Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
37 巻 , 8 号
選択された号の論文の24件中1~24を表示しています
Regular Articles
  • Jingjing Su, Hui Cheng, Dandan Zhang, Mei Wang, Chenye Xie, Yawen Hu, ...
    2014 年 37 巻 8 号 p. 1259-1268
    発行日: 2014/08/01
    公開日: 2014/08/01
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    5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs for various cancer treatments, but its chemo-drug resistance is a major obstacle in clinical settings. The anticancer effects of gambogenic acid (GNA) and its potential mechanisms have been well documented in the past few years. In this study, we determined the synergistic inhibitory effects of GNA and 5-FU on A549 human lung cancer cells. 5-FU combined with GNA inhibited the viability of A549 cells in a concentration-dependent manner. The mitochondrial tolerance of this two-kind of drugs combination treatment was stronger than a single-drug treatment. Combination treatment caused a morphological change of A549 cells. Flow cytometric evidence indicated that the combined treatment caused significant cell death, with the death rate of A549 cells treated with combination drugs showing a time-dependent manner. Furthermore, combination treatment of GNA and 5-FU showed up-regulated of caspase-3, caspase-9, bax, RIP1, apoptosis-inducing factor (AIF), voltage-dependent anion channel (VDAC), cytochrome c and cyclophilin D and down-regulated bcl-2. In conclusion, in addition to the activation of caspase-dependent apoptosis, the combination of GNA and 5-FU might also cause cell death of A549 cells by activating caspase-independent necroptosis. These mechanisms may be due to the toxicity of targeted toxin to mitochondria via the mitochondrial pathway.
  • Takayoshi Mamiya, Yuya Hasegawa, Masayuki Hiramatsu
    2014 年 37 巻 8 号 p. 1269-1273
    発行日: 2014/08/01
    公開日: 2014/08/01
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    In this study we investigated whether κ-opioid receptor stimulation by dynorphin A (1–13), a potent fragment of endogenous peptide, attenuated repeated stress-induced behavioral impairments in mice. In order to reduce the motivation to escape, mice were preexposed to inescapable electric footshock (day 0), and then dynorphin A (1–13) was administered to mice prior to the stress from the next day for 4 d (days 1–4). Dynorphin A (1–13) (1500 pmol/5 µL intracerebroventricular (i.c.v.)) attenuated the repeated stress-induced escape failures from the shock, and this improvement was inhibited by the pretreatment of nor-binaltorphimine (4.9 nmol/kg subcutaneously (s.c.)), a κ-opioid receptor antagonist. In the neurochemical experiments, we detected an increase in 5-hydroxyindoleacetic acid (5-HIAA) content, but not in serotonin (5-HT) content, and an increase in the 5-HIAA/5-HT ratio was observed in the amygdala of the group with footshock compared with the group without shock. Additionally, the changes in 5-HIAA content and the ratio were reversed by dynorphin A (1–13). However, there were no differences in 5-HT or 5-HIAA content or their ratios in the hippocampus among the three groups. These results suggest that dynorphin might alleviate the stress-induced behavioral impairments accompanied by regulation of the 5-HTergic system in the brain.
  • Yukinobu Kodama, Yumi Shiokawa, Tadahiro Nakamura, Tomoaki Kurosaki, K ...
    2014 年 37 巻 8 号 p. 1274-1281
    発行日: 2014/08/01
    公開日: 2014/08/01
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    We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and γ-polyglutamic acid (γ-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73–102 nm in particle size and 45–52 mV in ζ-potential. The silencing effect of siRNA/PEI complexes increased with an increase of PEI, and siRNA/PEI complexes with a charge ratio greater than 16 showed significant luciferase knockdown in a mouse colon carcinoma cell line regularly expressing luciferase (Colon26/Luc cells). However, strong cytotoxicity and blood agglutination were observed in the siRNA/Lipofectamine complex and siRNA/PEI16 complex. Recharging cationic complexes with an anionic compound was reported to be a promising method for overcoming these toxicities. We therefore prepared ternary complexes of siRNA with PEI (charge ratio 16) by the addition of γ-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of γ-PGA content, which decreased the ζ-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including γ-PGA with an anionic surface charge. The high incorporation of ternary complexes into Colon26/Luc cells was confirmed with fluorescence microcopy. Having achieved knockdown of an exogenously transfected gene, the ability of the complex to mediate knockdown of an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was assessed in B16-F10 cells. The ternary complex (siRNA/PEI16/γ-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.
