Biological and Pharmaceutical Bulletin

New Age Therapy for Alzheimer’s Disease by Neuronal Network Reconstruction

Chihiro Tohda

Released: October 01, 2016

p.1569-1575

Alzheimer’s disease (AD) is a recognized incurable neurodegenerative disorder. Clinically prescribed medicines for AD are expected to bring about only slight symptomatic improvement or a delay of its progression. Another strategy, amyloid β (Aβ) lowing agents, has not been successful at memory improvement. We have hypothesized that an improvement in cognitive function requires the construction of neuronal networks, including neurite regeneration and synapse formation; therefore, we have been exploring candidates for radical anti-AD drugs that can restore Aβ-induced neurite atrophy and memory impairment. Our studies found several promising drug candidates that may improve memory dysfunction in AD model mice. The main activity of these drugs is the restoration of damaged axons. Focusing on candidates based on the recovery of neurite atrophy in vitro certainly leads to positive effects on memory improvement also in vivo. This suggests that neuronal network reconstruction may importantly relate to functional recovery in the brain. When identifying the signaling mechanisms of exogenous compounds like natural medicine-derived constituents, molecules directly activated by the compound are hard to be identified. However, the drug affinity responsive target stability (DARTS) analysis may pave the way to an approach to determine the initial molecule of the signaling pathway. Exploring new drug candidates and clarifying their signaling pathways directly relating to neuronal network reconstruction may provide promising therapeutic strategies with which to overcome AD.

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Investigation of Appropriate Inhalation Technique for Mometasone Furoate Dry Powder Inhaler

Haruko Yokoyama, Kanako Ito, Hirokazu Mihashi, Yasuyuki Shiraishi, Risa Takayanagi, Yasuhiko Yamada

Released: October 01, 2016

p.1576-1580

The aim of this study was to establish an appropriate inhalation method with a mometasone furoate dry powder inhaler (MF-DPI). Utilizing a tone-based inhalation training device, we investigated the maximum peak inspiratory flow rate time (T_max PIFR) and peak inspiratory flow rate (PIFR) to determine whether either had an influence on lung deposition with use of an MF-DPI. A low tone indicated a PIFR of 28 L/min and a high tone that of 40 L/min, while 60 L/min was considered to be the standard. We established an inhalation profile in consideration of a human inhalation pattern, in which T_max PIFR was set at 0.5 s (T_max PIFR 0.5 s) and 2.5 s (T_max PIFR 2.5 s). The reference cut-off value derived with a cascade impactor test was used for evaluation of the rate of delivered dose in the lung, which was the amount of drug from stage 3 to 7 at all PIFRs. We then investigated the relationship of the fine particle fraction (FPF) with the claimed dose at T_max PIFR of 0.5 s and PIFR. There were no differences among the T_max PIFR values for the doses emitted from the device or for the rate of delivered doses in stages 3–7. However, FPF for the claimed dose at 40 L/min was significantly lower than that at 60 L/min, which was dependent on PIFR. Our results showed that PIFR but not T_max PIFR has an effect on lung deposition after inhalation with an MF-DPI.

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Evaluation for Peritoneal Injury at an Early Stage Using Dual Macromolecular Markers

Haruna Hirata, Shintaro Fumoto, Hirotaka Miyamoto, Mikiro Nakashima, Morio Nakayama, Koyo Nishida

Released: October 01, 2016

p.1581-1587

Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, making continuation of PD difficult. Therefore, it is necessary to evaluate peritoneal injury at an early stage and develop appropriate therapies. The aims of the present study were to evaluate peritoneal injury at an early stage and assess a drug for prevention of peritoneal injury using our previously developed novel evaluation method. Peritoneal injury was induced in model animals by intraperitoneal injection of methylglyoxal (MGO) for 1 to 5 consecutive days or chlorhexidine digluconate (CG) for 1 to 14 consecutive days. Tetramethylrhodamine-dextran (RD)-10 and fluorescein isothiocyanate-dextran (FD)-2000 were then injected into the peritoneal cavity and recovered after 120 min to evaluate peritoneal injury. The ratio of the concentration of RD-10 to FD-2000 (RD-10/FD-2000 ratio) significantly decreased in animals that had been treated with MGO or CG for 1 d. Moreover, the RD-10/FD-2000 ratio significantly increased in CG- and thalidomide-treated animals. The RD-10/FD-2000 ratio can be used to evaluate peritoneal injury at an early stage and assess the drug efficacy of thalidomide for prevention of peritoneal injury. This study will contribute to the development of therapeutic treatments for peritoneal injury.

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Preparation, in Vitro and in Vivo Evaluations of Compound Calculus Bovis Sativus and Ornidazole Film

Wei Li, Wenxi He, Ping Gao, Chengliang Zhang, Hongjiao Cai, Ying Chen, Dong Liu

Released: October 01, 2016

p.1588-1595

The aim of this study was to develop and to investigate a film of compound Calculus Bovis Sativus (CBS) and ornidazole film. A uniform mucoadhesive film was herein successfully obtained by a film-forming solusion containing insoluable drug. This film, as a valid adjunct for the treatment of oral mucosal ulcer, consisted of two main drugs (CBS, ornidazole) and three polymers (hydroxypropyl methyl cellulose, chitosan, poly(vinyl alcohol) (PVA)). The film was prepared with the film-forming suspension, using casting-solvent evaporation technique. The drug content, release behavior, swelling index and mucoadhesive properties of the film were detected. Then the effects of the prepared film on a glacial acetic acid-induced oral mucosal ulceration model of rabbits were evaluated. Moreover, the in vivo release of bilirubin and ornidazole in saliva were also detected in the oral mucosae of healthy volunteers. The films showed favorable in vitro drug release behaviors and swelling properties. Mucosal wounds in the animals were significantly relieved. With the films well tolerated, the salivary concentrations of ornidazole were maintained above the minimum inhibitory concentration against CBS for about 2 h. The compound CBS and ornidazole film functioned better than the film only containing CBS and ornidazole did. Therefore, it is a potentially efficient drug delivery system for the treatment of oral ulcers.

