Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 37 , Issue 11
Showing 1-22 articles out of 22 articles from the selected issue
Review
  • Hiroshi Kitagawa
    2014 Volume 37 Issue 11 Pages 1705-1712
    Published: November 01, 2014
    Released: November 01, 2014
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    Chondroitin sulfate (CS) chains constitute a class of glycosaminoglycans (GAGs). CS chains are distributed on the surfaces of virtually all cells and throughout most extracellular matrices; they are covalently attached to serine residues of core proteoglycan proteins. CS proteoglycans have been implicated as regulators of a variety of biological events, including cell–cell and cell–matrix adhesion, cell proliferation, morphogenesis, and neurite outgrowth. The functional diversity of CS proteoglycans is mainly attributed to the structural variability of the GAG chains, specifically the CS chains. Despite their relatively simple polysaccharide backbones, CS chains acquire remarkable structural variability via several types of enzymatic modifications, including sulfation. Moreover, the sulfation status of CS chains, chain length, number of CS chains per core protein, or combinations thereof can be finely tuned via CS biosynthetic machinery to specify the structure and function of CS proteoglycans. The term “sugar remodeling” refers to the experimental or therapeutic structural alteration of CS chains via perturbation of specific CS biosynthetic enzymes in cells or living organisms; sugar remodeling is a promising approach to the study of CS chain function. This review focuses on our recent findings regarding CS function which have resulted from studies involving sugar remodeling.
Regular Articles
  • Jia Qi, Xiao Han, Hai Tao Liu, Ting Chen, Jin Lian Zhang, Ping Yang, S ...
    2014 Volume 37 Issue 11 Pages 1713-1718
    Published: November 01, 2014
    Released: November 01, 2014
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    Heat shock protein 90 (HSP90) is a ubiquitous molecular chaperone involved in the proper conformation of many proteins. HSP90 inhibitors (17-dimethyl aminoethylamino-17-demethoxygeldanamycin hydrochloride [17-DMAG]) bind to and inactivate HSP90, suppressing some key signaling pathways involved in the inflammatory process. Since considerable evidence suggests that inflammation accounts for the progression of cerebral ischemic injury, we investigated whether 17-DMAG can modulate inflammatory responses in middle cerebral artery occluded (MCAO) mice. Male C57/BL6 mice were pretreated with 17-DMAG or vehicle for 7 d before being subjected to transient occlusion of middle cerebral artery and reperfusion. Mice were evaluated at 24 h after MCAO for neurological deficit scoring. Moreover, the mechanism of the anti-inflammatory effect of 17-DMAG was investigated with a focus on nuclear factor kappa B (NF-κB) pathway. 17-DMAG significantly reduced cerebral infarction and improved neurological outcome. 17-DMAG suppressed activation of microglia and decreased phosphorylation of inhibitory (I)κB and subsequent nuclear translocation of p65, which eventually downregulated expression of NF-κB-regulated genes. These results suggest that 17-DMAG has a promising therapeutic effect in ischemic stroke treatment through an anti-inflammatory mechanism.
  • Tomohisa Nakamura, Taketoshi Kushida, Naofumi Okamoto, Kenichi Oe, Ats ...
    2014 Volume 37 Issue 11 Pages 1719-1726
    Published: November 01, 2014
    Released: November 01, 2014
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    SKG/Jcl (SKG) mice spontaneously develop T cell-mediated autoimmune arthritis and may be an effective model for studying human rheumatoid arthritis. We sought to confirm that arthritis in SKG mice was caused by stem cell disorders. We induced systemic arthritis in normal C57/BL6 (B6) mice (H-2b type) by injecting lineage-negative (lin) immature cells isolated from bone marrow cells (BMCs) of SKG mice (H-2d type) directly into bone cavities. Twenty weeks later, we analyzed arthritis scores, hematoxylin–eosin (H–E) staining and tartrate-resistant acid phosphatase (TRAP) staining in ankle joints, H-2 type of hematolymphoid and osteoblast-like cells, serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and percentages of CD4+ T cells and osteoblast-like cells expressing receptor activator of nuclear factor kappa-B ligand (RANKL) in recipient mice. Donor-derived hematolymphoid cells and osteoblast-like cells had completely replaced donor-derived cells in the recipients (H-2b to H-2d). All recipients showed severe joint swelling with hyperemia and developed hypertrophic synovitis with lymphocytes accumulated around joints. All recipients also had higher TNF-α and IL-6 levels than untreated B6 controls. Furthermore, the percentages of CD4+ T cells and osteoblast-like cells expressing RANKL and the number of TRAP+ cells were greater in treated animals. Donor-derived hematolymphoid cells and osteoblast-like cells persisted in these recipients and promoted autoimmune arthritis and an increase in osteoclasts. Because autoimmune arthritis may be associated with abnormal lin immature cells, patients with intractable autoimmune arthritis may be treated by replacing these cells with direct injection of lin immature cells isolated from normal BMCs.