  • Xiang Zhou, Ping Gan, Lili Hao, Li Tao, Jia Jia, Bo Gao, Jiang-yun Liu ...
    2014 年 37 巻 8 号 p. 1282-1294
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Microglia activation-mediated neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and human immunodeficiency virus (HIV)-associated dementia. Inhibition of microglia activation may alleviate neurodegeneration under neuroinflammatory conditions. In the present study, we compared three flavone C-glycosides extracted from Trollius chinensis BUNGE using a cell-based assay to evaluate their antiinflammatory effects on microglial cells. The results showed that orientin-2″-O-galactopyranoside (OGA) significantly inhibited the production of nitric oxide and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated microglial cells. OGA also markedly inhibited the LPS-induced expression of TNF-α, interleukin-1β, inducible nitric oxide (NO) synthase, and cyclooxygenase-2, which was accompanied by suppression of the activation of nuclear factor (NF)-κB and the extracellular signal-regulated kinase (ERK) signal pathway. In addition, OGA decreased LPS-induced reactive oxygen species generation, which appears to be related to the activation of the NF-E2-related factor2 (NRF2)/ heme oxygenase-1 (HO-1) pathway in BV-2 microglial cells. Furthermore, OGA reduced the cytotoxicity of activated microglia toward HT-22 neuroblastoma cells in a co-culture system. Taken together, the present study demonstrated that the induction of HO-1-mediated inhibition of the NF-κB and ERK pathways contributes significantly to the antineuroinflammatory and neuroprotective effects elicited by OGA.
  • Seung-Heon Shin, Yee-Hyuk Kim, Jeong-Kyu Kim, Kwan-Kyu Park
    2014 年 37 巻 8 号 p. 1295-1300
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Bee venom (BV) has been used as an anti-inflammatory and immune modulating agent in Oriental medicine. This study used a mouse model to investigate the anti-allergic effect of BV, which is used in the treatment of various inflammatory diseases in traditional medicine. BV was obtained from the National Institute of Agricultural Science and Technology of Korea. Female BALB/C mice were sensitized by intraperitoneal injection of ovalbumin (OVA). BV was administered nasally prior to the intranasal instillation of OVA. Allergic behavior, serum OVA-specific immunoglobulin E (IgE), interleukin (IL)-4, IL-10, and interferon-gamma (INF-γ) levels in nasal lavage fluid were measured. Hematoxylin–eosin and periodic acid-Schiff staining were performed to evaluate histological change. BV attenuated nasal symptoms and inhibited the production of OVA-specific IgE and IL-4 in sensitized mice. The degree of inflammatory cell infiltration and goblet cell hyperplasia was attenuated by BV. Thus, BV effectively reduced allergic inflammation in a mouse model of allergic rhinitis, suggesting its potential as a useful therapeutic agent to treat allergic rhinitis.
  • Guoshuai Feng, Zhijian Zhang, Qingqing Bao, Zaijun Zhang, Libing Zhou, ...
    2014 年 37 巻 8 号 p. 1301-1307
    発行日: 2014/08/01
    公開日: 2014/08/01
    [早期公開] 公開日: 2014/05/28
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    The aims of this study were to investigate the effect of chinonin in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration in C57BL/6 mice and to examine the possible mechanisms. The neurotoxin MPTP was employed to create a subacute Parkinson’s disease (PD)-like model in C57BL/6 mice. Chinonin (10, 20, 40 mg/kg body weight) was intraperitoneally administered 0.5 h after MPTP (30 mg/kg) injection for 7 d consecutively. Chinonin showed neuroprotective effects in the MPTP-treated mice PD model by ameliorating motor impairment in the catwalk and open-field tests. Consistently, chinonin reduced loss of dopaminergic neurons in the substantia nigra and prevented depletion of dopamine and its metabolites 3-methoxy-4-hydroxy-phenylacetic acid and homovanillic acid in the striatum of mice. Compared with the MPTP group, in the chinonin plus MPTP groups significant increases of superoxide dismutase activity and glutathione levels were observed as well as a distinct reduction of lipid peroxidation product malondialdehyde in the striatum. Taken together, we propose that chinonin exerts neuroprotective effects in C57BL/6 mouse model of PD and these effects may be due to chinonin’s antioxidative property.