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Ligand Activity of Group 15 Compounds Possessing Triphenyl Substituent for the RXR and PPARγ Nuclear Receptors

Youhei Hiromori, Akiko Ido, Akira Aoki, Tomoki Kimura, Hisamitsu Nagase, Tsuyoshi Nakanishi

Released: October 01, 2016

p.1596-1603

We investigated the ability of group 15 compounds with a triphenyl substituent to bind to and activate human retinoic X receptor (RXR) and peroxisome proliferator-activated receptor (PPAR) γ and their ability to activate the receptor. Triphenylphosphine oxide (TPPO) transcriptionally activated both RXR and PPARγ. Triphenylbismuth (TPBi) transcriptionally activated PPARγ but not RXR. However, TPBi significantly inhibited RXR transcriptional activity induced by 9-cis retinoic acid (9cRA) and PPARγ transcriptional activity induced by rosiglitazone (Rosi). Triphenylarsine (TPAs) also significantly inhibited the 9cRA- and Rosi-induced transcriptional activity of both receptors, whereas TPAs alone had no effect on the transcriptional activity of RXR and PPARγ. Consistent with these results, TPAs and TPBi blocked the binding of [³H]9cRA to RXR and of [³H]Rosi to PPARγ in a competitive manner. However, contrary to the results of the reporter gene assay, TPPO did not compete with [³H]9cRA and [³H]Rosi for binding to RXR and PPARγ, respectively. Our findings indicate that 1) TPPO is a transcriptional activator—but not a ligand—of RXR and PPARγ; 2) TPBi is an antagonist of RXR and a partial agonist of PPARγ; and 3) TPAs is a dual antagonist of RXR and PPARγ. These results suggest that TPPO, TPAs, and TPBi are potential endocrine disrupters of the PPARγ–RXR signaling pathway.

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Acyl-glucuronide as a Possible Cause of Trovafloxacin-Induced Liver Toxicity: Induction of Chemokine (C-X-C Motif) Ligand 2 by Trovafloxacin Acyl-glucuronide

Ryo Mitsugi, Kyohei Sumida, Yoshiko Fujie, Robert H. Tukey, Tomoo Itoh, Ryoichi Fujiwara

Released: October 01, 2016

p.1604-1610

Trovafloxacin is an antibiotic that was withdrawn from the market relatively soon after its release due to the risk of hepatotoxicity. Trovafloxacin is mainly metabolized to its acyl-glucuronide by uridine 5′-diphosphate (UDP)–glucuronosyltransferase (UGT) 1A1. In this study, we examined whether the acyl-glucuronide is involved in the development of hepatotoxicity. A UGT1A1-induced cell model was developed and the toxicity of trovafloxacin acyl-glucuronide was evaluated. The UGT1A1-induced cell model was developed by treating HepG2 cells with chrysin for 48 h. Chemokine (C-X-C motif) ligand 2, a cytokine involved in drug-induced liver injury, was uniquely induced by trovafloxacin in the UGT1A1-induced HepG2 cells. Induction of UGT1A1 resulted in a decrease in cell viability. An in vivo animal study further demonstrated the importance of UGT1A1 in the trovafloxacin-induced liver toxicity. Although the complete mechanism of trovafloxacin-induced liver injury is still unknown, trovafloxacin acyl-glucuronide can be involved in the development of toxic reactions in vitro and in vivo.

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Synthesis of New N¹-Substituted-5-aryl-3-(3,4,5-trimethoxyphenyl)-2-pyrazoline Derivatives as Antitumor Agents Targeting the Colchicine Site on Tubulin

Salwa Elmeligie, Nadia Abdalla Khalil, Eman Mohamed Ahmed, Soha Hussein Emam, Sawsan Abo-Bakr Zaitone

Released: October 01, 2016

p.1611-1622

A series of pyrazoline derivatives 2a–e, 3a–e and 4a–e structurally related to combretastatin A4 (CA-4) were synthesized and characterized by spectroscopic means and elemental analyses. In these compounds, the cis double bond of CA-4 was replaced with the pyrazoline ring aiming to enhance the cytotoxic effects displayed by CA-4 and to prevent the cis/trans isomerization that is associated with inactivation of CA-4. The cytotoxic activity of all new compounds was investigated in vitro against MCF-7 and HCT-116 cell lines. The inhibition of tubulin polymerization by the most active compounds 3d, 4a and e was evaluated. The cytotoxicity of 4e was correlated with induction of apoptosis and caspase-3 activation in vitro thus indicating the apoptotic pathway of anticancer effect of these compounds. Furthermore, in vivo evaluation of the synthesized compounds was carried out against Ehrlich’s ascites carcinoma (EAC) solid tumor grown in mice. Compounds 2c, 3a and e showed significant reduction in tumor weight, and about 2–4 fold increase in caspase-3 expression.