  • Yoshihiko Shimokawa, Katsunori Sasahara, Noriaki Yoda, Katsuhiko Mizun ...
    2014 Volume 37 Issue 11 Pages 1727-1735
    Published: November 01, 2014
    Released: November 01, 2014
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    Delamanid is a new drug for the treatment of multidrug-resistant tuberculosis. Individuals who are co-infected with human immunodeficiency virus and Mycobacterium tuberculosis may require treatment with a number of medications that might interact significantly with the CYP enzyme system as inhibitors or inducers. It is therefore important to understand how drugs in development for the treatment of tuberculosis will affect CYP enzyme metabolism. The ability of delamanid to inhibit or induce CYP enzymes was investigated in vitro using human liver microsomes or human hepatocytes. Delamanid (100 µM) had little potential for mechanism-based inactivation on eight CYP isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Delamanid’s metabolites were noted to inhibit the metabolism of some CYP isoforms, but these effects were observed only at metabolite concentrations that were well above those observed in human plasma during clinical trials. Delamanid (≤10 µM) did not induce CYP1A2, CYP2C9, and CYP3A4 activities in human hepatocytes, and there were no increases in CYP1A2, CYP2B6, CYP2C9, and CYP3A4 mRNA levels. Taken together, these data suggest that delamanid is unlikely to cause clinically relevant drug–drug interactions when co-administered with products that are metabolized by the CYP enzyme system.
  • Yukiko Katagiri, Yasunori Miyazaki, Tomonobu Uchino, Yoshiyuki Kagawa
    2014 Volume 37 Issue 11 Pages 1736-1741
    Published: November 01, 2014
    Released: November 01, 2014
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    Goreisan suppository is prepared as a hospital preparation, and successfully used for the treatment of diarrhea and vomiting in young children with common cold. While clinical efficacy of the suppository has been reported, few studies have been carried out to clarify the preparation procedure and pharmacokinetics of the suppository. In this study, trans-cinnamic acid (CA) was used as a representative substance of goreisan constituents, and assayed by HPLC-UV. We investigated the properties of goreisan suppositories prepared using various sizes of pulverized goreisan extract granules, in vitro dissolution profiles using the reciprocating dialysis tube method, and pharmacokinetics in rabbits compared with those for goreisan enema. Mass and content uniformity tests on the suppositories of three size fractions, 0–75, 75–150, and 150–300 µm, showed good acceptance for all kinds of suppository. Storage stability at 4°C was maintained until 4 months. In vitro dissolution of CA from the suppository was proportional to time until 45 min, and slower than that from the enema. Finally, 80% of CA had dissolved at 60 min. Pharmacokinetic study in rabbits revealed that the area under the plasma concentration–time curve from 0 to 120 min (AUC0–120 min) of the suppository was twice that of the enema. Moreover, from a study in rabbits using CA injection and CA suppository, we revealed that CA was rapidly and well absorbed from the rectum, showing 84% absolute bioavailability. Thus, we illustrated the defined preparation procedure of the suppository and the superiority of the suppository over the enema. This study will support evidence that the suppository is fast-acting and efficacious in clinical use.
  • Mst. Naznin Ara, Takashi Matsuda, Mamoru Hyodo, Yu Sakurai, Noritaka O ...