  • Yuri Miura, Takatoshi Sato, Yoko Sakurai, Ryo Sakai, Wakako Hiraoka, T ...
    2014 年 37 巻 8 号 p. 1308-1314
    発行日: 2014/08/01
    公開日: 2014/08/01
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    O-Linked β-N-acetylglucosamine-modification (O-GlcNAcylation) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. However, the role of O-GlcNAcylation in the induction of heat shock proteins (Hsps) by arsenite remains unclear. We used O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of O-GlcNAcase, and glucosamine (GlcN), an enhancer of the hexosamine biosynthesis pathway, or O-GlcNAc transferase (OGT) short interfering RNA (siRNA) to enhance or suppress cellular O-GlcNAcylation levels, respectively, in HeLa cells. The exposure to arsenite increased O-GlcNAcylation and Hsp 70 levels in HeLa cells. However, the pre-treatment with PUGNAc or GlcN, which enhanced O-GlcNAcylation levels, decreased the arsenite-induced expression of Hsp 70. The pre-treatment with OGT siRNA, which suppressed O-GlcNAcylation levels, did not affect the induction of Hsp 70. We then examined the effects of O-GlcNAcylation on the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1), and found that neither the nuclear translocation nor phosphorylation of HSF1 was regulated by O-GlcNAcylation. Finally, Hsp 70 mRNA expression was induced by arsenite, whereas the addition of PUGNAc slightly suppressed its induction. These results indicate that O-GlcNAcylation is related to arsenite-induced Hsp 70 expression, and demonstrated that hyper-O-GlcNAcylation inhibited the induction of Hsp 70 via transcriptional factors instead of HSF1.
  • Bin-bin Yu, Shu-ying Dong, Mei-ling Yu, Guo-jun Jiang, Jie Ji, Xu-hui ...
    2014 年 37 巻 8 号 p. 1315-1322
    発行日: 2014/08/01
    公開日: 2014/08/01
    [早期公開] 公開日: 2014/05/29
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    Oxaliplatin is widely used in the treatment of variety of cancers, including cancer of the testis and colorectum. Gap junctions (GJs) can amplify the cytotoxicity of antinoeoplastic drugs through the bystander effect in different cancer cells. In this study, we demonstrate that total flavonoids of litsea coreana (TFLC), one extract from the dried leaves of litsea coreana leve, increase the cytotoxicity of oxaliplatin in mouse testicular cancer I-10 cells. We found that cell survival was substantially decreased only when functional GJs formed in I-10 cells. TFLC increased oxaliplatin cytotoxity (inducing cell death and apoptosis) by enhancing gap junction intercellular communication (GJIC) through elevated Cx43 protein expression. Furthermore, apoptosis-related protein (Bax, Bcl-2, caspase-3/9) results showed that the Bax/Bcl-2 ratio and activated caspase-3/9 increased when TFLC was used compared with treatment with oxaliplatin alone, which suggests that the mechanism of increased oxaliplatin-induced apoptosis was through the mitochondrial pathway. These results demonstrate that TFLC can enhance the cytotoxicity of oxaliplatin, and that these processes may be regulated in testicular tumor cells through GJ-mediated regulation of tumor cell apoptosis.
  • Yuki Tazawa, Ippei Usukubo, Kazuki Takada, Yoh Takekuma, Yoshihiro Shi ...
    2014 年 37 巻 8 号 p. 1323-1329
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Combination chemotherapy is often used to treat cancer. Many studies have shown schedule-dependent effects between anticancer drugs. Our previous studies showed that K-562 cells pretreated with non-cytotoxic concentrations of 4-hydroperoxycyclophosphamide (4-HPC), which is a preactivated analog of cyclophosphamide (CY), enhanced the cytotoxicity of etoposide (VP-16). The appearance of cellular resistance to anticancer drugs is a major problem in cancer chemotherapy. P-Glycoprotein (P-gp) plays an important role in drug resistance, and VP-16 is a substrate for this efflux pump. In the present study, we demonstrated schedule-dependent cytotoxicity of VP-16 and CY in P-gp-overexpressed K-562/P-gp cells. Cytotoxicity of VP-16 was enhanced in K-562/P-gp cells that were pretreated with a non-cytotoxic concentration of 4-HPC compared to that of cells not treated with 4-HPC. 4-HPC arrested the cell cycle at S phase. Cells in S phase are most sensitive to VP-16. The results suggest that cell cycle arrest by 4-HPC pretreatment may be responsible for the enhanced cytotoxicity of VP-16. The findings in this study should lead to improvements in clinical combination chemotherapy.
