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Shuang Wang, Mingsheng Wu, Dan Li, Mingli Jiao, Lan Wang, Haifeng Zhan ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1898-1906
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 27, 2012
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The goal of this project was to prepare long-acting lanreotide acetate poly(lactic-
co-glycolic acid) (PLGA) microspheres and to analyze the
in vivo and
in vitro release, safety and toxicology of these preparations. Long-acting lanreotide acetate PLGA microspheres that exhibited a 5-week slow-release period were prepared by a multiple-emulsion solvent evaporation method. Physical characterization, as well as the analysis of the
in vivo and
in vitro release, safety, acute toxicity and chronic toxicity of the lanreotide microspheres, were conducted in animal models in rats, guinea pigs, rabbits and beagle dogs. The lanreotide acetate PLGA microspheres prepared by multiple-emulsion solvent evaporation had smooth surfaces, uniform particle size and stable lanreotide loading.
In vivo and
in vitro experiments showed that the lanreotide acetate PLGA microspheres could continuously release lanreotide for 5 weeks. The safety of these long acting lanreotide microspheres was good in the following animal models: active systemic anaphylaxis test in guinea pigs, passive cutaneous anaphylaxis test in rats, hemolytic test in rabbits, local skin irritation test after subcutaneous administration in rabbits and muscle stimulation test in rabbits. Furthermore, no significant acute toxicity or chronic toxicity was observed after administration of lanreotide acetate PLGA microspheres in beagle dogs at dosages up to 22 mg/kg. The lanreotide acetate PLGA microspheres that were prepared in this study exhibited beneficial characteristics in apparent property and structural stability, as well as in release trends
in vivo and
in vitro.
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Bidur Bhandary, Geum-Hwa Lee, Anu Marahatta, Hak-Yong Lee, Sun-Young K ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1907-1913
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Supplementary material
Hyperlipidemia is a major contributor for atherosclerosis and hypolipidemic drugs such as statin are highly prescribed to treat elevated lipid level in plasma.
Rubus coreanus, which is widely cultivated in south eastern Asia, have been reported to show significant cholesterol lowering action in hyperlipidemic subjects. Our objective was to determine the cellular effect of
Rubus coreanus extract (RCE) on cholesterol biosynthesis in human hepatic cells (HepG2) and to elucidate the molecular mechanism by which it causes change in cholesterol metabolism. RCE treatment lowered cholesterol biosynthesis as well as secretion from HepG2 cells. This effect was associated with lowering the release of apolipoproteins from hepatic cells. RCE treatment also showed an increase in phosphorylation of foxhead box protein 01 (FoXo-1) and 5-adenosine monophosphate-activated protein kinase (AMPK), thus lowering expression of phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase, which might be a major pathway for cholesterol biosynthesis inhibition. Apart from this; RCE also lowered sterol regulatory element-binding protein-1 (SREBP-1) expression in HepG2 cells, showing a long term regulation of cholesterol biosynthesis activity. These results indicate that one of the anti-hyperlipidemic actions of RCE is due to inhibition of cholesterol biosynthesis in hepatic cells and provides first documentation of a hypolipidemic bio-molecular action of
Rubus coreanus.
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Chariya Hahnvajanawong, Supaluk Ketnimit, Kovit Pattanapanyasat, Natth ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1914-1925
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 22, 2012
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Cell cycle arrest is closely linked to apoptosis. Isomorellin—a caged xanthone isolated from
Garcinia hanburyi—induced apoptosis in cholangiocarcinoma (CCA) cell lines. To elucidate potential anticancer mechanisms, we investigated the effects of isomorellin on the growth, cell cycle progression, cell cycle regulated protein expression and nuclear factor-kappa B (NF-κB) activation of KKU-100 and KKU-M156 CCA cell lines; using sulforhodamine B assay, flow cytometry and Western blot analysis. The growth of both CCA cell lines was significantly inhibited by isomorellin treatment in a time- and dose-dependent manner. The respective IC
50 value of isomorellin for KKU-100 cells was 6.2±0.13, 5.1±0.11 and 3.5±0.25 µ
M at 24, 48 and 72 h. By comparison, the respective IC
50 value for KKU-M156 cells was 1.9±0.22, 1.7±0.14 and 1.5±0.14 µ
M at 24, 48 and 72 h. The growth inhibition of CCA cells by isomorellin was through the G0/G1 phase arrest mediated by inhibition of NF-κB activation, up-regulation of p53, p21 and p27 and down-regulation of cyclin D1, cyclin E, Cdk4 and Cdk2 protein levels. Our research suggests that isomorellin induces cell cycle arrest and apoptosis in CCA cell lines through p53 and the NF-κB-signaling pathway. The growth inhibitory potential of isomorellin was comparable to that of gambogic acid. Isomorellin shows potential as a therapeutic agent against human cholangiocarcinoma.