    2014 Volume 37 Issue 11 Pages 1742-1749
    Published: November 01, 2014
    Released: November 01, 2014
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    We describe herein the development of a high affinity and specific DNA aptamer as a new ligand for use in liposomal nanoparticles to target cultured mouse tumor endothelial cells (mTECs). Active targeted nanotechnology based drug delivery systems are currently of great interest, due to their potential for reducing side effects and facilitating the delivery of cytotoxic drugs or genes in a site specific manner. In this study, we report on a promising aptamer candidate AraHH036 that shows selective binding towards mTECs. The aptamer does not bind to normal cells, normal endothelial cells or tumor cells. Therefore, we synthesized an aptamer–polyethylene glycol (PEG) lipid conjugate and prepared aptamer based liposomes (ALPs) by the standard lipid hydration method. First, we quantified the higher capacity of ALPs to internalize into mTECs by incubating ALPs containing 1 mol%, 5 mol% and 10 mol% aptamer of total lipids and compared the results to those for unmodified PEGylated liposomes (PLPs). A confocal laser scanning microscope (CLSM) uptake study indicated that the ALPs were taken up more efficiently than PLPs. The measured Kd value of the ALPs was 142 nM. An intracellular trafficking study confirmed that most of the rhodamine labeled ALPs were taken up and co-localized with the green lysotracker, thus confirming that they were located in lysosomes. Finally, using an aptamer based proteomics approach, the molecular target protein of the aptamer was identified as heat shock protein 70 (HSP70). The results suggest that these ALPs offer promise as a new carrier molecule for delivering anti-angiogenesis drugs to tumor vasculature.
  • Mikio Saito, Manabu Abe, Tomoyasu Furukawa, Motohiro Yagi, Yoshihiro K ...
    2014 Volume 37 Issue 11 Pages 1750-1757
    Published: November 01, 2014
    Released: November 01, 2014
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    In vivo tests may be used for the diagnosis of allergy to iodinated contrast media (ICM); however, the tests do not provide definitive diagnosis and are associated with risks for patients. Diagnoses based on in vitro tests are limited, and there are almost no relevant studies. Herein, the authors examined involvement of allergic reaction from a multilateral standpoint in 39 patients suspected of having ICM allergies using leukocyte migration test (LMT). The positive rate of LMT was 44%. A comparison with the positive rate of LMT in drugs other than ICM (74%) indicated 30% difference, which was significantly low value, suggesting that there is poor involvement of these drugs in the allergic reaction. In LMT positives, 76% of hypersensitivity reactions were skin rash mainly erythema, and 18% was anaphylactic reactions. Cases considered as non-immediate hypersensitivity accounted for about 4 times as many as immediate-type hypersensitivity. In examination of relevancy between a history of drugs or food allergies, the incidence of ICM allergies was 35%. There is a high possibility that these adverse reactions were caused by pseudoallergy to drug. It was suggested that most hypersensitivity reactions were skin rash related to non-immediate hypersensitivity, and approximately 20% of the reaction was immediate anaphylactic reaction. Therefore attention should be paid not only to immediate-type hypersensitivity but also delayed reactions. Moreover, it was considered that patients with past history of drug or food allergies have a high potential for manifestation of the reactions.
  • Ping-ping Sun, Fang Yuan, Jing Xu, Ke Sai, Jie Chen, Su Guan
    2014 Volume 37 Issue 11 Pages 1758-1765
    Published: November 01, 2014
    Released: November 01, 2014
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    Cryptotanshinone (CT), isolated from the dried roots of Salvia militorrhiza, has been reported to have protective effects on myocardial and cerebral ischemia/reperfusion (I/R) injury both in vitro and in vivo. However, its effects and underlying mechanism on hepatic I/R injury remain unclear. To investigate its effects on hepatic I/R injury, thirty male Sprague-Dawley rats were randomized into 3 groups: a sham group, a vehicle-treated hepatic I/R group and a CT-treated (50 mg/kg) group. The hepatic I/R and CT-treated groups were subjected to 60 min of normothermic ischemia of the left lateral lobe of the liver, followed by 4 h of reperfusion. The animals were then sacrificed to collect the serum and the left liver lobe for assay. Hepatic function was protected, as evidenced by significantly reduced alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) levels in the CT-treated group as compared with I/R group. The terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) demonstrated significantly decreased apoptosis in the CT-administration animals. Western blotting demonstrated upregulation of the proapoptotic protein Bcl-2, as well as decreased levels of the activated form of caspase-3 and the cleaved form of its substrate, poly(ADP-ribose) polymerase (PARP) in the CT-treated group compared with those of the I/R group. In addition, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) was inhibited by CT. Our data suggest that CT attenuates hepatic I/R injury by inhibiting the intrinsic pathway of apoptosis, mediated partly through the inhibition of JNK and p38 MAPK phosporylation.