  • Yuji Yamakawa, Akinobu Hamada, Takashi Uchida, Daisuke Sato, Misato Yu ...
    2014 年 37 巻 8 号 p. 1330-1335
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Nilotinib, a second-generation tyrosine kinase inhibitor (TKI), has been approved for first-line chronic myeloid leukemia (CML) treatment. The improved clinical response of nilotinib over that of the first generation TKI, imatinib, has been thought to be a result of its high potency of inhibition of BCR-ABL kinase. This study aimed to characterize differences between nilotinib and imatinib in the intracellular accumulation and cytotoxic effect on the CML cell line K562. Accumulation of nilotinib in K562 cells was from 4.7- to 9.0-fold higher than that of imatinib. The cytotoxic effect of nilotinib on K562 cells was 14.2-fold higher than that of imatinib. Inhibition experiments in K562 cells, and examination of the cellular uptake using influx transporter-transfected human embryonic kidney (HEK) 293 cells, suggested that the influx transporters OCT1 and OATP1A2, which have been reported to mediate accumulation of imatinib in CML cells, contributed little to the uptake of nilotinib. Nilotinib was found to accumulate in imatinib-resistant K562 (K562/IM) cells overexpressing the efflux transporter P-glycoprotein (P-gp), although cytotoxic assays showed that K562/IM cells displayed 20000-fold greater resistance to nilotinib over the parent K562 cells. In conclusion, the present findings suggest that intracellular accumulation of nilotinib in CML cells contributes to its clinical response and efficacy in CML patients. Although nilotinib has been reported to be effective against imatinib-resistant ABL kinase mutants, the drug could not overcome imatinib resistance acquired by P-gp-overexpression. These results imply that classification of mechanisms of drug resistance is important for suitable strategies to treat imatinib-resistant CML patients.
  • Takeshi Chiba, Soichiro Kimura, Katsuo Takahashi, Yasunori Morimoto, A ...
    2014 年 37 巻 8 号 p. 1336-1340
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Serotonin (5-hydroxytryptamine; 5-HT) has an important physiological role in controlling lactation, namely, milk volume homeostasis, within mammary glands. The objectives of this study were to evaluate whether exogenous 5-HT can suppress β-casein expression, a differentiation marker, produced in human mammary epithelial cells, and to determine whether 5-HT can attenuate β-casein signaling via the prolactin (PRL) receptor (PRLr) and Janus kinase 2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL treatment increased the mRNA level of β-casein in the MCF-12A human mammary epithelial cell line, and the highest level occurred at days 7 and 14 of culture. In contrast, PRLr expression was not affected significantly by PRL treatment. PRL treatment in MCF-12A cells increased levels of β-casein and phosphorylated STAT5 (pSTAT5) proteins in a concentration-dependent manner, with a slight increase of STAT5 protein. β-Casein expression was inhibited by 0.1 mM 5-HT in a time-dependent manner. Additionally, treatment with 0.1 mM 5-HT for 72 h decreased protein levels of β-casein and pSTAT5, with a slight decrease in STAT5 levels. These results suggest that exogenous 5-HT can inhibit STAT5 phosphorylation, resulting in a decrease in β-Casein expression. In conclusion, we showed that exogenous 5-HT decreased β-casein expression in MCF-12A human mammary epithelial cells, and that 5-HT was responsible for inhibiting phosphorylation of STAT5, resulting in a decline in lactational function.
  • Hee Kyung Jo, Go Woon Kim, Kyung Ju Jeong, Do Yeon Kim, Sung Hyun Chun ...