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Tetsuya Ozeki, Yusuke Akiyama, Norimitsu Takahashi, Tatsuaki Tagami, T ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1926-1931
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Supplementary material
Production of drug nanoparticles is an effective strategy to enhance solubility and oral absorption of water-insoluble drugs. The handling of drug nanoparticles has been an important issue in drug formulation because nanoparticles easily aggregate each other and redispersion of these particles is very difficult. In the present study, we developed a unique two-solution mixing type spray nozzle that can prepare drug nanoparticles in microparticles in one step without any common solvent and surfactant, and then, the prepared formulation were evaluated. Ethylcellulose (EC) and mannitol (MAN) were used as a model polymer of water-insoluble compound and a water-soluble carrier, respectively. We characterized the EC/MAN microparticles produced by the novel spray nozzle when customizing the nozzle parts to mix EC and MAN solution. Relatively smaller EC nanoparticles (<110 nm) in MAN microparticles (approximately 3 µm) were obtained by changing the customizable parts in the nozzle. In addition, the core of EC nanoparticles (<50 nm) was also observed by atomic force microscopy. We also found that the mixing time in the nozzle parts affected the size and the standard deviation of EC nanoparticles. These results suggest that the size of EC nanoparticles in MAN microparticles is controllable by using this unique nozzle. After all, we could prepare MAN microparticles containing EC nanoparticles in one step by using the novel nozzle. The drug/MAN microparticles formulation produced by the nozzle may be useful for the handling of drug nanoparticles.
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Hironori Motoshige, Kyohei Oyama, Kiyoshi Takahashi, Koichi Sakurai
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1932-1940
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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This study demonstrated gemcitabine-induced cytotoxicity in the insulinoma cell line INS-1. Gemcitabine inhibited INS-1 cell proliferation and maintained consistent cell number for 24 h, and then caused apoptosis within 48 h of incubation. Since gemcitabine activates the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, which is involved in the resistance of pancreatic exocrine cancer to gemcitabine, we investigated the participation of this pathway in gemcitabine-induced cytotoxicity in INS-1 cells. LY294002 and wortmannin, two PI3-K inhibitors, significantly prevented gemcitabine-induced cytotoxicity in INS-1 cells, indicating that the PI3-K/Akt pathway is involved in gemcitabine-induced cytotoxicity. Gemcitabine-induced Akt phosphorylation in INS-1 cells was prevented by LY294002. Although gemcitabine induced cell cycle arrest at the G1 and early S phases, LY294002 did not inhibit the cell cycle. These data suggest that PI3-K activation does not influence gemcitabine-induced cell cycle arrest. In gemcitabine-treated cells, nuclear fragmentation and DNA ladder formation were observed. These findings suggest that gemcitabine induced apoptotic cell death in INS-1 cells through the activation of the PI3-K/Akt pathway.
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Sachiko Juman, Naomi Yasui, Katsumi Ikeda, Ai Ueda, Mariko Sakanaka, H ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1941-1946
Published: 2012
Released on J-STAGE: November 01, 2012
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Obesity is a condition in which excess body fat accumulates due to lipids producing adipocytes and an increased number of differentiated mature cells. Recently, new findings have shown that macrophages infiltrate into adipose tissues and produce various pro-inflammatory cytokines in obese subjects. The inflammatory changes induced by the cross-talk between adipocytes and macrophages are critical for the pathophysiology of obesity and thus of metabolic syndrome. Caffeic acid phenethyl ester (CAPE) is known to have many functions, including antibacterial, anticancer and anti-inflammatory properties, but there is no evidence of its effect on the inflammatory responses in hypertrophic adipocytes through stimulation by macrophages. We investigated the effect of CAPE on macrophages and hypertrophic adipocytes in this study. CAPE significantly suppressed the levels of lipopolysaccharide (LPS)-induced interleukin (IL)-1-beta, tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1 from a macrophage cell line, RAW264.7. Supernatants of stimulated RAW264.7 macrophages drastically increased mRNA levels of pro-inflammatory cytokines such as IL-6, MCP-1 and TNF-alpha in 3T3-L1 hypertrophic adipocytes. CAPE also significantly and dose-dependently reduced the gene expression of these cytokines. Our findings indicate that CAPE has inhibitory effects on the production of pro-inflammatory cytokines from LPS-stimulated RAW264.7 macrophages. In addition, CAPE suppressed gene expressions of cytokines under inflammatory conditions of hypertrophic adipocytes, suggesting that it may have the potential to suppress inflammation by macrophage infiltration into adipose tissue in obese patients.
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Pi-wen Zhao, David Yue-wei Lee, Zhong-ze Ma, Lian-sha Huang, Li-ping S ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1947-1955
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 28, 2012
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Antioxidant action is critical for maintaining the normal cardiovascular function and vascular endothelial cell is an important target of estrogen action through estrogen receptor (ER) pathway. This study is carried out to explore the antioxidant effect of carnosol in bovine aortic endothelial cells (BAECs)
via ER pathway. The ER subtype specific estrogenic effect of carnosol was further demonstrated by luciferase reporter gene assay in human embryonic kidney (HEK) 293 cells. Carnosol was extracted from Chinese medicine
Rosmarinus officinalis. ER positive BAECs were employed in cell proliferation assay and cell apoptosis tests. Oxidative stress by intracellular reactive oxygen species (ROS) were measured
via 2′7′-dichlorofluorescein (DCF) production. ERα and ERβ specific antagonists 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1
H-pyrazole (MPP) and 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-
a]pyrimidine-3-yl]phenol (PHTPP) were employed as tools in the experiment. ER negative HEK 293 cells were employed in luciferase reporter gene assay. The results indicate that carnosol can effectively attenuate H
2O
2 induced slowing down of cell growth and increasing of cell apoptosis. At the meantime, carnosol pretreating can also effectively reduce the H
2O
2 induced intracellular ROS elevation in BAECs. ERα and ERβ antagonist, especially ERα antagonist, can effectively decrease the above antioxidant effects of carnosol. The reporter gene analysis further demonstrates that the action of carnosol on inducing ERE dependent luciferase expression is realized
via ER pathway. The conclusion is that carnosol can exert antioxidant effects towards oxidative stress induced by H
2O
2 in BAECs. And such effects are realized
via ER, especially ERα pathway. The results contribute to explain the mechanism of cardiovascular protective function of carnosol in postmenopausal women.
