Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 42 , Issue 3
Showing 1-32 articles out of 32 articles from the selected issue
Current Topics - Drug Discovery: Recent Progress and the Future
Current Topics: Reviews
  • Yağız Anıl Çiçek, David C. Luther, Jessica A. Kretzmann, Vincent M. Ro ...
    2019 Volume 42 Issue 3 Pages 304-311
    Published: March 01, 2019
    Released: March 01, 2019
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    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized therapeutic gene editing by providing researchers with a new method to study and cure diseases previously considered untreatable. While the full range and power of CRISPR technology for therapeutics is being elucidated through in vitro studies, translation to in vivo studies is slow. To date there is no totally effective delivery strategy to carry CRISPR components to the target site in vivo. The complexity of in vivo delivery is furthered by the number of potential delivery methods, the different forms in which CRISPR can be delivered as a therapeutic, and the disease target and tissue type in question. There are major challenges and limitations to delivery strategies, and it is imperative that future directions are guided by well-conducted studies that consider the full effect these variables have on the eventual outcome. In this review we will discuss the advances of the latest in vivo CRISPR/Cas9 delivery strategies and highlight the challenges yet to be overcome.

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  • Tomoki Yamashita, Kazuo Takayama, Mika Hori, Mariko Harada-Shiba, Hiro ...
    2019 Volume 42 Issue 3 Pages 312-318
    Published: March 01, 2019
    Released: March 01, 2019
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    Orthotopic liver transplantation, rather than drug therapy, is the major curative approach for various inherited metabolic disorders of the liver. However, the scarcity of donated livers is a serious problem. To resolve this, there is an urgent need for novel drugs to treat inherited metabolic disorders of the liver. This requirement, in turn, necessitates the establishment of suitable disease models for many inherited metabolic disorders of the liver that currently lack such models for drug development. Recent studies have shown that human induced pluripotent stem (iPS) cells generated from patients with inherited metabolic disorders of the liver are an ideal cell source for models that faithfully recapitulate the pathophysiology of inherited metabolic disorders of the liver. By using patient iPS cell-derived hepatocyte-like cells, drug efficacy evaluation and drug screening can be performed. In addition, genome editing technology has enabled us to generate functionally recovered patient iPS cell-derived hepatocyte-like cells in vitro. It is also possible to identify the genetic mutations responsible for undiagnosed liver diseases using iPS cell and genome editing technologies. Finally, a combination of exhaustive analysis, iPS cells, and genome editing technologies would be a powerful approach to accelerate the identification of novel genetic mutations responsible for undiagnosed liver diseases. In this review, we will discuss the usefulness of iPS cell and genome editing technologies in the field of inherited metabolic disorders of the liver, such as alpha-1 antitrypsin deficiency and familial hypercholesterolemia.

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  • Tatsuya Fukuta, Takayuki Ishii, Tomohiro Asai, Naoto Oku
    2019 Volume 42 Issue 3 Pages 319-326
    Published: March 01, 2019
    Released: March 01, 2019
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    Ischemic stroke is one of the leading causes of severe disability and death. In clinical settings, tissue plasminogen activator (t-PA) for thrombolytic therapy is the only globally approved drug for the treatment of ischemic stroke. However, the proportion of patients who receive t-PA therapy is extremely limited due to its narrow therapeutic time window (TTW) and the risk of cerebral hemorrhage. Cerebral ischemia–reperfusion (I/R) injury is also a serious problem for patients’ outcomes. Hence, the development of more effective therapies has been desired to prolong the TTW of t-PA and prevent cerebral I/R injury. For delivering drugs into the brain, the blood–brain barrier (BBB) must be overcome since it limits drug penetration into the brain, leading to insufficient therapeutic efficacy. As a distinctive pathology after an ischemic stroke, it was reported that the vascular permeability of the BBB is increased around the ischemic region. We found that nano-sized liposomes can pass through the disrupted BBB and accumulate in the I/R region, and that delivery of neuroprotective agents using a liposomal drug delivery system (DDS) is effective for the treatment of cerebral I/R injury. Moreover, we have recently demonstrated that combination therapy with liposomal drugs and t-PA can suppress the deleterious effects of t-PA and extend its TTW in a rat ischemic stroke model. These findings indicate that applications of nanoparticle DDS technology could be a hopeful approach to drug development for ischemic stroke therapy. In this review, we introduce our findings on ischemic stroke treatment using liposomal DDS and recent advances from other research groups.

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  • Yoichi Naritomi, Seigo Sanoh, Shigeru Ohta
    2019 Volume 42 Issue 3 Pages 327-336
    Published: March 01, 2019
    Released: March 01, 2019
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    Predicting human pharmacokinetics (PK) such as clearance (CL) and volume of distribution (Vd) is a critical component of drug discovery. These predictions are mainly performed by in vitroin vivo extrapolation (IVIVE) using human biological samples, such as hepatic microsomes and hepatocytes. However, some issues with this process have arisen, such as inconsistencies between in vitro and in vivo findings; the integration of predicted CYP, non-CYP and transporter-mediated human PK; and the difficulty of evaluating very metabolically stable compounds. Various approaches to solving these issues have been reported. Allometric scaling using experimental animals has also often been used. However, this method has also shown many problems due to interspecies differences, albeit that various correction methods have been proposed. Another approach involves the production of chimeric mice with humanized liver via the transplantation of human hepatocytes into mice. The livers of these mice are repopulated mostly with human hepatocytes and express human drug-metabolizing enzymes and drug transporters, suggesting that these mice are useful for solving the issues of IVIVE and allometric scaling, and more reliably predicting human PK. In this review, we summarize human PK prediction methods using IVIVE, allometric scaling and chimeric mice with humanized liver, and discuss the utility of predicting human PK in drug discovery by comparing these chimeric mice with IVIVE and allometric scaling.