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Synergistic Anti-glioma Effects in Vitro and in Vivo of Enediyne Antibiotic Neocarzinostatin and Paclitaxel via Enhanced Growth Delay and Apoptosis-Induction

Gao Tianqin, Cheng Chunlei, Wang Jingjing

Released: October 01, 2016

[Advance Publication] Released: July 14, 2016

p.1623-1630

Neocarzinostatin (NCS) is a member of enediyne antibiotics with high anticancer potential. Our study was performed to explore the synergistic anti-glioma effects of NCS and paclitaxel (PTX) in vitro and in vivo. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicities of the drugs to human glioma cells U87MG and rat glioma cells C6 were evaluated. The results showed that the combinations of NCS and PTX can synergistically inhibit glioma cells survival. Cell apoptosis was detected by flow cytometry, and the results showed that the combinations of NCS and PTX synergistically enhanced apoptosis ratio of glioma cells. Western blot revealed that the cell signaling pathways of proliferation and apoptosis were synergistically regulated, in which Akt was synergistically inactivated, p53 was up-regulated with down-regulation of bcl-2. Meanwhile, with the subcutaneous model of U87MG cells and intracerebral implantation model of C6 cells, the combination strategy could synergistically delay the glioma growth and significantly prolong the survival of rats bearing orthotopic glioma. This study demonstrates that the combination of NCS and PTX can potentiate the effect on survival and apoptosis of glioma cells via suppression of Akt, bcl-2, and activations of p53; Meanwhile, the in vivo studies also confirmed that the combination of NCS and PTX synergistically inhibit the gliom growth. Our data about the combinational effects of NCS with PTX may provide an alternative strategy for glioma therapy.

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Ethanol Withdrawal-Induced Impaired Recognition Is Reversed by Chronic Exposure to Stress and the Acute Administration of Corticosterone in Mice

Hideaki Kato, Minoru Tsuji, Kazuya Miyagawa, Kotaro Takeda, Hiroshi Takeda

Released: October 01, 2016

p.1631-1637

The present study was designed to ascertain the effects of repeated exposure to stress and the acute administration of corticosterone (1, 3, 10 mg/kg, intraperitoneally (i.p.)) on the ethanol withdrawal-induced impairment of novel object recognition in mice. Mice were chronically treated with 3% ethanol for 7 d, with or without exposure to restraint stress for 1 h/d. A significant decrease in cognitive function was observed in the ethanol plus no stress group at 48 h after the discontinuation of ethanol treatment. This impaired recognition was recovered in the ethanol plus stress group. Moreover, we investigated the effects of acute pretreatment with corticosterone, which is a corticosteroid-type hormone produced in the cortex of the adrenal glands, on the impaired recognition after the discontinuation of ethanol treatment in mice. The impaired recognition in the 3% ethanol alone-treated group at 48 h after the discontinuation of ethanol treatment was recovered by treatment with the middle dose (3 mg/kg) of corticosterone, but not with the low or high doses (1, 10 mg/kg). These results suggest that chronic stress during the development of ethanol dependence may reduce the impaired recognition after the discontinuation of ethanol treatment. Moreover, acute pretreatment with the middle dose of corticosterone also recovered the impaired recognition after the discontinuation of ethanol treatment in mice. Adequate regulation of the hypothalamic–pituitary–adrenal (HPA) axis by corticosterone may improve the impaired recognition after the discontinuation of ethanol treatment.

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Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

Noritaka Nakamichi, Takahiro Ishimoto, Yoshihide Yamauchi, Yusuke Masuo, Yukio Kato

Released: October 01, 2016

p.1638-1645

The aim of the present study is to discover multidrug resistance-associated protein (MRP) inhibitors with neuroblastoma-selective cytotoxicity by means of fluorescence assay with a membrane-permeable fluorescent dye, Fluo-8 AM, based on our observation that gene expression of Mrp3 in neuroblastoma Neuro2a cells was remarkably higher than that in primary cultured cortical neurons, as determined by real-time PCR. Neuro2a cells showed minimal fluorescence upon incubation with Fluo-8 AM. However, blocking of Mrp3 efflux function by small interfering RNA (siRNA) transfection or inhibition with probenecid resulted in significant dye accumulation, observed as an increase of fluorescence. Interestingly, Mrp3 siRNA or probenecid treatment also resulted in increased cytotoxicity, as evidenced by decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-reducing activity of Neuro2a, with a concomitant increase in release of lactate dehydrogenase. On the other hand, primary cultured neurons exhibited higher fluorescence intensity after incubation with Fluo-8 AM regardless of addition of probenecid. Also, probenecid only minimally affected MTT-reducing activity. Thus, probenecid showed selective cytotoxicity towards Neuro2a cells. Based on these findings, we screened a series of established therapeutic agents for ability to induce Fluo-8 accumulation in Neuro2a cells. Several uricosuric and nonsteroidal anti-inflammatory drugs were identified, and these drugs were confirmed to decrease MTT-reducing activity selectively in Neuro2a. There was a negative linear correlation between Fluo-8 accumulation and cytotoxicity of these agents. Although the compounds identified here are insufficiently potent for practical application, further screening to discover higher-affinity MRP3 inhibitors using larger chemical libraries may uncover drug candidates with potent neuroblastoma-selective cytotoxicity.