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Hao Miao, LiuYa Zhao, ChunLi Li, QingHua Shang, Hui Lu, ZiJin Fu, Li W ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1956-1963
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Our study showed that Shikonin (SK) could provide an action against almost all
Candida albicans isolates tested. More importantly, to some Fluconazole (FCZ)-resistant
Candida albicans, the action of SK (MIC
80 value 4 µg/mL) was shown to be >16 times higher than that of FCZ (MIC
80 >64 µg/mL). To clarify the mechanism underlying this action, we performed a comparative study in untreated control
C. albicans and
C. albicans treated with SK. In this study, we found that SK treatment increased generation of endogenous reactive oxygen species (ROS) and decreased mitochondrial membrane potential. Furthermore, anti-oxidants
N-acetylcysteine (NAC) and glutathione (GSH) could reduce the antifungal activity of SK significantly in
C. albicans. Our analyses also identified 9 differentially expressed genes, which were related to glycolysis-related genes (
CDC19 and
HXK2), fermentation-related genes (
ALD5 and
ADH1), antioxidant defense-related genes (
SOD2 and
SOD5), thioredoxin reductase-related gene (
TRR1), mitochondrial respiratory electron transport chain-related gene (
MRF1) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidoreductase-related gene (
EBP1). These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the action of SK against
C. albicans.
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Zhenwei Zhang, Lei Miao, Weizhang Sun, Binghua Jiao, Bingui Wang, Li Y ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1964-1971
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 27, 2012
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Wentilactone B (EN-48-57) is one of the six derivatives separated from
Aspergillus wentii (EN-48). Of these derivatives, Wentilactone B exerted a more significant antibacterial and cytotoxic activity in several tumor cell lines. The present study demonstrates that Wentilactone B could efficiently induce SMMC-7721 cells apoptosis, but not normal hepatic cells, as measured by an inverted microscope, 4′,6-diamidino-2-phenylindole staining and flow cytometry. In addition, Wentilactone B could inhibit the metastasis of SMMC-7721 cells, which was detected by colony formation, scratch migration and a transwell assay, and could induce a series of intracellular events, including the down-regulation of CD44 and epidermal growth factor receptor proteins. In conclusion, Wentilactone B inhibited the growth of SMMC-7721 cells by triggering apoptosis and inhibiting metastasis.
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Masashi Morita, Saori Shinbo, Akiko Asahi, Tsuneo Imanaka
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1972-1979
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Very long chain fatty acid (VLCFA) metabolism in astrocytes is important for the maintenance of myelin structure in central nervous system. To analyze the contribution of the ABCD1-dependent and -independent pathways to VLCFA metabolism in astrocytes, we prepared human glioblastoma U87 cells with a silencing of ABCD1 and primary astrocytes from abcd1-deficient mice, and measured fatty acid β-oxidation in the presence or absence of a potent inhibitor of carnitine palmitoyltransferase I, 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA). In U87 cells, C24:0 β-oxidation was decreased to
ca. 70% of the control in the presence of POCA, and the activity was further decreased to
ca. 20% by the silencing of ABCD1. In mouse primary astrocytes, C24:0 β-oxidation was also decreased to
ca. 70% of the control in the presence of POCA. The C24:0 β-oxidation in Abcd1-deficient primary astrocytes was
ca. 60% of the wild-type cells and the activity was further decreased to
ca. 25% in the presence of POCA. Compared to human skin fibroblasts, in which VLCFA β-oxidation is not significantly inhibited by POCA, approximately one-third of the overall VLCFA β-oxidation was inhibited in both types of astrocytic cells. These results suggest that VLCFA is indeed β-oxidized in ABCD1-dependent pathway, but the ABCD1-independent peroxisomal and mitochondrial β-oxidation pathways significantly contribute to VLCFA β-oxidation in astrocytic cells.
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Shinya Hasegawa, Yotaro Ikeda, Masahiro Yamasaki, Tetsuya Fukui
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1980-1985
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Supplementary material
Acetoacetyl-CoA synthetase (AACS) is a ketone body-utilizing enzyme that converts acetoacetate to acetoacetyl-CoA in the cytosol and consequently provides acetyl units as the precursors for lipogenesis. To clarify the role of AACS in adipogenesis, we investigated the expression and localization of the AACS protein and the effect of AACS knockdown on 3T3-L1 differentiation. The protein expression of AACS is dramatically induced during 3T3-L1 differentiation and is localized in the cytoplasm of differentiated 3T3-L1 cells. Moreover, knockdown of AACS inhibits differentiation of 3T3-L1 cells and suppresses expression of the adipocyte markers, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). These results suggest that AACS has a crucial role in the mechanism of 3T3-L1 differentiation.