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  • Yoshiharu Sato, Ryo Matoba, Kikuya Kato
    2019 Volume 42 Issue 3 Pages 337-342
    Published: March 01, 2019
    Released: March 01, 2019
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    Liquid biopsy is a minimally invasive test for cancer genetic status based on circulating tumor DNA (ctDNA), circulating tumor cells, or other tumor-derived materials in blood plasma. Although the minimal invasiveness and time resolution are attractive features of liquid biopsy, the limited amount of ctDNA in plasma poses problems. Recent developments in digital PCR and next-generation sequencing (NGS)-based technology have improved the accuracy of liquid biopsy. In particular, molecular barcoding technology in NGS-based methods, i.e., tagging of molecular barcodes to cell-free DNA before amplification, reduces technical errors by validating the consensus of sequences originating from a single molecule, leading to marked improvement of the accuracy and detection limit. However, substitutions caused by DNA damage and somatic mutations originating from normal cells are still obstacles to the sensitive detection of mutations on ctDNA. Since there have been only a few clinical applications, a deeper understanding of ctDNA biology and more advanced analytical technology are needed for the practical application of liquid biopsy.

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  • Tesshu Hori, Masashi Fukutome, Chieko Koike
    2019 Volume 42 Issue 3 Pages 343-347
    Published: March 01, 2019
    Released: March 01, 2019
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    With an increasing number of identified causative genes, the widespread use of gene therapy is quickly becoming feasible. Once a target gene is selected, it is important to have a cell delivery method that is both specific and efficient. Cell type specificity and high efficiency is particularly important for the treatment of retinal degeneration, since viruses are efficient gene delivery vehicles for the nervous system, but often bring with them non-specific infections. In this review, we focus on adeno-associated virus (AAV). Over the last few decades, AAV has become a leading choice for safe gene delivery, in part due to its replication deficiency in cells without a helper virus. Here, we summarize the tropism of recombinant AAV (rAAV) for various types of mammalian retinal neurons in relation to capsid serotype and administration method. We also include our recent findings on an AAV serotype that AAV was specifically infected mouse cone photoreceptors when delivered by subretinal administration.

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Current Topics: Regular Article
  • Ryuji Watari, Motoharu Kakiki, Chihiro Yamasaki, Yuji Ishida, Chise Ta ...
    2019 Volume 42 Issue 3 Pages 348-353
    Published: March 01, 2019
    Released: March 01, 2019
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    In drug discovery, hepatocytes have been widely utilized as in vitro tools for predicting the in vivo hepatic clearance (CL) of drug candidates. However, conventional hepatocyte models do not always reproduce in vivo physiological function, and CYP activities in particular decrease quite rapidly during culture. Furthermore, conventional in vitro assays have limitations in their ability to predict hepatic CL of metabolically stable drug candidates. In order to accurately predict hepatic CL of candidate drugs, a new method of culturing hepatocytes that activates their functional properties, including CYP activities, is in high demand. In the previous study, we established a novel long-term culture method for PXB-cells® using a collagen vitrigel membrane (CVM) chamber, which can maintain CYP activity and liver specific functions at high levels for several weeks. In this study, the vitrigel culture method was applied to predictions of hepatic CL for 22 CYP typical substrates with low to middle CL, and the prediction accuracy by this method was assessed by comparing CL data between predicted (in vitro intrinsic CL using the dispersion model) and observed (in vivo clinical data) values. The results of this study showed that in vitro CL values for approximately 60% (13/22) and 80% (18/22) of the compounds were predicted within a 2- and 3-fold difference with in vivo CL, respectively. These results suggest that the new culture method using the CVM chamber and PXB-cells is a promising in vitro system for predicting human hepatic CL with high accuracy for CYP substrates, including metabolically stable drug candidates.

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Communication to the Editor
  • Yuko Yamakage, Hitomi Tsuiji, Takao Kohno, Himari Ogino, Takashi Saito ...
    2019 Volume 42 Issue 3 Pages 354-356
    Published: March 01, 2019
    Released: March 01, 2019
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    Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid β peptide (Aβ). Therefore, augmentation of Reelin activity may ameliorate Alzheimer’s disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aβ by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with “next-generation” AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aβ significantly in AppNL-F mice, which produce human wild-type Aβ. On the other hand, reducing ADAMTS-3 had no effect in AppNL-G-F mice, which produce the Arctic mutant Aβ (E22G) that forms protofibrils more efficiently than does wild-type Aβ. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aβ.