  • Zheng Li, Jian-Dong Jiang, Wei-Jia Kong
    2014 Volume 37 Issue 11 Pages 1766-1775
    Published: November 01, 2014
    Released: November 01, 2014
    [Advance publication] Released: September 05, 2014
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    Our previous studies showed that berberine (BBR) increases liver low-density lipoprotein (LDL) receptor expression in an extracellular signal-regulated kinase (ERK)-dependent manner. This study was designed to explore the upstream cellular signaling molecules recruited by BBR to activate the ERK mitogen-activated protein kinase (MAPK) cascade. Chemical inhibitors such as GW5074, manumycin A, and compound C or specific small interfering RNAs (siRNAs) were used in the blocking experiments; Western blot was used to determine the phosphorylation of kinases; real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the expression level of LDL receptor mRNA. Our results indicate that BBR increases p-Raf-1 (ser338) level time and dose dependently in HL-7702 cells, but has no influence on Ras activity; the stimulating activities of BBR on Raf-1 signaling and LDL receptor expression can be blocked by GW5074 completely, but not by manumycin A, a Ras inhibitor. BBR activates hepatic Raf-1 signaling and up-regulates LDL receptor expression in a rat model of hyperlipidemia with no impact on liver Ras activity. Importantly, our results show that the stimulating activities of BBR on hepatic Raf-1 signaling and LDL receptor expression are totally blocked by compound C, a selective inhibitor of AMP-activated protein kinase (AMPK), and also by silencing its expression with siRNA. Taken together, our results demonstrate for the first time that BBR up-regulates LDL receptor expression through Ras-independent, but AMPK-dependent Raf-1 activation in liver cells. Our study will help to elucidate the molecular pharmacology of BBR and provide new scientific evidence for its clinical application.
  • Yukako Ito, Mayu Taniguchi, Asuka Hayashi, Miku Anai, Shinya Morita, E ...
    2014 Volume 37 Issue 11 Pages 1776-1781
    Published: November 01, 2014
    Released: November 01, 2014
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    Dissolving microneedles (DMs) were applied to glucose monitoring in the dermal interstitial fluid (ISF) of rats and their potential as an alternative blood glucose monitoring device was evaluated. Sodium chondroitin sulfate was used to prepare DM array chips, which consisted of 300 DMs/cm2. The mean length of the DMs was 475±18 µm and the mean diameter of the basement was 278±8 µm. After DMs were inserted into the skin of the hair-removed rat abdomen, a wet unwoven cloth containing 10–30 µL of water was placed on the skin and ISF was extracted. By increasing the absorbed amount of water on the unwoven cloth from 10 to 30 µL, the extracted amount of glucose increased from 1.66±0.35 µg to 2.75±0.61 µg. Increasing the adhesion time of the wet unwoven cloth to the skin from 0.1 to 5.0 min, increased the amount of ISF glucose from 1.99±0.13 µg to 5.04±0.38 µg. The relation between the amount of glucose in ISF and blood glucose concentrations was examined. With increase in the adhesion time, the coefficient of determination, r2, increased from 0.501 to 0.750. The number of DMs also affected the relationship and values of the coefficient of determinations, r2 were: 0.340 (25 DMs), 0.758 (50 DMs), 0.763 (100 DMs), 0.774 (200 DMs), and 0.762 (300 DMs). These results indicate the usefulness of DMs as an alternative blood glucose monitoring device.
  • Ryuji Kato, Kota Shigemoto, Hiromasa Akiyama, Asaka Ieda, Yoshio Ijiri ...
