many people suffer from chilliness (hiesho) because of mental stress and an excessive use air-conditioning systems, which induces vasoconstriction in the peripheral skin. In this study, we focused on pyrazine derivatives, particularly compounds that are used as food flavoring materials, and investigated their effects on vascular function and blood flow. We examined the vasodilatory effect of pyrazine derivatives in the rat thoracic aorta and found 2-ethylpyrazine (2-EP) to be the most active pyrazine compound. Additionally, we found that 2-EP induces vasodilatation through the activities of endothelium-derived relaxing factors. 2-EP activates NO synthesis through the effect of endothelial NO synthase in the endothelium. As a result, cyclic GMP levels rise in smooth muscle cells and vasodilatation is induced. We also confirmed that 2-EP increases peripheral blood flow in rats. From these results, we concluded that 2-EP induces vasodilatation by inducing the release of NO and increasing peripheral blood flow.

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  • Satoshi Nakao, Haruna Hatahira, Sayaka Sasaoka, Shiori Hasegawa, Yumi ...
    Volume 40 (2017) Issue 12 Pages 2158-2165
    Released: December 01, 2017
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    Drug-induced photosensitivity (DIP) refers to the development of cutaneous disorders caused by the combined effects of different medications and light. The aim of this study was to obtain new information on drug risk comparisons and on DIP onset profiles, including seasonal variations, for clinically used prescription drugs. We analyzed reports of DIP recorded in the Japanese Adverse Drug Event Report (JADER) database using a reporting odds ratio (ROR). We also used Weibull proportional-hazards models for each drug to examine the patterns of DIP. The JADER database contains 430587 reports recorded from April 2004 to November 2016. The ROR values (95% confidence interval [CI]) of losartan/hydrochlorothiazide (HCTZ), valsartan/HCTZ, and ketoprofen were 214.5 (162.1–283.9), 104.7 (66.3–165.5), and 117.9 (76.6–181.5), respectively. For time-to-onset analysis, the median durations (interquartile range) for DIP caused by losartan/HCTZ, valsartan/HCTZ, and ketoprofen were 56 (41–78), 49 (38–88), and 8 (2–14) days, respectively. The lower limit of the 95% CI for the Weibull shape parameter β value for losartan/HCTZ was greater than 1. More than half of the reports of DIP onset following the administration of ketoprofen were recorded within 10 d of treatment initiation. The seasonal variation of photosensitivity reactions was shown to follow an annual sinusoidal pattern with a peak in April and May. Based on the results, losartan/HCTZ, valsartan/HCTZ, and ketoprofen should be used carefully in clinical practice to avoid DIP.

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    Editor’s picks

    Drug-induced photosensitivity (DIP) is a cutaneous adverse event caused by the combined effects of a medication and exposure to light. The article by Nakao et al.  evaluated the association between drugs and DIP by using the reporting odds ratio and time-to-onset analysis data from the Japanese Adverse Drug Event Report (JADER) database. More than half of the reports of DIP onset following ketoprofen administration were recorded within 10 days of initiation of treatment. The seasonal variation of DIP was shown to follow an annual sinusoidal pattern with peaks observed in April and May. The results of this study suggest close monitoring of patients taking suspected drugs, especially during the peak season of photosensitivity reactions.

  • Yusuke Kono, Hitomi Jinzai, Yota Kotera, Takuya Fujita
    Volume 40 (2017) Issue 12 Pages 2166-2174
    Released: December 01, 2017
    [Advance publication] Released: September 30, 2017
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    The present study aimed to investigate the effect of particle size (100, 500 nm), surface charge (cationic, neutral and anionic) and polyethylene glycol (PEG) modification of magnetic liposomes on their interaction with the human intestinal epithelial cell line, Caco-2. The cellular associated amount of all the magnetic liposomes was significantly increased by the presence of a magnetic field. The highest association and internalization into Caco-2 cells was observed with magnetic cationic liposomes. Moreover, small magnetic liposomes were more efficiently associated and taken up into the cells, than large ones. In contrast, PEG modification significantly attenuated the enhancing effect of the magnetic field on the cellular association of magnetic liposomes. We also found that magnetic cationic liposomes had the highest retention properties to Caco-2 cells. Moreover, the retention of large magnetic liposomes to the cells was much longer than that of small ones. In addition, magnetic cationic and neutral liposomes had relatively high stability in Caco-2 cells, whereas magnetic anionic liposomes rapidly degraded. These results indicate that the physicochemical properties and PEG modification of magnetic liposomes greatly influences their intestinal epithelial transport.