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Regular Articles
  • Wenxiang Xiong, Yaru Liang, Xue Li, Guosong Liu, Zhao Wang
    2019 Volume 42 Issue 3 Pages 357-364
    Published: March 01, 2019
    Released: March 01, 2019
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    Magnesium (Mg2+) is an endogenous cation that is involved in many essential biological reactions. Abnormal Mg2+ metabolisms in the body affect important physiological and pathological processes. However, most endogenous Mg2+ markers fail to represent body Mg2+ status; they are disadvantageous in terms of representational capacity, applied range, operational convenience, etc. In this article, we evaluated some of the most popular Mg2+ marker candidates. A logical model of the blood Mg2+ compartments was established, which consisted of unstable Mg2+ pools, representative Mg2+ pools, and conserved Mg2+ pools. These pools were based on the metabolic efficiency of Mg2+ in an acute Mg2+ intake test. The results of this study showed that only the erythrocyte intracellular ionized Mg2+ (RBC [Mg2+]i), a representative Mg2+ pool, could effectively represent abnormal body Mg2+ metabolisms in various conditions, including dietary Mg2+ adjustments, aging and metabolic syndrome. These results suggest that RBC [Mg2+]i might be a widely applicable marker of body Mg2+ levels. On unified technology platform and evaluation system, this research compared the representative capacities of RBC [Mg2+]i, plasma Mg2+ concentration (plasma [Mg2+]), erythrocyte intracellular total Mg (RBC [Mg]total) and Mg retention in rats and mice under various Mg2+-metabolism-related physiological and pathological conditions. Our technique for the direct quantitative analysis of RBC [Mg2+]i may prove valuable for basic physiological research, dietary Mg2+ regulation, as well as clinical monitoring/intervention of Mg2+-metabolism-related pathology.

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  • Fumiyoshi Kasahara, Takako Kato, Yoko Idota, Hitoe Takahashi, Chihaya ...
    2019 Volume 42 Issue 3 Pages 365-372
    Published: March 01, 2019
    Released: March 01, 2019
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    In this study, rats were fed a high-fat diet containing calcium alginate (Ca-Alg) for 5 weeks to examine the effects of Ca-Alg on lipid metabolism including triglyceride (TG) levels in the blood. We also investigated the mechanism of the TG-reducing effect of Alg in vitro. Rats were randomized into 5 groups: high-fat diet group (14% (w/w) lard, HF); three Ca-Alg-containing diet groups (2.5, 5 or 10% (w/w) Ca-Alg) and a resistant maltodextrin (RMD) diet group as a positive control (with 5% (w/w) RMD). The 10% Ca-Alg group showed a significant reduction of body weight increase from the 7th day. In addition, the increase of TG in blood was significantly suppressed, and the amount of TG excreted in feces was increased. Increase of body fat mass was in the order HF > RMD > Ca-Alg 2.5% > Ca-Alg 5% > Ca-Alg 10%, while the total weight of the extracted fat tissues was significantly reduced in the RMD, 5% and 10% Ca-Alg groups. Hepatic pathology showed clear circular vacuoles apparently representing TG accumulation in the HF group, while fewer vacuoles were seen in the Ca-Alg groups. The results of in vitro experiments indicated that Ca-Alg does not directly inhibit lipase activity, but may suppress absorption of TG by forming non-absorbable macromolecular micelles containing TG. These results suggest that Ca-Alg promotes excretion and suppresses absorption of TG, leading to reduced blood TG levels, and decreased hepatic and total body accumulation of fat. The findings should be helpful for designing future clinical trials.

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  • Kensuke Mohri, Hirofumi Misu, Hiroaki Takayama, Kiyo-aki Ishii, Akihir ...
    2019 Volume 42 Issue 3 Pages 373-378
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: December 28, 2018
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    A hepatokine is a collective term for liver-derived secretory factors whose previously-unrecognized functions have been recently elucidated. We have rediscovered selenoprotein P (SeP) and leukocyte cell-derived chemotaxin 2 (LECT2) as hepatokines that are involved in the development of insulin resistance and hyperglycemia. The aim of this study was to determine whether and, if so, how oral glucose loading alters the two hepatokines in humans. We measured concentrations of serum SeP and plasma LECT2 during 75 g oral glucose tolerance test (OGTT) (n = 20) in people with various degrees of glucose tolerance. In OGTT, concentrations of both serum SeP and plasma LECT2 decreased at 120 min compared with the baseline values, irrespective of the severity of glucose intolerance. Decrement of serum SeP during OGTT showed no correlations to the clinical parameters associated with insulin resistance or insulin secretion. In multiple stepwise regression analyses, plasma cortisol was selected as the variable to explain the changes in plasma concentrations of LECT2. The current data reveal the acute inhibitory actions of oral intake of glucose on circulating SeP and LECT2 in humans, irrespective of the severity of glucose intolerance. This study suggests that circulating SeP is regulated by the unknown clinical factors other than insulin and glucose during OGTT.

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  • Yulan Liao, Ho Jung Bae, Jiabao Zhang, Yubeen Kwon, Bokyung Koo, In Ho ...
    2019 Volume 42 Issue 3 Pages 379-388
    Published: March 01, 2019
    Released: March 01, 2019
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    Bee pollen consists of floral pollen mixed with bee secretions and nectar. It has been considered as a functional food and has different kinds of biologically active ingredients, such as flavonoids, polyphenols, phytosterols and minerals. However, its function in cognition has yet been investigated. In the present study, we investigated the ameliorating effect of bee pollen against scopolamine-caused cognitive impairment through the passive avoidance test, the Y-maze test and the Morris water maze test. In addition, Western blotting was employed to verify the effects of bee pollen on memory-related signaling molecules in the hippocampus. Bee pollen extract (100 or 300 mg/kg, per os (p.o.)) obviously reversed scopolamine-caused cognitive impairment in the passive avoidance test, ameliorated spontaneous alternation versus the scopolamine-treated group in the Y-maze test and prolonged swimming time in the target zone in the Morris water maze test. In addition, the phosphorylation levels of extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β), and the expression levels of brain-derived neurotrophic factor (BDNF) and tissue plasminogen activator (tPA) in the hippocampi, were increased in response to the treatment with bee pollen extract (100 or 300 mg/kg, p.o.). These results indicated that bee pollen ameliorates cognitive impairment induced by cholinergic blockade through the enhancing conversion of proBDNF to mature BDNF by tPA, probably, through the ERK-CREB pathway or Akt-GSK-3β signaling pathway and would be a useful agent for the treatment of cognitive dysfunction.