    2014 Volume 37 Issue 11 Pages 1782-1787
    Published: November 01, 2014
    Released: November 01, 2014
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    The expression levels of CYP and uridine diphosphate-glucuronosyl transferase (UGT) are lower in hepatocellular carcinoma cell lines than in human primary hepatocytes. However, a functional liver cell (FLC)-4 cell line that has a greater capacity to secrete liver-specific proteins than other hepatocellular carcinoma cells has recently been established. A three-dimensional culture using Engelbreth–Holm–Swan (EHS) gel induces the secretion of liver-specific proteins via the induction of hepatocyte nuclear factor-4α (HNF-4α). The aim of this study was to evaluate the mRNA expression of the enzymes CYP and UGT in FLC-4 and HepG2 cells in monolayer and three-dimensional cultures using EHS gel. The mRNA levels of HNF-4α, albumin, pregnane X receptor (PXR), constitutive androstane receptor (CAR), CYPs (1A2, 2E1, 2C8, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) were determined using real-time reverse transcription (RT) PCR. In a monolayer culture, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, and 1A9) were higher in FLC-4 cells than in HepG2 cells. In FLC-4 cells, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C8, 2C19, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) significantly increased in three-dimensional culture. FLC-4 cells cultured in EHS gel showed significantly increased expression levels of CYPs and UGTs. The results of this study suggest that human hepatocellular carcinoma FLC-4 cells cultured in EHS gel show potential for use in studying in vitro drug metabolism.
  • Shanjun Tan, Wenkui Yu, Zhiliang Lin, Qiyi Chen, Jialiang Shi, Yi Dong ...
    2014 Volume 37 Issue 11 Pages 1788-1794
    Published: November 01, 2014
    Released: November 01, 2014
    [Advance publication] Released: August 30, 2014
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    Ginsenoside Rb1 (GRb1), one of the principle active components of Panax ginseng, has been reported to reduce inflammation in various diseases. In the present study, we investigated whether GRb1 has an anti-inflammatory effect on postoperative ileus (POI) and further contributes to the recovery of gastrointestinal motility. POI was induced in rats by intestinal manipulation. The POI rats received 5, 10 and 20 mg/kg GRb1 orally via gavage four times before and after surgery. Gastrointestinal motility was assessed by charcoal transport. Systemic inflammation was assessed by serum tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 concentrations, whereas intestinal inflammation was assessed by the activity of myeloperoxidase, and concentrations and gene expression of TNF-α, IL-1β, IL-6 and IL-10 in the ileum tissue. The results revealed that GRb1 increased rat gastrointestinal transit with POI. The increased levels of systemic and intestinal inflammatory parameters in POI rats were also reduced by GRb1. In addition, GRb1 reduced systemic and intestinal inflammation and increased the gastrointestinal transit of POI rats in a dose-dependent manner, and with significance at doses of 10 and 20 mg/kg. These results suggest that GRb1 has a potent anti-inflammatory effect on POI and further contributes to the recovery of gastrointestinal motility. GRb1 may be a promising treatment for POI prophylaxis.
  • Takashi Kawaguchi, Kanako Azuma, Takuhiro Yamaguchi, Satoru Iwase, Tad ...
    2014 Volume 37 Issue 11 Pages 1795-1802
    Published: November 01, 2014
    Released: November 01, 2014
    [Advance publication] Released: September 09, 2014
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    With the shift of a large proportion of cancer chemotherapy recipients to ambulatory care, the role of hospital pharmacists has changed, and their provision of information is essential care for cancer patients. There is little research on pharmacist–patient relations, particularly about pharmacist counselling, in Japan. To meet patients’ needs, pharmacist counselling should be optimized. Here, breast cancer patients’ preferences for pharmacist counselling were assessed using a discrete choice experiment. Bayesian nonlinear optimal methodology was employed to obtain six attributes (attitude of pharmacist, quality of information, explanation of side effects, frequency of pharmacist counselling before starting chemotherapy, cost of pharmacist counselling, and follow-up with the pharmacist after starting chemotherapy) of two to three levels each. The attributes and levels were used to create 12 hypothetical scenarios that were divided into two questionnaires of six choice sets each. Two hundred eighty participants were randomly assigned to complete one of these questionnaires (blocks). Attributes were analyzed by conditional logit model to determine significant predictors of patient preferences. The responses of 278 patients to 1667 scenarios were analyzed. Attitude of pharmacist, quality of information, cost of pharmacist counselling, and follow-up with the pharmacist after starting chemotherapy were significant predictors of patient preferences, with quality of information receiving the highest priority. Thus patients receiving pharmacist counselling before starting chemotherapy prefer to interact with a pharmacist with a friendly, interested attitude who provides individualized information. Further research is needed to elucidate the information that Japanese patients consider most important and to enhance pharmacist–patient communication.