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Lorena Maione-Silva, Kamilla Amaral David Rocha, Lidiane Correia de Ol ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1986-1990
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 30, 2012
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Genistein (GEN) has potential advantages for topical skin delivery, but no literature data are available for its quantitation in different skin layers, such as the stratum corneum (SC). Therefore, a simple, rapid, selective and sensitive bioanalytical method was developed and validated for GEN quantitation in porcine skin samples following
in vitro permeation studies. GEN was assayed by HPLC with UV-Vis detection (270 nm) using 0.5% acetic acid in water–
n-propanol–acetonitrile (50 : 2 : 48, v/v/v) as mobile phase (flow-rate of 1.0 mL/min). Specificity was demonstrated since endogenous skin components did not interfere with GEN peak. Standard analytical curve was linear over the concentration range (0.1–60 µg/mL) and the lower limit of quantitation was determined for different skin layers (100 ng/mL). GEN recovery from skin layers ranged from 95.57 to 97.57%. Permeation studies were carried out using an automated vertical diffusion cell apparatus. No fluctuation on the amount of GEN retained in the SC was observed over time, but increasing amounts of the drug were found in deeper layers of the skin. The method was reliable and reproducible for the quantitation GEN in skin samples enabling the determination of the cutaneous penetration profile of this drug in permeation experiments.
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Qingmei Tian, Hongsheng Bi, Yan Cui, Dadong Guo, Xiaofeng Xie, Weihua ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1991-1996
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Qingkailing injection is a well-known composite formula of traditional Chinese medicine and is commonly used in clinical practice, which could offer immunomodulatory effect from our clinical experience on uveitis treatment by Qingkailing. We did the experiment in order to investigate the curative effect and mechanism of Qingkailing injection to rat experimental autoimmune uveitis (EAU). EAU was induced in Lewis rats by immunization IRBP1177–1191 in complete Freund’s adjuvant in multi-point. We found that Qingkailing injection can alleviate autoimmune uveitis in rats, inhibit the differentiation toward Th1 and Th17 effector cells and the relevant cytokines secretion. The therapeutic effect may also be regulated through increased secretion of interleukin (IL)-10.
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Pornthip Waiwut, Myoung-Sook Shin, Satoru Yokoyama, Ikuo Saiki, Hiroak ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
1997-2003
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Gomisin A, a dibenzocyclooctadiene lignan isolated from the fruit of
Schisandra chinensis, has been reported as an anti-cancer substance. In this study, we investigated the effects of gomisin A on cancer cell proliferation and cell cycle arrest in HeLa cells. Gomisin A significantly inhibited cell proliferation in a dose-dependent manner after 72 h treatment, especially in the presence of tumor necrosis factor-α (TNF-α), due to cell cycle arrest in the G1 phase with the downregulation of cyclin D1 expression and Retinoblastoma (RB) phosphorylation. In addition, gomisin A in combination with TNF-α strongly suppressed the expression of signal transducer and activator of transcription 1 (STAT1). Inhibition of STAT1 pathways by a small-interfering RNA against STAT1 and AG490 Janus kinase (JAK) kinase inhibitor AG490 reduced the cyclin D1 expression and RB phosphorylation, indicating that JAK-mediated STAT1 activation is involved in gomisin A-induced G1 cell cycle arrest.
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Tao Fan, Wei Long Jiang, Jian Zhu, Yu Feng Zhang
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
2004-2009
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 22, 2012
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Stroke is the third leading cause of death in industrialized countries and the most important cause of acquired adult disability. Many evidences suggest that inflammation accounts for the progression of cerebral ischemic injury. Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignin isolated from certain plants, has shown anti-inflammatory activity against diabetes and Alzheimer’s disease. In this study, we tested whether arctigenin can protect middle cerebral artery occluded (MCAO) rats. Male Sprague-Dawley rats were pretreated with arctigenin or vehicle for 7 d before being subjected to transient occlusion of middle cerebral artery and reperfusion. Rats were evaluated at 24 h after MCAO for neurological deficit scoring. Furthermore, the mechanism of the anti-inflammatory effect of arctigenin was investigated with a focus on inflammatory cells, proinflammatory cytokines, and transcriptional factors. Arctigenin significantly reduced cerebral infarction and improved neurological outcome. Arctigenin suppressed the activation of microglia and decreased the expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α. These results revealed that arctigenin has a promising therapeutic effect in ischemic stroke treatment through an anti-inflammatory mechanism.