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  • Masato Takeuchi, Sayuri Nakano, Sachiko Tanaka-Mizuno, Chika Nishiyama ...
    2019 Volume 42 Issue 3 Pages 389-393
    Published: March 01, 2019
    Released: March 01, 2019
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    Warfarin is a drug used for anticoagulation management, with a narrow therapeutic range and multiple drug–drug interactions. Adherence and proper use of concomitant medication are thus fundamental to the efficacy and safety of warfarin therapy. In 2012, we retrospectively analyzed data from three large-scale pharmacy chains in Japan. We included all adults (≥ 20 years old) with at least one record of warfarin dispensation. We examined patient demographic data, adherence as measured by medication possession ratio (MPR), and co-dispensation focusing on the number of concomitant dispensations and concurrent use of medications that increase bleeding risk. Thresholds of underadherence and overadherence were set at <0.9 and >1.1, considering the narrow therapeutic window. We reviewed 443007 warfarin dispensation records of 71340 individuals (median age, 73 years; 62% male). The MPR was 1.0 (interquartile range: 0.96–1.0), and underadherence and overadherence was found in 16.3 and 1.9% of individuals, respectively. The median number of co-dispensed drugs was eight at each pharmacy encounter, which did not differ by age group. Drugs associated with a high bleeding risk were dispensed in 40.0% of encounters and accounted for 16.4% of all co-dispensed drugs. In summary, we found optimal overall adherence, as assessed by MPR, among our Japanese study population, even when defining a strict cut-off value. However, polypharmacy was common in all age groups and medications with a high bleeding risk profile were often co-dispensed with warfarin. Future research addressing how these dispensation patterns affect patient outcome is warranted.

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  • Li-Hong Mei, Gao Yang, Fang Fang
    2019 Volume 42 Issue 3 Pages 394-400
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: December 27, 2018
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    The photodynamic therapy (PDT) depends on the presence of molecular oxygen. Thus, the efficiency of PDT is limited in anoxic regions of tumor tissue and vascular shutdown. It is reported the use of hyperbaric oxygen (HBO) may enhance the efficiency of PDT. However, there are rarely studies about utilizing HBO plus PDT for treatment with human squamous cell carcinoma (SCC). Therefore, this study aimed to investigate and compare the therapeutic effect of combined therapy and PDT alone treatment. Multiple cellular and molecular biology techniques were used in the current study such as CCK-8, Western blotting, flow cytometry, monodansylcadaverine (MDC) staining and immunofluorescence assay. The results of combination index indicated that HBO combination with PDT synergistically inhibited A431 cells proliferation in vitro. In addition, we found that HBO significantly enhanced PDT-induced cell apoptosis via increasing the active caspase-3, active caspase-9, Apaf-1 and Bax levels and down-regulating Bcl-2. Meanwhile, the result of MDC and immunofluorescence assay confirmed that HBO increased PDT-induced autophagosome formation in A431 cells. Interestingly, autophagy inhibitor 3-methyladenine (3-MA) further increased combination-induced cell apoptosis by increasing the levels of active-caspase 9 and Apaf-1. Our results showed that HBO combined with PDT markedly induced A431 cells apoptosis and autophagy. Nevertheless, autophagy play a pro-survival role against apoptosis. Thus, HBO combination with PDT may constitute a promising approach to treat human squamous cell carcinoma in the future.

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  • Takumi Iwasawa, Takahisa Shinomiya, Nozomi Ota, Natsumi Shibata, Kenya ...
    2019 Volume 42 Issue 3 Pages 401-410
    Published: March 01, 2019
    Released: March 01, 2019
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    Ridaifen (RID)-B is an analog derived from tamoxifen (TAM). TAM has an antitumor effect by acting as an antagonist to estrogen receptor (ER). However, TAM is known to also induces apoptosis in cancer cells that do not have ER. We clarified that RID-B induces cell death at a lower concentration than TAM, and causes ER-independent apoptosis and autophagy. Based on the results of previous studies, we assumed that RID-B had a unique target different from ER and examined structural activity correlation to determine what kinds of structural features are related to RID-B activity. As a result, we found there was activity even without one of phenyl groups (Ar3) in RID-B and revealed that two pyrrolidine side chains peculiar to RID-B are related to the action. Furthermore, analogs with shorter alkyl side chains induced autophagy, but analogs with certain length of alkyl side chains induced apoptosis. Also, although there is no doubt that RID-B induces apoptosis by causing mitochondrial injury, our results suggested that such injury induced mitochondria-selective autophagy. We revealed that RID-B induce mitophagy and that this mitophagy is a defense mechanism against RID-B. Our results suggest that autophagy was induced against apoptosis caused by mitochondrial dysfunction in RID-B, so the combination of autophagy inhibitor and anticancer-drug can be effective for cancer treatment.