  • Chuanying Xu, Shunying Zhu, Mingyuan Wu, Yan Zhao, Wei Han, Yan Yu
    2014 Volume 37 Issue 11 Pages 1803-1810
    Published: November 01, 2014
    Released: November 01, 2014
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    Osteoarthritis (OA) is a worldwide disease in aged people, causing not only physical suffering to the patients themselves, but also a great burden on their families and on society. Here we used a mouse OA model induced by destabilization of the medial meniscus (DMM), and studied the therapeutic effect of recombinant human midkine (rhMK) on this OA model. Our results indicated that the DMM surgery induced mechanical allodynia and locomotor activity obstacles, together with cartilage injury in the C57BL/6 mice. The rhMK treatment mitigated the OA related mechanical allodynia, improved locomotor activity capacity, and prevented degradation of the cartilage. Considering the safety issue of rhMK used as a biologic, we also inspected the main organs in the rhMK treated mice throughout the process and found no pathological change. These results suggest that rhMK could be used as a biologic to treat OA or OA related pain.
  • Kazuaki Matsumoto, Naoko Kanazawa, Akari Shigemi, Kazuro Ikawa, Norifu ...
    2014 Volume 37 Issue 11 Pages 1811-1815
    Published: November 01, 2014
    Released: November 01, 2014
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    The antimicrobial agents vancomycin and metronidazole have been used to treat Clostridium difficile infections (CDIs). However, it remains unclear why patients are at risk of treatment failure and recurrence. Therefore, this study retrospectively examined 98 patients with CDIs who were diagnosed based on the detection of toxin-positive C. difficile to determine the risk factors affecting drug treatment responses and the recurrence of CDI. No significant difference was observed in the cure rate or dosage between the vancomycin and metronidazole groups. The 90-d mortality rate and total number of drugs associated with CDIs, including antiinfective agents used within 2 months before the detection of toxin-positive C. difficile, were significantly lower in the treatment success group than in the failure group. The total number of antiinfective agents and gastric acid-suppressive agents used during CDI therapy was also significantly lower in the success group than in the failure group. The period from the completion of CDI therapy to restarting the administration of anticancer agents and steroids was significantly longer in patients without than in patients with recurrence. These results indicate that the total number of drugs associated with CDIs should be minimized to reduce the risk of CDIs, that not only antibiotics but also gastric acid-suppressive agents should be discontinued during CDI therapy to increase therapeutic efficacy, and that the use of anticancer agents and steroids should be delayed as long as possible after patients are cured by CDI therapy to prevent recurrence.
Notes
  • Hiroshi Fukasawa, Hideaki Muratake, Marina Nagae, Kiyoshi Sugiyama, Ko ...
    2014 Volume 37 Issue 11 Pages 1816-1819
    Published: November 01, 2014
    Released: November 01, 2014
    [Advance publication] Released: September 10, 2014
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    Pregabalin, (S)-3-isobutyl-γ-aminobutyric acid (GABA), is a widely used adjuvant therapy for patients with neuropathic pain, which is defined as chronic pain caused by lesions or diseases of the somatosensory nervous system. However, dizziness and somnolence (sleepiness) are common dose-limiting side effects, probably due to excessive sedative effects on higher centers of the central nervous system (CNS) which are involved in the anticonvulsant and analgesic actions of pregabalin. We speculated that transdermal delivery would minimize centrally mediated side effects. To test this idea, we evaluated the analgesic effects of pregabalin delivered through the transdermal route in animal models of neuropathic pain. Transdermally administered pregabalin increased the pain thresholds in response to mechanical stimuli in a partial sciatic nerve ligation model in rats and a spinal nerve ligation model in mice, and surprisingly also in normal animals. It is noteworthy that simple transdermal application of an aqueous solution of pregabalin is effective. This could be a useful treatment option to avoid or minimize the CNS-mediated side effects of orally administered pregabalin.