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Perturbed Calcineurin-NFAT Signaling Is Associated with the Development of Alzheimer’s Disease

Masashi Asai, Aimi Kinjo, Shoko Kimura, Ryotaro Mori, Takashi Kawakubo, Keiro Shirotani, Sosuke Yagishita, Kei Maruyama, Nobuhisa Iwata

Released: October 01, 2016

p.1646-1652

Down syndrome (DS), the most common genetic disorder, is caused by trisomy 21. DS is accompanied by heart defects, hearing and vision problems, obesity, leukemia, and other conditions, including Alzheimer’s disease (AD). In comparison, most cancers are rare in people with DS. Overexpression of dual specificity tyrosine-phosphorylation-regulated kinase 1A and a regulator of calcineurin 1 located on chromosome 21 leads to excessive suppression of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, resulting in reduced expression of a critical angiogenic factor. However, it is unclear whether the calcineurin-NFAT signaling pathway is involved in AD pathology in DS patients. Here, we investigated the association between the calcineurin-NFAT signaling pathway and AD using neuronal cells. Short-term pharmacological stimulation decreased gene expression of tau and neprilysin, and long-term inhibition of the signaling pathway decreased that of amyloid precursor protein. Moreover, a calcineurin inhibitor, cyclosporine A, also decreased neprilysin activity, leading to increases in amyloid-β peptide levels. Taken together, our results suggest that a dysregulation in calcineurin-NFAT signaling may contribute to the early onset of AD in people with DS.

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Liver-Specific Silencing of Lipin1 Reduces Fat Mass as Well as Hepatic Triglyceride Biosynthesis in Mice

Kazuaki Kajimoto, Erina Suemitsu, Yusuke Sato, Yu Sakurai, Hideyoshi Harashima

Released: October 01, 2016

p.1653-1661

Lipin1, a bifunctional protein, regulates fatty acid utilization in the triglyceride biosynthesis pathway. In the current study, using a liver-specific in vivo short interfering RNA (siRNA) delivery system, we examined the pathological and physiological roles of hepatic Lipin1 in the development of insulin resistance and the maintenance of systemic energy homeostasis. Liver-specific silencing of Lipin1 expression was achieved by the systemic administration of siRNA against Lpin1 mRNA (siLpin1)-loaded lipid nanoparticles (LNPs) to wild type mice at 3–4 d intervals for 25 d. The siLpin1-treated mice showed normal blood glucose levels and insulin sensitivity, however, triglyceride (TG) levels were reduced in liver and peripheral blood of them. The knockdown of hepatic Lipin1 in mice led to marked decrease in adipose tissue mass and adipocyte diameters in epididymal and inguinal fat depots without the undesired silencing of Lipin1 in adipose tissue. In summary, we report for the first time that the down-regulation of hepatic Lipin1 expression leads to less adiposity as well as a decrease in TG level in the liver and blood circulation, without any alterations in the glucose tolerance and blood glucose levels. Our findings may provide new insights into the physiological roles of hepatic Lipin1 in systemic energy homeostasis.

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Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization

Yoshimi Okamoto-Uchida, Ryosuke Nakamura, Yumiko Matsuzawa, Megumi Soma, Hiroshi Kawakami, Akiko Ishii-Watabe, Tomoko Nishimaki-Mogami, Reiko Teshima, Yoshiro Saito

Released: October 01, 2016

p.1662-1666

The physicochemical nature of allergen molecules differ from the liquid phase to the solid phase. However, conventional allergy tests are based on the detection of immunoglobulin (Ig)E binding to immobilized allergens. We recently developed an in vitro allergy testing method using a luciferase-reporting humanized rat mast cell line to detect IgE crosslinking-induced luciferase expression (EXiLE test). The aim of the present study was to evaluate the effects of antigen immobilization on the results of different in vitro allergy tests using two anti-ovalbumin (OVA) antibodies (Abs), E-C1 and E-G5, with different properties in the OVA-induced allergic reaction. Both Abs showed clear binding to OVA with an enzyme-linked immunosorbent assay and by BIAcore analysis. However, only E-C1 potentiated EXiLE response for the liquid-phase OVA. On the other hand, OVA immobilized on solid-phase induced EXiLE responses in both E-C1 Ab- and E-G5 Ab-sensitized mast cells. Western blotting of OVA indicated that E-C1 Ab binds both to OVA monomers and dimers, unlike E-G5 Ab, which probably binds only to the OVA dimer. These results suggest that antigen immobilization enhanced IgE crosslinking ability through multimerization of allergen molecules in the solid phase, resulting in an increase in false positives in IgE binding-based conventional in vitro allergy tests. These findings shed light on the physicochemical nature of antigens as an important factor for the development and evaluation of in vitro allergy tests and suggest that mast cell activation-based allergy testing with liquid-phase allergens is a promising strategy to evaluate the physiological interactions of IgE and allergens.