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  • Hidesuke Fukazawa, Mari Fukuyama, Yoshitsugu Miyazaki
    2019 Volume 42 Issue 3 Pages 411-416
    Published: March 01, 2019
    Released: March 01, 2019
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    Many bacteria encode tyrosine kinases that are structurally unrelated to their eukaryotic counterparts and are termed BY-kinases. Two BY-kinases, CapB1 and CapB2, have been identified in the Staphylococcus aureus genome. Although CapB1 and CapB2 share more than 70% homology, earlier studies with purified enzymes did not find any evident kinase activity in CapB1, whereas CapB2 was autophosphorylated on a C-terminal tyrosine cluster in the presence of the kinase modulator proteins CapA1 or CapA2. For the convenient analysis of BY-kinases, we attempted to express CapB2 in an active form in a mammalian cell line. To this end, the C-terminal activation domain of CapA1 was attached to the N-terminus of CapB2, and the resulting CapA1/CT-CapB2 chimera was further fused with various tags and transfected into HEK293T cells. Immunoblotting analyses showed that when fluorescent protein tags were attached to the N-terminus, CapA1/CT-CapB2 was both expressed and tyrosine phosphorylated in HEK293T cells. Mutation of the ATP-binding lysine abrogated tyrosine phosphorylation, indicating that tyrosine phosphorylation was catalyzed by the transfected bacterial kinase and not by endogenous cellular enzymes. Unexpectedly, mutation of the C-terminal tyrosine cluster did not abolish autophosphorylation. Further analyses revealed that CapA1/CT-CapB2 phosphorylated not only itself but also the attached fluorescent protein tag. Several domains and residues important for tyrosine kinase activity were identified from the production of various mutants. We also present data that CapB1, which was previously thought to be catalytically inert, may possess intrinsic kinase activity.

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  • Kenichi Nakamura, Shu Zhu, Katsuko Komatsu, Masao Hattori, Makoto Iwas ...
    2019 Volume 42 Issue 3 Pages 417-423
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: January 10, 2019
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    Puerarin (daidzein 8-C-glucoside) is an isoflavone C-glucoside contained in the roots of Pueraria lobata OHWI. We have previously isolated the human intestinal bacterium, strain PUE, which metabolizes puerarin to daidzein, though the enzyme which cleaves C-glycosidic bond has not been clarified. Here, we identified one of the intermediates of enzymatic puerarin C-deglycosylation reaction as 3″-oxo-puerarin (1): C-3 in the glucose moiety connecting to hydroxyl is oxidized to ketone group. 1 was easily isomerized to the mixture of 1, 2″-oxo-puerarin (2a) and cyclic acetal (2b) of 2a in non-enzymatic condition. We identified the putative puerarin-metabolizing operon of strain PUE composed of 8 genes (dgpA–H). Among them, DgpB–C complex was expressed in Escherichia coli, which cleaved the C-glycosidic bond in 1 but not puerarin. These results suggested that the puerarin C-deglycosylation reaction is a two-step enzymatic reaction, including the oxidation reaction at C-3″ in puerarin to give 1, and the subsequent C-deglycosylation of 1 to provide daidzein.

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  • Ji-Sun Shin, Hee-Soo Han, Seung-Bin Lee, Da-bin Myung, Keunsuk Lee, Su ...
    2019 Volume 42 Issue 3 Pages 424-431
    Published: March 01, 2019
    Released: March 01, 2019
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    Hydrangea serrata (THUNB.) SER. (Hydrangeaceae) leaves have been used as herbal teas in Korea and Japan. The objective of this study was to identify anti-photoaging compounds in aqueous EtOH extract prepared from leaves of H. serrata and their effects on UVB-irradiated Hs68 human foreskin fibroblasts. Phytochemical study on H. serrata leaves led to the isolation and characterization of ten compounds: hydrangenol, thunberginol A, thunberginol C, hydrangenoside A, hydrangenoside C, cudrabibenzyl A, 2,3,4′-trihydroxystilbene, thunberginol F, quercetin 3-O-β-D-xylopyranosyl (1-2)-β-D-galactopyranoside, quercetin 3-O-β-D-xylopyranosyl (1-2)-β-D-glucopyranoside. Cudrabibenzyl A, 2,3,4′-trihydroxystilbene, quercetin 3-O-β-D-xylopyranosyl (1-2)-β-D-galactopyranoside, quercetin 3-O-β-D-xylopyranosyl (1-2)-β-D-glucopyranoside were firstly isolated from H. serrata. We estimated the effects of 10 compounds on cell viability and production of pro-collagen Type I, matrix metalloproteinase (MMP)-1, and hyaluronic acid (HA) after UVB irradiation. Of these compounds, hydrangenol showed potent preventive activities against reduced cell viability and degradation of pro-collagen Type I in UVB-irradiated Hs68 fibroblasts. Hydrangenol had outstanding inductive activities on HA production. It suppressed mRNA expression levels of MMP-1, MMP-3, hyaluronidase (HYAL)-1, HYAL-2, cyclooxygenase-2 (COX-2), interleukin (IL)-6, IL-8, and IL-1β in UVB-irradiated Hs68 fibroblasts. When Hs68 fibroblasts were exposed to hydrangenol after UVB irradiation, UVB-induced reactive oxygen species (ROS) production was suppressed. Hydrangenol also inhibited the activation of activator protein-1 (AP-1) and signal transduction and activation of transcription 1 (STAT-1) by downregulating phosphorylation of p38 and extracellular signal-regulated kinase (ERK). Our data indicate that hydrangenol isolated from H. serrata leaves has potential protective effects on UVB-induced skin photoaging.