  • Ryohei Aoyagi, Megumi Funakoshi-Tago, Yosuke Fujiwara, Hiroomi Tamura
    2014 Volume 37 Issue 11 Pages 1820-1825
    Published: November 01, 2014
    Released: November 01, 2014
    [Advance publication] Released: September 11, 2014
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    Recent epidemiological studies showed that coffee consumption is associated with a lower risk of type 2 diabetes, presumably due to suppression of excess fat accumulation in adipocytes. However, the mechanism underlying the effect of coffee on adipocyte differentiation has not been well documented. To elucidate the mechanism, we investigated the effect of coffee on the differentiation of mouse preadipocyte 3T3-L1 cells. Coffee reduced the accumulation of lipids during adipocytic differentiation of 3T3-L1 cells. At 5% coffee, the accumulation of lipids decreased to half that of the control. Coffee also inhibited the expression of the peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor controlling the differentiation of adipocytes. Furthermore, coffee reduced the expression of other differentiation marker genes, aP2, adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), and lipoprotein lipase (LPL), during adipocyte differentiation. Major bioactive constituents in coffee extracts, such as caffeine, trigonelline, chlorogenic acid, and caffeic acid, showed no effect on PPARγ gene expression. The inhibitory activity was produced by the roasting of the coffee beans.
  • Hikaru Tanaka, Yukiko Takahashi, Shogo Hamaguchi, Naoko Iida-Tanaka, T ...
    2014 Volume 37 Issue 11 Pages 1826-1830
    Published: November 01, 2014
    Released: November 01, 2014
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    The effects of terfenadine and pentamidine on the human ether-a-go-go related gene (hERG) channel current and its intracellular trafficking were evaluated. Green fluorescent protein (GFP)-linked hERG channels were expressed in HEK293 cells, and the membrane current was measured by an automated whole cell voltage clamp system. To evaluate drug effects on channel trafficking to the cell membrane, the fraction of channel present on the cell membrane was quantified by current measurement after drug washout and confocal microscopy. Terfenadine directly blocked the hERG channel current but had no effect on trafficking of hERG channels to the cell membrane after application in culture medium for 2 d. In contrast, pentamidine had no direct effect on the hERG channel current but reduced trafficking of hERG channels. The two drugs inhibited hERG channel function through different mechanisms: terfenadine through direct channel blockade and pentamidine through inhibition of channel trafficking to the cell membrane. Combined use of automated voltage clamp and confocal microscopic analyses would provide insights into the mechanisms of drug-induced QT-prolongation and arrhythmogenesis.
  • Tomofumi Okuno, Eri Honda, Tomohiro Arakawa, Hirofumi Ogino, Hitoshi U ...
    2014 Volume 37 Issue 11 Pages 1831-1837
    Published: November 01, 2014
    Released: November 01, 2014
    [Advance publication] Released: August 30, 2014
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    The aim of the present study was to clarify the mechanism underlying the inhibition of cell proliferation in human lung cancer A549 cells by selenium (Se) compounds. Methylseleninic acid (CH3SeO2H, abbreviated as MSA), a synthetic Se compound, is a direct precursor of active methylselenol (CH3SeH) and is considered to be one of beneficial agents for cancer prevention and therapy. Sodium selenite (Na2SeO3), an inorganic Se form, is utilized in clinical Se supplementation. MSA markedly inhibited the growth of A549 cells at a concentration of 2.5×10−6 mol/L for 1 d. On Day 1, Na2SeO3 also inhibited A549 cell growth at the concentration of 7.5×10−6 mol/L. These compounds induced cell cycle arrest at the G1 phase and apoptosis under the inhibitory condition. Reduced glutathione (GSH) is critical to MSA or Na2SeO3 metabolism. The depletion of intracellular GSH suppressed Na2SeO3-induced G1 arrest, but promoted Na2SeO3-induced apoptosis. Therefore, Na2SeO3 appears to have directly induced apoptosis. In contrast, the MSA-induced G1 arrest was ameliorated by a marked decrease in GSH content. Additionally, the depletion of GSH slightly suppressed MSA-induced apoptosis. The difference in inhibitory effects between MSA and Na2SeO3 may be due to this variation in GSH-related metabolism. After exposure of A549 cells to MSA, the GSH content was significantly decreased. These results indicate that because MSA-induced G1 arrest and apoptosis induction are enhanced by GSH, the maintenance of GSH is essential for the effective anticancer action of MSA in A549 cells.
  • Rina Yagasaki, Tsutomu Nakahara, Hiroko Ushikubo, Asami Mori, Kenji Sa ...