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  • Xinpeng Li, Ami Kawamura, Yusuke Sato, Masaki Morishita, Kosuke Kusamo ...
    Volume 40 (2017) Issue 12 Pages 2175-2182
    Released: December 01, 2017
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    In this study, the effects of N-acyl taurates (NATs) on the intestinal absorption of curcumin (CUR), a water-insoluble and poorly absorbed compound, were examined in rats. Sodium methyl lauroyl taurate (LMT) and sodium methyl cocoyl taurate (CMT) were the most effective in increasing the solubility and intestinal absorption of CUR. The intestinal membrane toxicity of the NATs was also evaluated by measuring the activity of lactate dehydrogenase (LDH), a toxicity marker. NATs did not increase the activity of LDH, suggesting that they may be safely administered orally. We further elucidated the absorption-enhancing mechanisms of NATs by using Caco-2 cells. In cellular transport studies, LMT and CMT reduced the transepithelial electrical resistance value of Caco-2 cells and increased the transport of 5(6)-carboxyfluorescein and CUR. Hence, the intestinal absorption enhancement by LMT and CMT was attributed to the synergistic effect of higher solubility and greater permeability of the cell layer towards CUR in the presence of the surfactants. In summary, co-administration of CUR with either LMT or CMT is a simple and effective method to enhance oral delivery of CUR.

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  • Yuma Yamada, Laila Burger, Eriko Kawamura, Hideyoshi Harashima
    Volume 40 (2017) Issue 12 Pages 2183-2190
    Released: December 01, 2017
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    While Coenzyme Q10 (CoQ10) is thought to be effective for the treatment of a variety of diseases, it limits its cellular uptake. Because of the hydrophobic nature of CoQ10, it is reasonable to assume that it could be encapsulated within a liposomal carrier. Several reports regarding the packaging of CoQ10 in liposomes have appeared, but detailed investigations of the preparation of CoQ10 encapsulated liposomes have not been reported. As a result, information regarding the optimal method of packaging CoQ10 in liposomes is not available. In this study, several types of liposomes were prepared using different methods and their characteristics were compared. Since CoQ10 is mainly located in the inner mitochondrial membrane, a liposome that targets mitochondria, a MITO-Porter, was used as a model liposome. It was possible to incorporate high levels of CoQ10 into the carrier. Transmission electron microscopy analyses showed that an empty MITO-Porter and the CoQ10-MITO-Porter were structurally different from one another. Even though significant structural differences were observed, mitochondrial delivery was not affected in mitochondrial disease fibroblast cells, as evidenced by confocal laser scanning microscopy observations. The results reported herein suggest that the CoQ10-MITO-Porter might be a suitable candidate for the potential medical therapy of mitochondria-related diseases.