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Malik Suliman Mohamed Mustafa, Yoshitaka Nakajima, Hiroshi Oyama, Nobu ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
2010-2016
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Supplementary material
Oligopeptidase B (OPB; EC 3.4.21.83) from 2 Gram-negative bacteria,
Stenotrophomonas maltophilia (Stm) and
Serratia marcescens (Sem), and the Gram-positive bacterium
Rhodococcus erythropolis (Re) were cloned and characterized to clarify their activities and substrate specificities using peptidyl-MCA substrates containing Arg or Lys. The cloned enzymes, Stm, Sem and ReOPBs, in addition to
Escherichia coli OPB (EcOPB) were expressed using a pET expression system. Although the Stm and SemOPBs share 45% sequence identity to each other and up to 60% identity with respect to their catalytic domains, their activities towards MCA substrates were quite different. StmOPB is approximately 100–500 times more active than SemOPB and 3–30 times more active than EcOPB. The activity of ReOPB is comparable to that of StmOPB and it shares 40% and 36% identity to StmOPB and SemOPB, respectively. Some features of Stm, Re and EcOPBs are similar to those of previously cloned OPBs, which were also strongly inhibited by substrates, but SemOPB differs from all other OPBs in that it is not inhibited by substrates; even substrates containing double arginine at 35 µ
M did not inhibit SemOPB. On the other hand, the same substrates at only 5 µ
M inhibited the activity of the Stm, Re, and EcOPB. This phenomenon was not observed with substrates containing single or double lysine.
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Sayako Maruyama, Noriko Ohkita, Minoru Nakayama, Eiko Akaboshi, Takehi ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
2017-2022
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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RecQ5 is a member of the RecQ family of DNA helicases. There are 5 RecQ members in humans. Defects in 3 of them,
i.e., BLM, WRN, and RTS, cause Bloom, Werner, and Rothmund–Thomson syndromes, respectively. RECQL1 and RECQL5 have not been associated with any human disease, and their precise roles are unknown. Our previous study suggests that the lack of RecQ5, which is the
Drosophila homolog of RECQL5, leads to the accumulation of DNA double-stranded breaks (DSBs). It is possible that RecQ5 is involved in DSB repair. However, little is known about this possible function of RecQ5 in DSB repair. Here, we report that Rad51 protein, which plays a critical role in DSB repair, interacted with RecQ5
in vitro and
in vivo in
Drosophila. The Rad51 protein interacted with the C-terminal region of RecQ5, as shown by the yeast two-hybrid method. Moreover, the C-terminal region of the RecQ5 protein and the central region of Rad51 interacted directly and specifically when examined by the glutathione-
S-transferase pull-down method. Consistent with these results, when RecQ5 and Rad51 were co-expressed in
Drosophila cells in culture, they became co-localized in nuclei and could be co-immunoprecipitated. Furthermore, RecQ5-deficient flies (
recq5) were more sensitive to the chemotherapeutic agent cisplatin compared with wild-type ones. Also, Rad51 mutants (
rad51) were more sensitive to cisplatin, with sensitivity similar to that of
recq5 rad51 double mutants. These data suggest that RecQ5 and Rad51 in
Drosophila functioned for survival after the flies had been treated with cisplatin.
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You Jung Kim
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
2023-2027
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: September 03, 2012
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Hyperin and quercetin are phenolic compounds present in fruits and vegetables that have been reported to possess strong anti-oxidative properties. Although increasing evidence strongly suggests that antioxidants suppress the melanin synthesis that is causally associated with oxidative stress, the protective actions of hyperin and quercetin against oxidative stress-induced melanogenesis have not been fully explored. To elucidate the suppressive effects of hyperin and quercetin on oxidative stress and melanin synthesis, peroxynitrite (ONOO
−) scavenging activity was measured
in vitro as were quantifications of melanin content, intracellular total RS, ONOO
−, superoxide (
•O
2), nitric oxide (NO
•), catalase activity and the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. Results showed that
in vitro, hyperin and quercetin reduced ONOO
−. Additionally, hyperin and quercetin suppressed total RS, ONOO
−,
•O
2, and NO
•, catalase activity, and melanin synthesis, while they boosted the GSH/GSSG ratio in B16F10 melanoma cells (B16 cells). Therefore, I propose that hyperin and quercetin have a powerful capacity to modulate oxidative stress-induced melanogenesis.
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Mamiko Kajiwara, Takahiro Ueno, Noboru Fukuda, Hiroyuki Matsuda, Toshi ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
2028-2035
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: September 05, 2012
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Fcγ receptors I and III are thought to be involved in the development of lupus nephritis. Expression of Fc receptor common gamma chain (FcRγ) is necessary for the stable expression of Fcγ receptors I and III. The aim of this study was to develop a novel agent for the treatment of immune complex related renal disease using a gene regulator, pyrrole(Py)–imidazole(Im) (PI) polyamide, targeting the mouse FcRγ gene promoter. Two PI polyamides targeting FcRγ promoters were designed and synthesized. The effect of the PI polyamides on FcRγ mRNA expression was evaluated in J774.A cells by real-time polymerase chain reaction (PCR), and CD16/32 protein expression was determined by immunocytochemical analysis and flow cytometry. The effects of these polyamides on FcRγ gene expression and CD16/32 protein expression were evaluated in mouse peripheral blood mononuclear cells (PBMCs). One milligram per kilogram body weight of PI polyamide was injected
via the tail vein every 2 d for 1 week and PBMCs were collected and analyzed. PI polyamide showed a specific binding to the target DNA in a gel mobility shift assay. Treatment of J774.A cells with 1.0 µ
M PI polyamide 1 significantly reduced FcRγ mRNA expression and CD16/32 surface protein expression in J774.A cells. Similarly, PI polyamide significantly decreased expression of FcRγ mRNA and CD16/32 in the PBMCs of C57B6 mice. PI polyamide designed to bind the FcRγ promoter decreased FcRγ gene and CD16/32 protein expression. PI polyamide targeting the FcRγ gene may be a novel gene regulator for the prevention of lupus nephritis or other immune complex-related disease.