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Anti-HSV Activity of Kuwanon X from Mulberry Leaves with Genes Expression Inhibitory and HSV-1 Induced NF-κB Deactivated Properties

Fang Ma, Wenwei Shen, Xiaoqi Zhang, Manmei Li, Yifei Wang, Yuxiao Zou, Yaolan Li, Hui Wang

Released: October 01, 2016

p.1667-1674

Six stilbene derivatives isolated from Mulberry leaves including Kuwanon X, Mulberrofuran C, Mulberrofuran G, Moracin C, Moracin M 3′-O-b-glucopyranoside and Moracin M were found to have antiviral effects against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) at different potencies except for Mulberrofuran G. Kuwanon X exhibited the greatest activity against HSV-1 15577 and clinical strains and HSV-2 strain 333 with IC₅₀ values of 2.2, 1.5 and 2.5 µg/mL, respectively. Further study revealed that Kuwanon X did not inactivate cell-free HSV-1 particles, but inhibited cellular adsorption and penetration of HSV-1 viral particles. Following viral penetration, Kuwanon X reduced the expression of HSV-1 IE and L genes, and decreased the synthesis of HSV-1 DNA. Furthermore, it was demonstrated that Kuwanon X inhibited the HSV-1-induced nuclear factor (NF)-κB activation through blocking the nuclear translocation and DNA binding of NF-κB. These results suggest that Kuwanon X exerts anti-HSV activity through multiple modes and could be a potential candidate for the therapy of HSV infection.

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Inhibitory Effects of Oligostilbenoids from the Bark of Shorea roxburghii on Malignant Melanoma Cell Growth: Implications for Novel Topical Anticancer Candidates

Hiroyuki Moriyama, Mariko Moriyama, Kiyofumi Ninomiya, Toshio Morikawa, Takao Hayakawa

Released: October 01, 2016

p.1675-1682

Human malignant melanomas remain associated with dismal prognosis due to their resistance to apoptosis and chemotherapy. There is growing interest in plant oligostilbenoids owing to their pleiotropic biological activities, including anti-inflammatory, antioxidant, and anticancer effects. Recent studies have demonstrated that resveratrol, a well-known stilbenoid from red wine, exhibits cell cycle-disrupting and apoptosis-inducing activities on melanoma cells. The objective of our study was to evaluate the anti-melanoma effect of oligostilbenoids isolated from the bark of Shorea roxburghii. Among the isolates, four resveratrol oligomers, i.e., (−)-hopeaphenol, vaticanol B, hemsleyanol D, and (+)-α-viniferin, possessed more potent antiproliferative action than did resveratrol against SK-MEL-28 melanoma cells. Cell cycle analysis revealed that (−)-hopeaphenol, hemsleyanol D, and (+)-α-viniferin arrested cell division cycle at the G1 phase, whereas vaticanol B had little effect on the cell cycle. In addition, cell proliferation assay also revealed that (+)-α-viniferin induced DNA damage followed by induction of apoptosis in SK-MEL-28 cells, which was confirmed by an increased expression of γ-H2AX and cleaved caspase-3, respectively. The compounds vaticanol B, hemsleyanol D, and resveratrol significantly increased the expression of p21, suggesting that they are able to block cell cycle progression. Moreover, these oligostilbenoids downmodulated cylin D1 expression and extracellular signal-regulated kinase (ERK) activation. Furthermore, hemsleyanol D, (+)-α-viniferin, and resveratrol significantly decreased the expression of cyclin B1, which could also suppress cell cycle progression. The present study thus suggests that these plant oligostilbenoids are effective as therapeutic or chemopreventive agents against melanoma.

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CYP2A13 Genetic Polymorphisms in Relation to the Risk of Bladder Cancer in Japanese Smokers

Masaki Kumondai, Hiroki Hosono, Kazuhiko Orikasa, Yoichi Arai, Tomio Arai, Haruhiko Sugimura, Seiichiro Ozono, Takayuki Sugiyama, Tatsuya Takayama, Takamitsu Sasaki, Noriyasu Hirasawa, Masahiro Hiratsuka

Released: October 01, 2016

p.1683-1686

Tobacco-specific nitrosamines including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), which can be activated by the metabolic enzyme CYP2A13, are potent procarcinogens. Smoking plays a role in carcinogenesis in the human bladder, which expresses CYP2A13 at a relatively high level. Numerous genetic polymorphisms of CYP2A13 causing amino acid substitution might reduce CYP2A13 metabolic activity toward NNK and NNN, resulting in decreased susceptibility to bladder cancer. The aim of this study was to reveal any association between bladder cancer development and CYP2A13 genetic polymorphisms in Japanese smokers. The CYP2A13 genotype of each subject (163 bladder cancer patients and 161 controls) was determined by next-generation sequencing (NGS) of the full CYP2A13 gene. All samples were genotyped for five CYP2A13 variant alleles (CYP2A13*2, *3, *4, *6, *7). Based on biological logistic regression, the odds ratio (95% confidence interval) for the CYP2A13*1/*2 genotype was 0.34 (0.17–0.69). Thus, CYP2A13 genetic polymorphisms might play important roles in the development of bladder cancer in Japanese smokers.