    2014 Volume 37 Issue 11 Pages 1838-1842
    Published: November 01, 2014
    Released: November 01, 2014
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    Ocular pathologic angiogenesis is a causative factor for retinopathy of prematurity, diabetic retinopathy, and age-related macular degeneration. In the present study, we examined the effects of rapamycin and everolimus, inhibitors of mammalian target of rapamycin (mTOR), on retinal pathologic angiogenesis in mice with oxygen-induced retinopathy (OIR), an animal model of proliferative ischemic retinopathy. Mice were exposed to 80% oxygen from postnatal day (P) 7 to P10, and were then brought into room air and subcutaneously injected with rapamycin and everolimus. The neovascular tufts, the size of the central avascular zone, and the immunoreactivity for phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTOR activity, were evaluated in flat-mounted retinas. Retinal neovascular tufts and vascular growth in the avascular zone were observed in P15 mice with OIR. In addition, intense immunoreactivity for pS6 was detected in the neovascular tufts and in endothelial cells located at the vascular–avascular border. Both rapamycin and everolimus reduced the extent of retinal neovascular tufts and pS6 immunoreactivity, but they also increased the size of the avascular zone. Thus, activation of the mTOR pathway in endothelial cells contributes to retinal pathologic angiogenesis, and mTOR inhibitors that target proliferating endothelial cells are promising candidates as anti-angiogenic agents for the treatment of vasoproliferative retinal diseases.
  • Hiroko Kobayashi, Naomi Motoyoshi, Tadashi Itagaki, Norio Inokuchi
    2014 Volume 37 Issue 11 Pages 1843-1847
    Published: November 01, 2014
    Released: November 01, 2014
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    Here, we determined the sequence of a cDNA encoding a guanylic acid-specific ribonuclease (RNase He1) from Hericium erinaceus that exhibits high sequence identity (59%) with RNase Po1, an enzyme with anti-cancer activity and which is found in Pleurotus ostreatus. RNase He1 and RNase Po1 have similar structures and heat stabilities; hence, RNase He1 may also have potential as an anti-cancer agent. Therefore, we initiated structure-function studies to further characterize the enzyme. Based on the RNase Po1 structure, RNase He1 is predicted to form 3 disulfide bonds involving Cys7–Cys98, Cys5–Cys83, and Cys47–Cys81 linkages. The Cys5Ala mutant exhibited no RNase activity, whereas the Cys81Ala mutant retained RNase activity, but had reduced heat stability. Therefore, the Cys5–Cys83 bond in RNase He1 is essential for the structure of the RNase active site region. Similarly, the Cys47–Cys81 bond helps maintain the conformational stability of the active site region, and may contribute to the greater heat stability of RNase He1.
  • Satoshi Endo, Yuki Arai, Toshiyuki Matsunaga, Akira Ikari, Ossama El-K ...
    2014 Volume 37 Issue 11 Pages 1848-1852
    Published: November 01, 2014
    Released: November 01, 2014
    JOURNALS FREE ACCESS FULL-TEXT HTML
    Five rabbit aldo-keto reductases (AKRs) that participate in the reduction of drug ketones and endogenous ketosteroids have recently been cloned and characterized. Among them, AKR1C30 and AKR1C31 show the highest amino acid sequence identity of 91%, but markedly differ in their substrate specificity and inhibitor sensitivity. AKR1C30 reduces two drugs (ketotifen and naloxone) and 17-keto-5β-androstanes, whereas AKR1C31 does not reduce the two drugs, but is active towards loxoprofen and various 3/17/20-ketosteroids. In addition, AKR1C30 is selectively inhibited by carbenoxolone, valproic acid and phenobarbital. Residues A54 and R56 are located adjacent to the catalytic residue Y55 of AKR1C30. To clarify the determinants for the substrate specificity and inhibitor sensitivity of AKR1C30, we performed the mutagenesis of A54 and R56 to the corresponding residues (L and Q) of AKR1C31. The A54L mutation produced an enzyme that had almost the same substrate specificity as AKR1C31 and decreased the sensitivity to the inhibitors except for carbenoxolone. The R56Q mutation decreased the affinity for only carbenoxolone among the substrates and inhibitors. Thus, the difference in the properties between the two enzymes can be attributed to their residue difference at positions 54 and 56.
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