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Ji-Young Park, Hyung Jae Jeong, Jang Hoon Kim, Young Min Kim, Su-Jin P ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
2036-2042
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: September 03, 2012
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Supplementary material
The papain-like protease (PL
pro), which controls replication of the severe acute respiratory syndrome coronavirus (SARS-CoV), has been identified as a potential drug target for the treatment of SARS. An intensive hunt for effective anti-SARS drugs has been undertaken by screening for natural product inhibitors that target SARS-CoV PL
pro. In this study, diarylheptanoids
1–
9 were isolated from
Alnus japonica, and the inhibitory activities of these compounds against PL
pro were determined. Of the isolated diarylheptanoids, hirsutenone (
2) showed the most potent PL
pro inhibitory activity, with an inhibitory concentration (IC
50) value of 4.1 µ
M. Structure–activity analysis showed that catechol and α,β-unsaturated carbonyl moiety in the molecule were the key requirement for SARS-CoV cysteine protease inhibition.
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Takafumi Naito, Takashi Osawa, Naoya Suzuki, Toshiya Goto, Akira Takad ...
Article type: Regular Article
2012 Volume 35 Issue 11 Pages
2043-2049
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Contamination of the external surface of anticancer drug vials supplied to hospital pharmacies has been recognized as a potential health hazard. The aim of this study was to investigate the levels of contamination on the exterior surface of vials containing platinum anticancer drugs in Japan. Platinum contamination on the exterior surface of vials containing cisplatin or carboplatin was examined using products commercially available in Japan. Cisplatin vials from 42 batches (2 drug contents, 10 products and 5 manufacturers) and carboplatin vials from 28 batches (3 drug contents, 7 products and 3 manufacturers) were used. Five vials were randomly sampled from each batch. Exterior contamination levels of 0.070–144 ng/vial as cisplatin and 0.21–1630 ng/vial as carboplatin were detected. Significant differences in the levels of contamination among the batch numbers were observed in 6 of 10 cisplatin products and 6 of 7 carboplatin products. Significant differences in the levels of contamination were observed in 3 cisplatin products with different contents of drug within the vials and 1 carboplatin product with different contents of drug within the vials. Significant differences in the contamination levels among the cisplatin manufacturers but not carboplatin manufacturers were observed. The degree of contamination of the carboplatin products was significantly higher than that of the cisplatin products. In conclusion, external contamination was confirmed in all cisplatin and carboplatin vials tested. The degree of contamination was different among different batch numbers, drug contents, manufacturers, and platinum anticancer drug.
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Zhenzhen Ni, Zhengbing Zhuge, Wenlu Li, Huimin Xu, Zhongmiao Zhang, Ha ...
Article type: Note
2012 Volume 35 Issue 11 Pages
2050-2053
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 31, 2012
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To investigate the inhibitory effects of hydroxysafflor yellow A (HSYA) on the protein glycation
in vitro. Using bovine serum albumin (BSA)-glucose assay, BSA-methylglyoxal (MGO) assay, and
N-acetylglycyl-lysine methyl ester (G.K.) peptide-ribose assay, inhibitory effects of HSYA were investigated. Advanced glycation end products (AGEs) production was assessed by AGEs-specific fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In BSA-glucose assay, HSYA concentration dependently decreased AGEs formation, with maximum inhibitory effects at 1 m
M by 95%. Further more, HSYA also showed significant inhibitory effects on MGO-medicated protein modification and subsequent cross-linking of proteins. Finally, when co-incubated with G.K. peptide and ribose, HSYA exhibited its antiglycation effects, and the maximum inhibitory effects of HSYA at 1 m
M were 84%. Overall, our present study provides the first evidence of the antiglycation effects of HSYA on AGEs formation
in vitro.
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Hiroki Ozawa, Yoshiko Sonoda, Saori Kato, Erika Suzuki, Ryotaro Matsuo ...
Article type: Note
2012 Volume 35 Issue 11 Pages
2054-2058
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: August 20, 2012
JOURNAL
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Supplementary material
Endogenous sulfatide, such as 3-sulfated galactosylceramide (3-sulfatide) has been reported to be involved in neuronal development and regulation of tumor cell metastasis. Recently, a new 6-sulfated glucosylceramide (6-sulfatide) has been isolated from the ascidian,
Ciona intestinalis. To determine the antitumor function of the new sulfatide, we examined the effects of synthetic 6-sulfatide and 3-sulfatide on the metastatic features of a murine melanoma cell line, B16F10. Both sulfatides significantly inhibited the adhesion of melanoma cells onto fibronectin-coated tissue plates and, the motility and invasion of the cells, with 6-sulfatide showing stronger inhibitory activities. In addition, both sulfatides inhibited α
5-, and β
1- but not α
v- or β
3-integrin expression. Furthermore, these sulfatides inhibited the activation of focal adhesion kinase, Akt, and extracellular signal-regulated kinase signaling pathways, which are thought to be important for cell migration and invasion. Therefore, these sulfatides may serve as promising drug candidates for the treatment of cancer metastasis.