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  • Shuiqing Li, Xingyang Xue, Xiaolu Yang, Sujuan Zhou, Shumei Wang, Jian ...
    2019 Volume 42 Issue 3 Pages 432-441
    Published: March 01, 2019
    Released: March 01, 2019
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    Moutan Cortex charcoal has been used to ameliorate blood heat symptoms and treat pathologic hemorrhage down the ages. Although well known as an agent with the effect of astringency and hemostasis, its active ingredients and action mechanism remain unclear. In the present study, molecular docking technology was employed to screen the potential hemostatic compounds in Moutan Cortex charcoal and their target proteins. Protein–protein-interaction (PPI) analysis was performed to explain the functions and enrichment pathways of the target proteins. The results showed that a total of 25 compounds were estimated as active constituents targeting multiple proteins related to hemostatic diseases, including 5 proteins (SERPINC1, FVIII, FX, FII and FXII) that were considered as the key targets. Then the drug-target (D-T) network was constructed to analyze the underlying hemostatic mechanism of Moutan Cortex charcoal, followed by a hierarchical cluster analysis (HCA) for compounds clustering, and a coagulation screening test for compound verification on their coagulation activities, with the results indicating that M15 (5-Tetradecenoic acid) and M31 (1-Monolinolein) might be the key compounds contributing to the hemostasis effect of Moutan Cortex charcoal by involving in the pathways related to complement, coagulation cascades and the platelet activation, particularly by activating FVIII, FX, FII and FXII and inhibiting SERPINC1. This study has demonstrated that Moutan Cortex charcoal may work as a hemostatic through the interaction between multiple-compounds and multiple-proteins, which provides the basis for further researches on the hemostasis mechanism of Moutan Cortex charcoal.

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  • Xinjia He, Meilan Xue, Shu Jiang, Weiwei Li, Jinming Yu, Shuai Xiang
    2019 Volume 42 Issue 3 Pages 442-447
    Published: March 01, 2019
    Released: March 01, 2019
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    Fucoidan is an active component of seaweed, and could inhibit proliferation and induce apoptotic cell death in several tumor cells. However, the function of fucoidan in breast cancer is largely unknown. In the present study, we evaluated the anti-cancer potential of fucoidan in human breast cancer MCF-7 cells. Adult Sprague–Dawley rats were randomized to receive fucoidan (200 or 400 mg/kg·body weight per day) or normal saline via gastric gavage for 3 consecutive days. Serum samples were prepared from these rats, and used for subsequent experiments to examine the potential effects in MCF-7 cells. Cell viability was determined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was examined with Hoechst33258 staining and flow cytometry. Cell migration and invasion were measured by wound scratch assay and Transwell assay, respectively. Western blot and enzyme-linked immunosorbent assay (ELISA) were used to examine the expression of secretory E-cadherin and matrix metalloproteinase-9 (MMP-9). Conditioned serum from fucoidan-treated rats significantly suppressed cell proliferation and enhanced apoptosis. Cell migration and invasion were also significantly decreased. Observed effects of conditioned serum were associated with upregulation of E-cadherin and downregulation of MMP-9. Conditioned serum of rats treated with fucoidan could inhibit the proliferation and promote apoptosis of MCF-7 cells. Cell invasion and migration were inhibited, possibly via decreased epithelial–mesenchymal transition (EMT) process. Fucoidan may be a promising therapeutic agent for human breast cancers.

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  • Mari Ishigami-Yuasa, Hisao Ekimoto, Hiroyuki Kagechika
    2019 Volume 42 Issue 3 Pages 448-452
    Published: March 01, 2019
    Released: March 01, 2019
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    Combination therapy is often an effective strategy to treat cancer. In this study, we examined the growth-inhibitory effects of Am80 (tamibarotene), a specific retinoic acid receptor (RAR) α/β agonist, in combination with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), or a DNA methyl transferase (DNMT) inhibitor, 5-aza-2′-deoxycytidine, on androgen receptor (AR)-positive and AR-negative prostate cancer cell lines (LNCaP and PC-3, respectively). We found that the combination therapy of SAHA and Am80 showed an enhanced growth-inhibitory effect on LNCaP cells. Further studies with various HDAC isotype-selective inhibitors showed that SAHA and KD5170 (a selective class I and II HDAC inhibitor) each increased the RARα protein level in LNCaP cells. Our results indicate that the target of the enhancing effect belongs to the Class IIb HDACs, especially HDAC6. Dual targeting of Class IIb HDAC and RARα may be a candidate therapeutic strategy for prostate cancer.

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    Editor’s picks

    The growth-inhibitory effects of Am80 (tamibarotene), a specific retinoic acid receptor (RAR) α/β agonist, in combination with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA) on androgen receptor positive or negative prostate cancer cell lines were investigated. Ishigami-Yuasa et al. found that the combination therapy of Am80 and SAHA showed an enhanced growth-inhibitory effect on LNCaP cells. Studies with various HDAC isotype-selective inhibitors indicated that the Class IIb HDACs, especially HDAC6, had significant roles in the enhanced effect of the combination. Thus, dual targeting of Class IIb HDAC and RARα would be useful therapeutic strategy for prostate cancer.