    2014 年 37 巻 8 号 p. 1341-1351
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Beneficial effect of eugenol on fatty liver was examined in hepatocytes and liver tissue of high fat diet (HFD)-fed C57BL/6J mice. To induce a fatty liver, palmitic acid or isolated hepatocytes from HFD-fed Sprague-Dawley (SD) rats were used in vitro studies, and C57BL/6J mice were fed HFD for 10 weeks. Lipid contents were markedly decreased when hepatocytes were treated with eugenol for up to 24 h. Gene expressions of sterol regulatory element binding protein 1 (SREBP1) and its target enzymes were suppressed but those of lipolysis-related proteins were increased. As a regulatory kinase for lipogenic transcriptional factors, the AMP-activated protein kinase (AMPK) signaling pathway was examined. Protein expressions of phosphorylated Ca2+-calmodulin dependent protein kinase kinase (CAMKK), AMPK and acetyl-CoA carboxylase (ACC) were significantly increased and those of phosphorylated mammalian target of rapamycin (mTOR) and p70S6K were suppressed when the hepatocytes were treated with eugenol at up to 100 µM. These effects were all reversed in the presence of specific inhibitors of CAMKK, AMPK or mTOR. In vivo studies, hepatic triglyceride (TG) levels and steatosis score were decreased by 45% and 72%, respectively, in eugenol-treated mice. Gene expressions of fibrosis marker protein such as α-smooth muscle actin (α-SMA), collagen type I (Col-I) and plasminogen activator inhibitor-1 (PAI-1) were also significantly reduced by 36%, 63% and 40% in eugenol-treated mice. In summary, eugenol may represent a potential intervention in populations at high risk for fatty liver.
  • Hitoshi Ueno, Hitomi Kajihara, Hajime Nakamura, Tomofumi Okuno, Fumito ...
    2014 年 37 巻 8 号 p. 1352-1358
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Although supplementation with the selenocompound, sodium selenite has been shown to stimulate the concanavalin A-induced T-cell mitogenic response, the mechanisms responsible remain unclear. This study was conducted to evaluate the relationships between the induction of apoptosis, formation of tumor necrosis factor (TNF)-alpha and reactive oxygen species (ROS), activation of apoptosis signal-regulating kinase (ASK) 1 and the thioredoxin (Trx) system when mitogenesis was stimulated by selenite. TNF-alpha was dose-dependently released by mouse splenocytes treated with selenite, and apoptosis was induced when TNF-alpha was added at the indicated concentrations. However, supplementation with selenite at low concentrations inhibited the accumulation of ROS with the increased expression of Trx reductase 1 and induction of apoptosis in wild-type splenocytes, and also at high concentrations in Trx-1-transgenic mouse splenocytes. The suppression of apoptosis was accompanied by a decrease in the expression of phospho-ASK1. These results suggest that the stimulation of T-cell mitogenesis by selenite may be partly attributed to the inhibited accumulation of ROS due to a reduced Trx-1/TR1 system, the inactivation of ASK1, and the suppression of apoptosis.
  • Tomoharu Yokooji, Hitomi Nouma, Hiroaki Matsuo
    2014 年 37 巻 8 号 p. 1359-1365
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Ingested proteins are absorbed from the intestinal lumen via the paracellular and/or transcellular pathways, depending on their physicochemical properties. In this study, we investigated the absorption pathway(s) of ovalbumin (OVA), an egg white-allergen, as well as the mechanisms of aspirin-facilitated OVA absorption in rats. In situ intestinal re-circulating perfusion experiments showed that the absorption rate of fluorescein isothiocyanate (FITC)-labeled OVA in the distal intestine was higher than that for a marker of non-specific absorption, FITC-dextran (FD-40), and that colchicine, a general endocytosis inhibitor, suppressed OVA absorption. In the distal intestine, bafiromycin A1 and phenylarsine oxide inhibited the OVA absorption rate, whereas mehyl-β-cyclodextrin exerted no significant effects. Thus, OVA is preferentially absorbed from the distal intestine via the paracellular and receptor- and clathrin-mediated endocytic pathways. Furthermore, aspirin increased OVA absorption in the presence or absence of colchicine, indicating that aspirin facilitated OVA absorption by inducing intestinal barrier disruption and paracellular permeability.
  • Ligang Jie, Hongyan Du, Qingchun Huang, Song Wei, Runyue Huang, Weifen ...