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Sustained Release of Mitomycin C from Its Conjugate with Single-Walled Carbon Nanotubes Associated by Pegylated Peptide

Takahisa Ohta, Yasuhiko Hashida, Fumiyoshi Yamashita, Mitsuru Hashida

Released: October 01, 2016

p.1687-1693

A novel sustained release formulation of mitomycin C (MMC) was developed by employing single-walled carbon nanotubes (SWCNTs) wrapped by designed peptide with polyethylene glycol (PEG) modification (pegylation) as a nano-scale molecular platform. The amino groups of polycationic and amphiphilic H-(-Cys-Trp-Lys-Gly-)(-Lys-Trp-Lys-Gly-)₆-OH [CWKG(KWKG)₆] peptide associated with SWCNTs were modified using PEG with 12 units (PEG₁₂) to improve the dispersion stability of the composite. Then thiol groups of peptide were conjugated with MMC using N-ε-maleimidocaproic acid (EMCA) as a linker via transformation of aziridine group of MMC. The obtained SWCNTs-CWKG(KWKG)₆-(PEG)₁₂-C₆-MMC composites particularly that with 13.6% PEG modification extent of amino groups, showed good dispersion stability both in water and in a cell culture medium for 24 h. The release of MMC from SWCNTs-CWKG(KWKG)₆-(PEG)₁₂-C₆-MMC was confirmed to follow first-order kinetics being accelerated by the pH increase in good agreement with the results observed for MMC-dextran conjugate with the same conjugation structure. The SWCNTs-CWKG(KWKG)₆-(PEG)₁₂ composite exhibited a considerably low cytotoxicity against cultured human lung adenocarcinoma epithelial cell line (A₅₄₉). In contrast, SWCNTs-CWKG(KWKG)₆-(PEG)₁₂-C₆-MMC demonstrated delayed but relatively corresponding antitumor activity with free MMC at the same concentration. The results suggested the potential role of SWCNTs-CWKG(KWKG)₆-(PEG)₁₂ as a carrier for a controlled release drug delivery system (DDS).

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Clostridium perfringens α-Toxin Impairs Lipid Raft Integrity in Neutrophils

Masaya Takehara, Teruhisa Takagishi, Soshi Seike, Kyohei Oishi, Yoshino Fujihara, Kazuaki Miyamoto, Keiko Kobayashi, Masahiro Nagahama

Released: October 01, 2016

p.1694-1700

Clostridium perfringens type A, a Gram-positive, anaerobic bacterium, causes gas gangrene. Recently, we reported that C. perfringens α-toxin blocked neutrophil differentiation in an enzyme activity-dependent manner to impair host innate immunity, which should be crucial for the pathogenesis of C. perfringens. However, the detailed mechanism remains unclear. Lipid rafts have been reported to be platforms for signaling molecules involved in the regulation of cell differentiation in many different cell types. In this study, we found that cell surface expression of a lipid raft marker, GM1 ganglioside, decreased in association with neutrophil differentiation by flow cytometry analysis and morphological observation. In vitro treatment of isolated mouse bone marrow cells with α-toxin or an α-toxin variant lacking phospholipase C and sphingomyelinase activities revealed that α-toxin increased the cell surface expression of GM1 ganglioside in an enzyme activity-dependent manner. C. perfringens infection also increased GM1 ganglioside levels in bone marrow myeloid cells. Moreover, treatment of bone marrow cells with methyl-β-cyclodextrin, a lipid raft-disrupting agent, impaired neutrophil differentiation. Together, our results suggest that the integrity of lipid rafts should be properly maintained during granulopoiesis, and α-toxin might perturb lipid raft integrity leading to the impairment of neutrophil differentiation.

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Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities

Hirotaka Amano, Daichi Kazamori, Kenji Itoh

Released: October 01, 2016

p.1701-1707

Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01–1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21–45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003–0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1′-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19–49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug–drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC₅₀>1 mM) on the activities of five major isoforms of human CYP in vitro.

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Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate

Wataru Matsuzaki, Hiromasa Takahashi, Hiroyuki Nakamura, Toshihiko Murayama

Released: October 01, 2016

p.1708-1717

Ceramide kinase (CerK) and ceramide-1-phosphate (C1P) are involved in various cellular functions, while regulation of the enzyme activity has not been well elucidated. We herein investigated the effects of several glycerophospholipids on human recombinant CerK activity with CaCl₂ and MgCl₂ by measuring the formation of fluorescent labeled C1P in vitro. CerK activities were 44.1±11.4 (pmol/µg/min) with vehicle, 137±29 with 2 mM CaCl₂, and 144±32 with 2 mM MgCl₂ in the glycerol/albumin buffer. The addition of glycerophospholipids such as phosphatidylcholine, phosphatidylinositol (PI), PI 4,5-bisphosphate (PI(4,5)P₂), and phosphatidic acid had no effect on CerK activity with CaCl₂, although PI(4,5)P₂ and phosphatidic acid bound to CerK in the lipid–protein overlay assay. The addition of cardiolipin (diphosphatidylglycerol) at concentrations up to 0.1 µM increased, whereas those more than 1 µM decreased CerK activity with CaCl₂/MgCl₂. In the lipid–protein overlay assay, cardiolipin bound to CerK and CerK lacking pleckstrin homology (PH) domain, but not PH domain of CerK, in CaCl₂-independent manner. Cardiolipin also bound to CerK in the multilamellar vesicle binding assay. A deviation from the normal range of cellular cardiolipin, both the decrease by phospholipase D6 expression and increase by an exogenous addition of the lipid, negatively regulated C1P formation in intact HepG2 cells. Our results revealed that cardiolipin bound to CerK and regulated the formation of C1P in vitro and in cells.