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  • Hiroyuki Oyama, Izumi Morita, Yuki Kiguchi, Tomomi Morishita, Sakiko F ...
    Volume 40 (2017) Issue 12 Pages 2191-2198
    Released: December 01, 2017
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    Cortisol levels in bodily fluids represent a useful index for pituitary−adrenal function, and thus practical anti-cortisol antibodies are required. We have studied “antibody-breeding” approaches, which involve in vitro evolution of antibodies to improve their antigen-binding performances. Here, we produced an antibody fragment to measure serum cortisol levels with over 30-fold enhanced affinity after single mutagenesis and selection steps. A mouse anti-cortisol antibody, Ab-CS#3, with insufficient affinity for practical use, was chosen as the prototype antibody. A “wild-type” single-chain Fv fragment (wt-scFv; Ka, 3.4×108 M−1) was prepared by bacterial expression of a fusion gene combining the VH and VL genes for this antibody. Then, random point mutations were generated separately in VH or VL by error-prone PCR, and the resulting products were used to assemble scFv genes, which were displayed on filamentous phages. Repeated panning of the phage library identified a mutant scFv (scFv#m1-L10) with an over 30-fold enhanced affinity (Ka 1.2×1010 M−1). Three amino acid substitutions (Cys49Ser, Leu54Pro, and Ser63Gly) were observed in its VL sequence. In a competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose–response curves with measuring range ca. 0.03–0.6 ng/assay cortisol, midpoint of which (0.15 ng/assay) was 7.3-fold lower than that of wt-scFv. Although cortisone, 11-deoxycortisol, and prednisolone showed considerable cross-reactivity, the mutant scFv should enable sensitive routine cortisol assays, except for measurement after metyrapone or high-dose of prednisolone administrations. Actually, cortisol levels of control sera obtained with the scFv-based ELISA were in the reference range.

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Notes
  • Mika Hosokawa, Shota Tanaka, Kumiko Ueda, Seigo Iwakawa
    Volume 40 (2017) Issue 12 Pages 2199-2204
    Released: December 01, 2017
    [Advance publication] Released: September 28, 2017
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    Limited information is currently available on how to apply epigenetic modifiers to current colorectal cancer (CRC) chemotherapy. The purpose of this study is to clarify the schedule-dependent effects of combined treatment with conventional anticancer drugs and epigenetic modifiers in human CRC cells. Cytotoxicity in 4 CRC cell lines (SW480, HT29, SW48, and HCT116) was measured using the WST-8 assay. As epigenetic modifiers, 3 DNA methyltransferase (DNMT) inhibitors such as decitabine (DAC), azacytidine (AC), and zebularine (Zeb), and 3 histone deacetylase (HDAC) inhibitors including trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and valproic acid (VPA) were used. Combination effects were analyzed by the isobologram method. SW480 cells showed the lowest sensitivity to the anticancer drugs 5-fluorouracil, SN-38 (the active form of irinotecan), and oxaliplatin. In SW480 cells, epigenetic modifiers other than VPA showed the most significant synergistic effects when used before anticancer drugs, while VPA showed synergistic effects in co- or post-treatment. In the 3 other CRC cells, synergistic effects were less frequent and weaker. The dose of anticancer drugs may be reduced by combining epigenetic modifiers in SW480 cells, which are less sensitive to anticancer drugs, unlike the more sensitive HT29, SW48, and HCT116 cell lines. These results provide useful information for understanding how to incorporate epigenetic modifiers into current CRC chemotherapy.

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  • Baek-Soo Han, Nguyen Van Minh, Ha-Young Choi, Jung-Su Byun, Won-Gon Ki ...
    Volume 40 (2017) Issue 12 Pages 2205-2211
    Released: December 01, 2017
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    The methanol extract of the roots and stems of Daphne genkwa and its constituents yuanhuacin (1) and genkwanine N were previously reported to have Nurr1 activating effects and neuroprotective effects in an animal model of Parkinson’s disease (PD). In this study, four more daphnane-type diterpenes (acutilonine F (2), wikstroemia factor M1 (3), yuanhuadine (5), and yuanhuatine (6)) and two phorbol-type diterpenes (prostratin Q (4) and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (7)) were isolated as Nurr1 activating compounds from the D. genkwa extract. Consistent with their higher Nurr1 activating activity, compounds 1, 4, 5, and 7 exhibited higher inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial BV-2 cells with an IC50 (µM) of 1–2, which was 15–30 times more potent than that of minocycline (29.9 µM), a well-known anti-neuroinflammatory agent. Additionally, these diterpenes reduced expression and transcription of LPS-induced pro-inflammatory cytokines in BV-2 cells. Thus, the daphnane-type and phorbol-type diterpenes had anti-neuroinflammatory activity with Nurr1 activation and could be responsible for the anti-PD effect of the roots and stems of D. genkwa.

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