    2014 年 37 巻 8 号 p. 1366-1372
    発行日: 2014/08/01
    公開日: 2014/08/01
    [早期公開] 公開日: 2014/06/11
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    Tanshinone IIA (Tan IIA), a phytochemical derived from the roots of Salvia miltiorrhiza BUNGE, has been documented with anti-tumor, pro-apoptotic, and anti-inflammatory activities. Salvia miltiorrhiza has long been used to treat rheumatoid arthritis (RA). Apoptosis induction of RA-fibroblast-like synoviocytes (FLS) was suggested to be a potential therapeutic approach for RA. The aim of this study was to investigate whether Tan IIA promotes apoptosis in RA-affected FLS. In this study, the viability of an immortalized FLS cell line derived from RA patients was assessed by 3-(4,5-dimethylthiazol-2-yl)-5,3-carboxymethoxyphenyl-2,4-sulfophenyl-2H-tetrazolium (MTS) assay after Tan IIA treatment. Apoptosis was measured by terminal deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay and flow cytometry. Cell cycle was evaluated by flow cytometry. The expressions of mitochondrial apoptosis-related molecules, including Bcl-2, Bax, mitochondrial cytochrome c (Cyt-c), cytosolic Cyt-c, apoptotic protease activating factor 1 (Apaf-1), procaspase-9, procaspase-3, caspase-9, and caspase-3 were determined by Western blotting. Our data demonstrate that Tan IIA induced apoptosis of RA-FLS, blocked the cell cycle in the G2/M phase, and regulated the protein expression of Bcl-2, Bax, and Apaf-1, the release of mitochondrial Cyt-c, and the activation of caspase-9 and caspase-3. The results support the conclusion Tan IIA treatment likely induces apoptosis of RA-FLS through blockade of the cell cycle in the G2/M phase and a mitochondrial pathway. These data suggest that Tan IIA may have therapeutic potential for RA.
  • Maleenart Petchsangsai, Theerasak Rojanarata, Praneet Opanasopit, Tana ...
    2014 年 37 巻 8 号 p. 1373-1382
    発行日: 2014/08/01
    公開日: 2014/08/01
    [早期公開] 公開日: 2014/06/13
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    The objective of the present work was to investigate the effects of 3 combinatorial techniques (microneedle (MN), electroporation (EP), and sonophoresis (SN)) on the in vitro skin permeation of the hydrophilic macromolecular compound fluorescein isothiocyanate-dextran (FD-4; molecular weight (MW) 4.4 kDa). Assessment of the in vitro skin permeation of FD-4 was performed in porcine skin. MN, EP, and SN were used as physical enhancement methods, given the potential of their various mechanisms. The total cumulative amount of FD-4 that permeated through treated skin using 2 or 3 combined methods, i.e., MN+EP, MN+SN, EP+SN, and MN+EP+SN, was investigated. Microconduits created by MN alone and in combination with the other techniques were observed under confocal laser scanning microscopy (CLSM). The histology of the treated skin was examined. In vitro skin permeation experiments revealed that the total cumulative amount of FD-4 that permeated porcine skin using 3 combined techniques (MN+EP+SN) was greater than the amount observed using a single method or 2 combinations (MN+EP, MN+SN, SN+EP). The histological images indicate no noticeable damage in the skin treated with all of the enhancement methods. These results suggest that MN+EP+SN may serve as a potentially effective combination strategy to transdermally deliver various hydrophilic macromolecules without causing structural alterations or skin damage.
  • Hiroki Kinouchi, Hirokazu Arimoto, Kenzo Nishiguchi, Masako Oka, Hidek ...
    2014 年 37 巻 8 号 p. 1383-1389
    発行日: 2014/08/01
    公開日: 2014/08/01
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    In the present study, we examined the interaction of antimicrobial agents with four model lipid membranes that mimicked mammalian cell membranes and Gram-positive and -negative bacterial membranes and analyzed the binding kinetics using our surface plasmon resonance (SPR) technique. The selective and specific binding characteristics of antimicrobial agents to the lipid membranes were estimated, and the kinetic parameters were analyzed by application of a two-state reaction model. Reproducible analysis of binding kinetics was observed. Vancomyicn, teicoplanin, erythromycin, and linezolid showed little interaction with the four lipid membranes in the SPR system. On the other hand, vancomycin analogues showed interaction with the model lipid membranes in the SPR system. The selective and specific binding characteristics of vancomycin analogues to the lipid membranes are discussed based on data for in vitro antibacterial activities and our data on the binding affinity of the D-alanyl-D-alanine terminus of a pentapeptide cell wall obtained by SPR. The mechanism of antibacterial activity against Staphylococcus aureus and vancomycin-resistant enterococci could be evaluated using the binding affinity obtained with our SPR techniques. The results indicate that the SPR method could be widely applied to predict binding characteristics, such as selectivity and specificity, of many antimicrobial agents to lipid membranes.