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Influence of Hot Spring Water on Fatty Acid Composition of Skin Surface Lipids in Hairless Mouse Model of Atopic Dermatitis

Kazuhiko Nishimura, Naoki Aoyanagi, Eiji Uchino, Yutaka Itabashi

Released: October 01, 2016

p.1718-1722

When hairless NCN24 mice with atopic dermatitis (AD) were sprayed with a petroleum-containing alkaline salt spring water rich in metaboric acid and sodium bicarbonate, AD symptoms diminished. Reversed-phase HPLC with fluorescence detection (HPLC/FD) and online MS revealed that fatty acid (FA) composition of the skin surface lipids was similar to that in non-AD mice compared with that in AD mice. Strong negative correlations were noted between the levels of total serum immunoglobulin E (IgE) and palmitoleic acid and between the levels of total serum IgE and branched-hexadecanoic acid. Conversely, a strong positive correlation was noted between the levels of total serum IgE and linoleic acid. The present study demonstrates that the petroleum-containing spring water alters the FA composition of skin surface lipids in AD mice, which can be used as an index to evaluate inflammation.

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Effect of Long-Term Diabetes on Serotonin-Mediated Contraction in Carotid Arteries from Streptozotocin-Induced Diabetic Male and Female Rats

Shun Watanabe, Takayuki Matsumoto, Makoto Ando, Shota Kobayashi, Maika Iguchi, Kumiko Taguchi, Tsuneo Kobayashi

Released: October 01, 2016

p.1723-1727

An accumulating body of evidence suggests that males and females differ in vascular function in arteries under pathophysiological states. In this study, we tested whether there was a sex difference associated with serotonin (5-hydroxytryptamine, 5-HT)-mediated contraction in the carotid arteries of long-term streptozotocin (STZ)-induced diabetic rats [viz. 23 or 24 weeks after STZ (65 mg/kg, intravenously (i.v.)) injection starting at 8 weeks old of rats]. In the control group, the 5-HT- and high-K⁺-induced contractions were greater in females than in males. In both sexes, treatment with STZ led to a decrease of 5-HT-induced contraction in carotid arteries compared to controls. In STZ-induced diabetic rats, the carotid arterial 5-HT-induced contraction was greater in female rats than in diabetic male rats. The high-K⁺-induced contraction was greater in diabetic female rats than in either age-matched female controls or diabetic male rats. Expression of the 5-HT_2A receptor, which is the main receptor for 5-HT-induced contraction in rat carotid arteries, was similar among the four groups. These results suggest that decreased 5-HT-induced carotid arterial contraction is seen in both sexes under long-term STZ-induced diabetic conditions. Further, this reduction seems to be weaker in females than in males. This alteration of 5-HT-induced contraction may be partly associated with increased voltage-dependent Ca²⁺ channel activity.

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Substrate Specificity of Equine and Human Influenza A Virus Sialidase to Molecular Species of Sialic Acid

Tadanobu Takahashi, Saori Unuma, Sawako Kawagishi, Yuuki Kurebayashi, Maiko Takano, Hiroki Yoshino, Akira Minami, Takashi Yamanaka, Tadamune Otsubo, Kiyoshi Ikeda, Takashi Suzuki

Released: October 01, 2016

p.1728-1733

Most equine influenza A viruses (IAVs) show strong binding to glycoconjugates containing N-glycolylneuraminic acid (Neu5Gc) as well as N-acetylneuraminic acid (Neu5Ac). Therefore, the progeny of equine IAV is thought to be released from the infected cell surface through removal of sialic acids by the viral sialidase. In the present study, equine IAV sialidases showed significantly lower substrate affinity than that of human IAV sialidases to artificial and natural Neu5Gc-conjugated substrates. The substrate specificity of equine IAV sialidases is in disagreement with their binding specificity to molecular species of sialic acid. The results suggest that substrate specificity of equine IAV sialidase for Neu5Ac, rather than for Neu5Gc, is important for an advantage at the early infection stage and the process of progeny virus release from the surface of infected cells.

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Anti-MUC1 Aptamer/Negatively Charged Amino Acid Dendrimer Conjugates for Targeted Delivery to Human Lung Adenocarcinoma A549 Cells

Marie Masuda, Shigeru Kawakami, Wassana Wijagkanalan, Tadaharu Suga, Yuki Fuchigami, Fumiyoshi Yamashita, Mitsuru Hashida

Released: October 01, 2016

p.1734-1738

We previously developed a negatively charged amino acid dendrimer to address the safety concerns associated with the constituent unit of these systems, which resulted in the formation of a sixth-generation glutamic acid-modified dendritic poly(L-lysine) system (KG6E). The aim of this study was to develop a nanocarrier for targeted drug delivery into cancer cells. In this study, we have synthesized a conjugate material consisting of anti-mucin 1 (MUC1) aptamer (anti-MUC1 apt) and KG6E (anti-MUC1 apt/KG6E) for targeted drug delivery to human lung adenocarcinoma A549 cells, which express high levels of the MUC1. The anti-MUC1 apt/KG6E was efficiently internalized by the A549 cells and subsequently transported to the endosomal and lysosomal compartments. In contrast, the cellular association of the sequence scrambled aptamer/KG6E conjugate (scrambled apt/KG6E) was much lower than that of the anti-MUC1 apt/KG6E in A549 cells. These results suggest that our newly developed anti-MUC1 apt/KG6E can be internalized in A549 cells via a MUC1 recognition pathway.