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Masaki Ino, Yoshibumi Shimizu, Tamotsu Tanaka, Akira Tokumura
Article type: Note
2012 Volume 35 Issue 11 Pages
2059-2063
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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The aim of this study was to investigate the effect of fasting on
in vivo plasma levels of lysophosphatidic acid (LPA), a physiologically important lysophospholipid mediator. We assayed to measure activities of an LPA-producing enzyme (lysophospholipase D) and LPA-degrading enzyme activities (lysophspholipase A, lipid phosphate phosphatase) in rat plasma or blood, by measuring choline, fatty acid and inorganic phosphate, respectively. Both LPA and its precursor lysophosphatidylcholine (LPC) were quantified by liquid chromatography-tandem mass spectrometry. Fasting of rats for 24 h decreased plasma concentrations of oleoyl-, linoleoyl-, arachidonoyl- and docosahexaenoyl-LPAs, but not palmitoyl- and stearoyl-LPAs, possibly due to decreased levels of corresponding LPCs in the plasma and elevated lipid phosphate phosphatase activity for LPAs in the blood. Our results indicate that the
in vivo circulating levels of LPAs in rats are affected by fasting.
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Hidesuke Fukazawa, Atsuko Masumi
Article type: Note
2012 Volume 35 Issue 11 Pages
2064-2068
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
Advance online publication: September 07, 2012
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The bromodomain and extraterminal (BET) family is a group of chromatin-binding proteins characterized by two bromodomains, an extraterminal (ET) domain, and several other conserved regions of unknown function. In humans, the BET family consists of four members, BRD2, BRD3, BRD4 and BRDT, that all normally localize to the nucleus. We identified a 12-amino acid stretch in the inter-bromodomain region that is perfectly conserved among the BET family members. We deleted these residues and expressed the mutant proteins in HEK293T cells to investigate the function of this motif. We found that the deletion of this motif alters the localization of BET proteins. Mutated BRD3 and BRD4 were excluded from the nucleus, and BRDT was found to be diffused throughout the nucleus and cytoplasm. Although the mutant BRD2 remained predominantly in the nucleus, a punctate distribution was also observed in the cytosol. It has been reported that a conserved motif between the second bromodomain and the ET domain serves as a nuclear localization signal for BRD2. Nevertheless, BET mutants lacking the reported nuclear localization signal motif but retaining the 12-amino acid stretch resided in the nucleus. Furthermore, these mutants were diffused throughout the cytoplasm when the 12 residues were removed. These results indicate that the conserved amino acid stretch in the inter-bromodomain region of the BET family functions as a nuclear localization signal.
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Masayuki Okamoto, Masahiro Fuchigami, Takeshi Suzuki, Nobuhide Watanab ...
Article type: Note
2012 Volume 35 Issue 11 Pages
2069-2074
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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C–C chemokine ligand 2 (CCL2)/its receptor (CCR2) axis is considered as an important signaling pathway in inflammatory diseases. TLK-19705 is a novel CCR2 antagonist, (1-(1,3-dimethyl-1-
H-pyrazolo[3,4-
b]pyridine-5-carbonyl)-3-(4-fluoro-3-(trifluoromethyl)phenyl)urea), and the inhibitory activity was antagonized by the third extracellular loop peptide of CCR2. We examined in this study the effects of TLK-19705 on diabetic nephropathy and atherosclerosis in mouse models. Treatment with TLK-19705 (30 mg/kg/d) for 8 weeks ameliorated urinary albumin–creatinine ratio in
db/
db mice. In addition, TLK-19705, given at 10 mg/kg/d for 8 weeks, significantly reduced the areas of atherosclerotic lesion in apolipoprotein E knockout mice. In conclusion, the results of this study indicate not only considerable therapeutic potential of CCR2 antagonists for diabetic nephropathy and atherosclerosis, but also that TLK-19705 would serve as a powerful tool in mechanistic investigation of these inflammatory diseases.
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Midori Soda, Satoshi Endo, Toshiyuki Matsunaga, Hai-Tao Zhao, Ossama E ...
Article type: Note
2012 Volume 35 Issue 11 Pages
2075-2080
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, was recently identified as both diagnostic marker and therapeutic target in the treatment of several types of cancer. In this study, we have examined AKR1B10 inhibition by five xanthone derivatives, components of pericarps of mangosteen, of which α- and γ-mangostins show potential anti-cancer properties. Among the five xanthones, γ-mangostin was found to be the most potent competitive inhibitor (inhibition constant, 5.6 n
M), but its 7-methoxy derivative, α-mangostin, was the second potent inhibitor (inhibition constant, 80 n
M). Molecular docking of the two mangostins in AKR1B10 and site-directed mutagenesis of the putative binding residues revealed that Phe123, Trp220, Val301 and Gln303 are important for the tight binding of γ-mangostin, and suggested that the 7-methoxy group of α-mangostin impairs the inhibitory potency by altering the orientation of the inhibitor molecule in the substrate-binding site of the enzyme.