Notes
  • Seung Bo Yang, Sang Min Lee, Ji-Hae Park, Tae Hoon Lee, Nam-In Baek, H ...
    2014 年 37 巻 8 号 p. 1390-1396
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Cynanchum wilfordii is one of most widely used medicinal plants in Oriental medicine for the treatment of various conditions. In the present study, we isolated cynandione A (CA) from an extract of Cynanchum wilfordii roots (CWE) and investigated the effects of CA on the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced BV-2 microglial cells. CWE and CA significantly decreased LPS-induced nitric oxide production and the expression of iNOS in a concentration-dependent manner, while they (CWE up to 500 µg/mL and CA up to 80 µM) did not exhibit cytotoxic activity. Results from reverse transcription-polymerase chain reaction (RT-PCR) analysis and enzyme-linked immunosorbent assay (ELISA) showed that CA significantly attenuated the expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β in LPS-stimulated BV-2 cells. Furthermore, CA inhibited the phosphorylation of inhibitor kappa B-alpha (IκB-α) and translocation of nuclear factor-kappa B (NF-κB) to the BV-2 cell nucleus, indicating that CWE and CA may have effective anti-inflammatory activities via NF-κB inactivation in stimulated microglial cells.
  • Ki Yong Lee, Ok-Nam Bae, Shelley Weinstock, Mounzer Kassab, Arshad Maj ...
    2014 年 37 巻 8 号 p. 1397-1401
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Asiatic acid (AA) is a pleiotropic neuroprotective agent that has been shown to attenuate infarct volume in mouse and rat models of focal ischemia and has a long clinically relevant therapeutic time-window. Because in a future trial AA would be administered with tissue-plasminogen activator (t-PA), the only approved acute stroke therapy, we sought to determine the effect of AA when co-administered with t-PA in a rat focal embolic stroke model. Male rats were treated with AA (75 mg/kg) alone, low-dose t-PA (2.5 mg/kg) alone, or a combination of AA and low-dose t-PA at 3 h after inducing embolic stroke. AA significantly reduced infarct volume whereas low-dose t-PA alone did not reduce infarct volume compared with vehicle. Significantly, combination treatment further enhanced reduction of infarct volume versus AA alone. Treatment with AA reduced cytochrome c (CytoC) and apoptosis-inducing factor (AIF) release from brain mitochondria after ischemia. AA was also neuroprotective against L-glutamate-induced toxicity in primary cortical neurons. In summary, combination treatment with AA and low-dose t-PA at 3 h after embolic stroke reduces infarct volume, improves neurological outcome, and provides neuroprotection. The neuroprotective effects of AA were partially associated with reduction of AIF and CytoC release.
  • Yoshitaka Kanayama, Chizuru Aoki, Nobuyuki Kawai, Morio Sato, Yasuo Sa ...
    2014 年 37 巻 8 号 p. 1402-1405
    発行日: 2014/08/01
    公開日: 2014/08/01
    [早期公開] 公開日: 2014/06/13
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    The purpose of this study was to clarify the adsorption of cisplatin on regenerative-medicine (RM) gelatin sponge, and to verify the relationship between the cisplatin release pattern of cisplatin-adsorbed RM gelatin sponge and the dissolving time of RM gelatin sponge. We tested various RM gelatin sponges, one with a molecular weight of 50000 Daltons (RM-50 gelatin sponge) that is 100% saline soluble at 24 h, RM-50-120 (heated at 120°C) that is 54.3% saline soluble at 24 h, and RM-50-140 (heated at 140°C) that is 15.8% saline soluble at 24 h. We investigated the production of cisplatin-adsorbed RM gelatin sponge and measured free cisplatin released from cisplatin-adsorbed RM gelatin sponge. There was no significant difference in the weight of adsorbed cisplatin among the RM-50, RM-50-120, and RM-50-140. The results mean that cisplatin adsorbs onto RM gelatin sponge irrespective of heating temperature. The average adsorbed weight of cisplatin per gram of RM gelatin sponge was 29.3 mg, which was approximately five times more than that per g previously reported for Gelpart (non-soluble gelatin sponge, clinically available). Cisplatin release in the RM-50 gelatin was the most rapid at only 1 h after incubation; it was released gradually and increasingly in the RM-50-120 gelatin, and released slowly in the RM-50-140 gelatin for 24 h incubation. Cisplatin-adsorbed RM gelatin sponge released cisplatin proportional to the dissolving time of RM gelatin sponge, indicating that the cisplatin release time can be controlled by heating for sterilization of RM gelatin sponge.