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Identification of a Selective SIRT2 Inhibitor and Its Anti-breast Cancer Activity

Asad Ali Shah, Akihiro Ito, Akiko Nakata, Minoru Yoshida

Released: October 01, 2016

p.1739-1742

SIRT2 is a member of the human sirtuin family of proteins and possesses nicotinamide adenine dinucleotide (NAD)-dependent lysine deacetylase activity. SIRT2 has been involved in various cellular processes including gene transcription, genome constancy, and the cell cycle. In addition, SIRT2 is deeply implicated in diverse diseases including cancer. In this study, we identified a small molecule inhibitor of SIRT2 with a structure different from known SIRT2 inhibitors by screening from a chemical library. The hit compound showed a high selectivity toward SIRT2 as it only inhibited SIRT2, and not other sirtuins including SIRT1 and SIRT3 or zinc-dependent histone deacetylases (HDACs) including HDAC1 and HDAC6, in vitro. The compound increased the acetylation level of eukaryotic translation initiation factor 5A (eIF5A), a physiological substrate of SIRT2, and reduced cell viability of human breast cancer cells accompanied with a decrease in c-Myc expression. These results suggest that the compound is cellular effective and has potential for development as a therapeutic agent against breast cancers by specific inhibition of SIRT2.

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Volume And Issue List
Biological and Pharmaceutical Bulletin

Predecessor

Biological and Pharmaceutical Bulletin Vol. 39(2016) No. 10

Review

New Age Therapy for Alzheimer’s Disease by Neuronal Network Reconstruction

Regular Articles

Investigation of Appropriate Inhalation Technique for Mometasone Furoate Dry Powder Inhaler

Evaluation for Peritoneal Injury at an Early Stage Using Dual Macromolecular Markers

Preparation, in Vitro and in Vivo Evaluations of Compound Calculus Bovis Sativus and Ornidazole Film

Ligand Activity of Group 15 Compounds Possessing Triphenyl Substituent for the RXR and PPARγ Nuclear Receptors

Acyl-glucuronide as a Possible Cause of Trovafloxacin-Induced Liver Toxicity: Induction of Chemokine (C-X-C Motif) Ligand 2 by Trovafloxacin Acyl-glucuronide

Synthesis of New N¹-Substituted-5-aryl-3-(3,4,5-trimethoxyphenyl)-2-pyrazoline Derivatives as Antitumor Agents Targeting the Colchicine Site on Tubulin

Synergistic Anti-glioma Effects in Vitro and in Vivo of Enediyne Antibiotic Neocarzinostatin and Paclitaxel via Enhanced Growth Delay and Apoptosis-Induction

Ethanol Withdrawal-Induced Impaired Recognition Is Reversed by Chronic Exposure to Stress and the Acute Administration of Corticosterone in Mice

Screening to Identify Multidrug Resistance-Associated Protein Inhibitors with Neuroblastoma-Selective Cytotoxicity

Perturbed Calcineurin-NFAT Signaling Is Associated with the Development of Alzheimer’s Disease

Liver-Specific Silencing of Lipin1 Reduces Fat Mass as Well as Hepatic Triglyceride Biosynthesis in Mice

Different Results of IgE Binding- and Crosslinking-Based Allergy Tests Caused by Allergen Immobilization

Anti-HSV Activity of Kuwanon X from Mulberry Leaves with Genes Expression Inhibitory and HSV-1 Induced NF-κB Deactivated Properties

Inhibitory Effects of Oligostilbenoids from the Bark of Shorea roxburghii on Malignant Melanoma Cell Growth: Implications for Novel Topical Anticancer Candidates

CYP2A13 Genetic Polymorphisms in Relation to the Risk of Bladder Cancer in Japanese Smokers

Sustained Release of Mitomycin C from Its Conjugate with Single-Walled Carbon Nanotubes Associated by Pegylated Peptide

Clostridium perfringens α-Toxin Impairs Lipid Raft Integrity in Neutrophils

Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities

Effects of Glycerophospholipids on Ceramide Kinase Activity: Cardiolipin-Affected Cellular Formation of Ceramide-1-phosphate

Notes

Influence of Hot Spring Water on Fatty Acid Composition of Skin Surface Lipids in Hairless Mouse Model of Atopic Dermatitis

Effect of Long-Term Diabetes on Serotonin-Mediated Contraction in Carotid Arteries from Streptozotocin-Induced Diabetic Male and Female Rats

Substrate Specificity of Equine and Human Influenza A Virus Sialidase to Molecular Species of Sialic Acid

Anti-MUC1 Aptamer/Negatively Charged Amino Acid Dendrimer Conjugates for Targeted Delivery to Human Lung Adenocarcinoma A549 Cells

Identification of a Selective SIRT2 Inhibitor and Its Anti-breast Cancer Activity

System Maintenance

Announcement From J-STAGE

Journal Tools

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Volume And Issue List Biological and Pharmaceutical Bulletin

Predecessor

Biological and Pharmaceutical Bulletin Vol. 39(2016) No. 10

Review

Regular Articles

Notes

System Maintenance

Announcement From J-STAGE

Journal Tools

Share this Title

Volume And Issue List
Biological and Pharmaceutical Bulletin