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Hiroko Shibata, Chikako Yomota, Toru Kawanishi, Haruhiro Okuda
Article type: Note
2012 Volume 35 Issue 11 Pages
2081-2087
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Liposomes are of great interest as drug delivery vehicles, and studies have focused on understanding how the physical and chemical characteristics of liposomes can be modified to improve their
in vivo behavior. In a previous study, we found that the slightly negatively-charged liposomes aggregate only in the culture medium of human umbilical vein endothelial cells, whereas the liposomes modified with polyethylene glycol (PEG) (PEGylated) did not aggregate. In the present study, we investigated the underlying mechanism of this phenomenon. Firstly, it was found that heparin in the culture medium is one of the factors that cause aggregation of the non-PEGylated liposomes. Since the addition of ethylenediaminetetraacetic acid (EDTA) prevented the aggregation, metal ions, such as Ca
2+ and Mg
2+, in the culture medium could also be important in driving the aggregation. In the presence of heparin, higher concentrations of Ca
2+ or Mg
2+ increased the particle size of the non-PEGylated liposomes, although no change in the particle size of PEGylated liposomes was observed. Under conditions in which aggregation occurred, we measured the binding and uptake of liposomes by macrophages
in vitro. The binding and uptake of non-PEGylated liposomes were significantly increased with increasing Ca
2+ concentrations, whereas those of PEGylated liposomes were unchanged. While the formation of aggregations of cationic or anionic liposomes has been reported previously, there are few reports addressing the aggregation of slightly negatively-charged or neutral liposomes. Thus, our data provide useful insights on the effect of PEGylation on liposomal aggregation and
in vivo behavior.
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Takeo Yamaguchi, Yohei Iwata, Shingo Miura, Kazumasa Kawada
Article type: Note
2012 Volume 35 Issue 11 Pages
2088-2091
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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Supplementary material
Recently, we have found that pressure-induced hemolysis is enhanced by inhibiting water transport
via aquaporin-1 (AQP1), as seen in
p-chloromercuribenzoate (
pCMB)-treated erythrocytes. So, using this method we reinvestigated the functions as AQP1 inhibitors of drugs and chemicals such as acetazolamide, sodium nitroprusside, tetraethylammonium ions (TEA
+), and dimethylsulfoxide (DMSO). The values of hemolysis at 200 MPa were almost unaffected by acetazolamide or sodium nitroprusside, decreased by TEA
+, and increased significantly by DMSO. Furthermore, the erythrocytes were exposed to
pCMB in the presence of TEA
+ or DMSO. The enhancement effect of
pCMB on pressure-induced hemolysis was unaffected by TEA
+ but attenuated by DMSO. Taken together, these results suggest that, of drugs and chemicals examined here, DMSO only is an AQP1 inhibitor, but the effect of DMSO is small compared with
pCMB.
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2,3,22,23-Tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene, an Acyclic Triterpenoid Isolated from the Seeds of Alpinia katsumadai, Inhibits Acyl-CoA : Cholesterol Acyltransferase Activity
Soon-Yong Choi, Moon Hee Lee, Jung Ho Choi, Young Kook Kim
Article type: Note
2012 Volume 35 Issue 11 Pages
2092-2096
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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In order to isolate a cholesterol-lowering compound from
Alpinia katsumadai, an inhibitor for acyl-CoA : cholesterol acyltransferase (ACAT), an enzyme responsible for the cholesterol ester formation in liver, was purified, its chemical structure was determined, and
in vivo and
in vitro inhibition activities were performed. In a high fat diet mouse model, we discovered that the ethanol extract of
Alpinia katsumadai reduced plasma cholesterol, triglyceride, and low density lipoprotein (LDL) levels. An acyclic triterpenoid showing ACAT inhibitory activity was isolated from the extract of seeds of
A. katsumadai. By NMR spectroscopic analysis of its
1H-NMR,
13C-NMR,
1H–
1H correlation spectroscopy, heteronuclear multiple bond connectivity (HMBC), hetero multiquantum coherence (HMQC) and nuclear Overhauser effect, chemical structure of 2,3,22,23-tetrahydroxyl-2,6,10,15,19,23-hexamethyl-6,10,14,18-tetracosatetraene (
1), were elucidated. The acyclic triterpenoid was found to be responsible for the ACAT inhibition activities of rat liver microsomes with IC
50 values of 47.9 µ
M. It also decreased cholesteryl ester formation with IC
50 values of 26 µ
M in human hepatocyte HepG2 cell. The experimental study revealed that the ethanol extract of
A. katsumadai has a hypolipemic effect in high fat diet mice, and the isolated acyclic triterpenoid has ACAT inhibition activity, showing a potential novel therapeutic approach for the treatment of hyperlipidemia and atherosclerosis.
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Motoki Hino, Hideaki Ichihara, Yoko Matsumoto, Ryuichi Ueoka
Article type: Note
2012 Volume 35 Issue 11 Pages
2097-2101
Published: November 01, 2012
Released on J-STAGE: November 01, 2012
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New-type three-component cationic hybrid liposomes (HLs) composed of dimyristoylphosphatidylcholine (DMPC), polyoxyethylene(21)dodecyl ether (C
12(EO)
21) and
O,
O′-ditetradecanoyl-
N-(α-trimethylammonioacetyl) diethanolamine chloride (2C
14ECl) were produced. Cationic HLs were smaller and more stable than pure DMPC liposomes. It is noteworthy that cationic HLs could remarkably inhibit the growth of human colon cancer (HCT116) cells along with apoptosis
in vitro for the first time in this study.
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