  • Yun-Ju Kwon, Mi-Jin Sohn, Hyun-Ju Kim, Won-Gon Kim
    2014 年 37 巻 8 号 p. 1406-1410
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Dihydrofolate reductase (DHFR) has been confirmed to be a novel target for antibacterial drug development. In this study, we determined that a fungal metabolite from Stachybotrys sp. FN298 can inhibit the DHFR of Staphylococcus aureus. Its structure was identified as a lactone form of stachybotrydial using mass spectrometry and nuclear magnetic resonance analysis. This compound inhibited S. aureus DHFR with a half-maximal inhibitory concentration of 41 µM. It also prevented the growth of S. aureus and methicillin-resistant S. aureus (MRSA) with a minimum inhibitory concentration of 32 µg·mL−1. To our knowledge, this is the first description of a DHFR inhibitor of microbial origin. The inhibitory function of the lactone form of stachybotrydial highlights its potential for development into a new broad-spectrum antibacterial agent and as an agent against MRSA.
  • Yuri Tawaraya, Mamoru Hyodo, Mst Naznin Ara, Yuma Yamada, Hideyoshi Ha ...
    2014 年 37 巻 8 号 p. 1411-1415
    発行日: 2014/08/01
    公開日: 2014/08/01
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    The use of mitochondria-based systematic evolution of ligands by exponential enrichment (SELEX) was explored. Mitochondria were isolated from rat liver and confirmed intact by respiratory control index. Isolated mitochondria and a 2′-F RNA random library were mixed and the bound RNAs collected. The counter selection was applied with nucleus and unbound RNAs were collected. After 7 rounds of selection, two sequences (Mitomer1 and Mitomer2) were verified to bind to mitochondria and the truncated Mitomer2 (short Mitomer2) showed better binding to isolated mitochondria than Mitomer1.
  • Keiko Kumagai, Mai Akakabe, Masayuki Tsuda, Masashi Tsuda, Eri Fukushi ...
    2014 年 37 巻 8 号 p. 1416-1421
    発行日: 2014/08/01
    公開日: 2014/08/01
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    Hyperpolarization of stable isotope-labeled substrates and subsequent NMR measurement of the metabolic reactions allow for direct tracking of cellular reactions in vitro and in vivo. Here, we report the hyperpolarization of 13C6-glucose-d7 and evaluate its use as probes to observe glucose flux in cells. We measured the lifetime of the polarized signal governed by the spin–lattice relaxation time T1. 13C6-Glucose-d7 exhibited a T1 that was over ten times as long as that of 13C6-glucose, and metabolic NMR studies of hyperpolarized 13C6-glucose-d7 using tumor cell lysate led to observation of the resonances due to phosphorylated fluctofuranoses generated through aerobic glycolysis.
  • Daisuke Kondoh, Saori Yamamoto, Tatsunosuke Tomita, Koyomi Miyazaki, N ...
    2014 年 37 巻 8 号 p. 1422-1427
    発行日: 2014/08/01
    公開日: 2014/08/01
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    The circadian clock is a cell-autonomous endogenous system that generates circadian rhythms in the behavior and physiology of most organisms. We previously reported that the harmala alkaloid, harmine, lengthens the circadian period of Bmal1 transcription in NIH 3T3 fibroblasts. Clock protein dynamics were examined using real-time reporter assays of PER2::LUC to determine the effects of harmine on the central clock in the suprachiasmatic nucleus (SCN). Harmine significantly lengthened the period of PER2::LUC expression in embryonic fibroblasts, in neuronal cells differentiated from neuronal progenitor cells and in SCN slices obtained from PER2::LUC mice. Although harmine did not induce the transient mRNA expression of clock genes such as Per1, Per2 and Bmal1 in embryonic fibroblasts, it significantly extended the half-life of PER2::LUC protein in neuronal cells and SCN slices. Harmine might lengthen the circadian period of the molecular clock by increasing PER2 protein stability in the SCN.
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