  • Shiho Nagata, Tetsuro Marunouchi, Kouichi Tanonaka
    2019 Volume 42 Issue 3 Pages 453-461
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: January 10, 2019
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    Protein quality control (PQC) in the heart plays an important role to maintain cellular protein homeostasis. Impairment of PQC may cause the development of heart failure. It is well known that histone deacetylase 6 (HDAC6) is an essential enzyme for regulating the cellular PQC response. In this study, we aimed at examining the association between HDAC6 and the chaperone system and the effects of HDAC6 inhibition in the development of heart failure following myocardial infarction (MI). MI was induced by coronary artery ligation. Coronary artery-ligated and sham-operated rats were divided into groups that were orally administered suberoylanilide hydroxamic acid (SAHA) or vehicle from the 2nd to 8th week after the operation. The cardiac function and protein expression levels in the viable left ventricle were analyzed by echocardiography, Western blotting, and immunohistochemistry at the 2nd and 8th weeks after the operation. The deacetylase activity of HDAC6 was markedly elevated during the development of heart failure after MI. In the failing heart, a decrease in heat-shock protein (HSP) contents and an accumulation of ubiquitinated proteins were observed, indicating PQC dysfunction. Inhibition of HDAC6 activity by SAHA treatment enhanced the translocation of heat-shock transcription factor 1 to the nucleus and induced the expression of HSP, resulting in maintenance of cellular protein homeostasis. The cardiac pump function after MI was also improved by SAHA administration. Our findings suggest that inhibition of HDAC6 activity is a novel approach for the treatment of heart failure following MI.

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  • Lanqing Meng, Jun Lin, Qing Huang, Ping Liang, Jianmin Huang, Chongdon ...
    2019 Volume 42 Issue 3 Pages 462-467
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: December 26, 2018
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    Panax notoginseng saponins (PNS) have been widely used in China to treat stroke. Accumulating evidence has found that microRNA (miR)-155 plays critical roles in the pathology of ischemic stroke. Here we investigated whether PNS plays a protective effect against oxygen–glucose deprivation/reoxygenation (OGD/R)-induced focal inflammation and injury in SH-SY5Y cells by regulating miR-155 expression. Treatment with PNS at a concentration less than 160 µg/mL had no effect on the proliferation of SH-SY5Y cell. In OGD/R-induced SH-SY5Y cells, 160 µg/mL PNS treatment promoted cell proliferation and cell cycle progression, as well as decreased inhibited apoptosis and miR-155 expression. However, overexpression of miR-155 attenuated the promotion effects of PNS on cell proliferation and cell cycle, apoptosis inhibition in OGD/R-induced SH-SY5Y cells. Moreover, 160 µg/mL PNS treatment decreased the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in OGD/R-induced SH-SY5Y cells, whereas overexpression of miR-155 reversed PNS-induced decreases in the levels of IL-1β, IL-6, and TNF-α in OGD/R-treated SH-SY5Y cells. In conclusion, PNS attenuated OGD/R-induced injury in human undifferentiated SH-SY5Y cells by regulating the expression of inflammatory factors through miR-155.

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  • Kenji Goto, Keiichi Hiramoto, Kazuya Ooi
    2019 Volume 42 Issue 3 Pages 468-474
    Published: March 01, 2019
    Released: March 01, 2019
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    Skin dryness is a characteristic of rheumatoid arthritis (RA) model mice. However, the mechanism underlying the induction of dry skin by RA is unclear. We hypothesized that T helper (Th)2 and Th17 cells mediate this process. A mouse model of DBA/1JJmsSlc collagen-induced arthritis was treated with Th2 or Th17 cell inhibitor, and transepidermal water loss (TEWL) and the expression of markers associated with allergic reaction and inflammation were evaluated. TEWL and plasma levels of thymic stromal lymphopoietin, interleukin (IL)-6 and -17, and tumor necrosis factor (TNF)-α were increased in the arthritis mouse model compared to that in control mice. Administration of Th2 cell inhibitor abolished the increase in TEWL, IL-6, and TNF-α levels, whereas Th17 cell inhibitor reversed TEWL and decreased IL-17 level. Th2 and Th17 cells contribute to the induction of dry skin, but via distinct mechanisms.

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  • Xingyu Zhao, Xiaoxing Song, Jierui Zhao, Wenhe Zhu, Jiancheng Hou, Yan ...
    2019 Volume 42 Issue 3 Pages 475-480
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: December 28, 2018
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    Human papillomaviruses (HPVs), such as HPV 16 and HPV 18 are related to cervical cancer. Therefore, it is important to inhibit HPV-positive cervical cancer for treating cervical cancer. This study is aiming at investigating the proposed molecular mechanism, which underlies the antineoplastic potential of the aqueous extract of juglone of HPV-positive cervical cancer cells. According to the results, it is showed that, juglone prohibited HPV positive cervical cancer cells’ growth through dose-dependent way. Nevertheless, when pin 1 was knocked down, the proliferation inhibition reduced. The detection of apoptosis and cell cycle also illustrated that juglone influenced HPV positive cells. Western blot expressed the influence mechanism that it affected the B-cell lymphoma 2 (Bcl-2) family and later activated the Caspase-depended apoptosis way. It is contributable for this study to understand the mechanism of inhibiting HPV positive cells by juglone and it also provides an effective strategy for the application of it in the future.

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  • Mai Nagasaka, Kaori Tsuzuki, Yu Ozeki, Muneshige Tokugawa, Nobumichi O ...
    2019 Volume 42 Issue 3 Pages 481-488
    Published: March 01, 2019
    Released: March 01, 2019
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    Lysine-specific demethylase 1 (LSD1/KDM1A) is a histone demethylase and specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 (H3K4). The LSD1-mediated demethylation of H3K4 promotes the assembly of the c-Myc-induced transcription initiation complex. Although LSD1 and c-Myc are both strongly expressed in human cancers, the mechanisms by which their activities are coordinated remain unclear. We herein demonstrated that LSD1 is a direct target gene of c-Myc. The knockdown of c-Myc decreased the expression of LSD1 in several cancer cell lines. We identified two non-canonical E-boxes in the proximal promoter region of the LSD1 gene. A chromatin immunoprecipitation assay showed that c-Myc bound to these E-boxes in the LSD1 promoter. Importantly, LSD1 mRNA expression correlated with c-Myc expression in human acute myeloid leukemia (AML), glioblastoma, stomach adenocarcinoma, and prostate adenocarcinoma. The present results suggest that LSD1 is induced by c-Myc and forms a positive feedback mechanism in transcription reactions by c-Myc.

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  • Niraj Chaudhary, Keiko Ueno-Shuto, Tomomi Ono, Yuko Ohira, Kenji Watan ...
    2019 Volume 42 Issue 3 Pages 489-495
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: January 10, 2019
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    Cystic fibrosis (CF), the most common lethal inherited disorder caused by mutation in the gene encoding the CF transmembrane regulator (CFTR), is characterized by chronic inflammation that ultimately leads to death from respiratory failure. In CF patients, up-regulation of toll-like receptor-2 (TLR2), a pattern recognition receptor that senses CF-pathogenic bacteria Staphylococcus aureus peptidoglycan (PGN), in airway epithelial cells is observed, and enhanced proinflammatory responses towards PGN may result in detrimental effects in CF patients. Here, we showed that curcumin, a well known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in CF bronchial epithelial cell line, CFBE41o- cells. Strong suppression of TLR2 gene and protein expression was observed at more than 40 µM of curcumin treatment in CFBE41o- cells. Consistent with decreased expression of TLR2, PGN-dependent interleukin-8 (IL-8) gene up-regulation was markedly reduced by 40 µM of curcumin treatment. Strong reductions of TLR2 gene expression and function were also observed in primary human CF bronchial epithelial cells, but not in human non-CF primary cells. Interestingly, curcumin treatment decreased nuclear expression of transcription factor specificity protein 1 (SP1), a factor that is critical for increased basal TLR2 expression in CF cell line and primary cells. Finally, curcumin-dependent SP1 reduction was diminished by anti-oxidant N-acetylcystein (NAC) and proteasomal inhibitor MG-132, suggesting the crucial roles of oxidative and proteasomal degradation pathways. Taken together, our study shows that curcumin down-regulates TLR2 gene expression and function in CF bronchial epithelial cells possibly by accelerating SP1 degradation via an oxidative process.

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Notes
  • Dyah Aninta Kustiarini, Toshiaki Nishigaki, Hiroyuki Kanno, Hideto To
    2019 Volume 42 Issue 3 Pages 496-500
    Published: March 01, 2019
    Released: March 01, 2019
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    Morinda citrifolia L., known as noni, originated from Indonesia exhibits various pharmacological activities including anti-inflammatory properties. However, the validity of noni fruit juice as a treatment for rheumatoid arthritis (RA), an autoimmune disorder, has not been confirmed yet. Therefore, the main purpose of this research was to evaluate the efficacy of noni fruit juice (INFJ) made in Indonesia using SKG mice as an animal model of RA, which shows the resembling characteristics of human RA patients. Furthermore, the safety of INFJ was examined by repeated dose experiments in mice. INFJ was mixed with water at 50% and administered to SKG mice sensitized with mannan, free access for 4 weeks. Arthritis scores of fore- and hind-leg joints were measured and the joints were histopathologically examined. The sub-acute and sub-chronic toxicities of INFJ were evaluated using BALB/c mice. The arthritic scores were significantly lower from the 7 d after sensitization in the INFJ group than the control group. Histopathological examinations of the joints revealed inhibition of severity of RA. In both toxicity studies, INFJ did not show any toxicities. INFJ exhibited anti-arthritic activity in arthritic and histopathological examinations of the joints in SKG mice. Present study was the first report where noni juice may be effective against RA. The dose of noni juice showing efficacy against RA was confirmed safe from repeated dose studies in mice.

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  • Atsuyoshi Okada, Keiichiro Suzuki, Keisuke Hara, Moeko Kojina, Tetsuya ...
    2019 Volume 42 Issue 3 Pages 501-506
    Published: March 01, 2019
    Released: March 01, 2019
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    The mechanism underlying the increased pharmacological effects of phenobarbital in rats with glycerol-induced acute renal failure (ARF) was examined. In the experiments, a surgical cannula was inserted in the lateral ventricle of the rats for phenobarbital infusion, and the ARF induction was performed by intramuscular administration of 50% glycerol. The onset time of anesthesia by phenobarbital was determined with the tail flick method. In addition, cerebral microsomes were prepared from excised cerebral cortices of sham and ARF rats, and the cerebral expression of the γ-aminobutyric acid (GABA)A receptor and two cation-chloride transporters, KCC2 and NKCC1, was evaluated by Western blotting, as their functions are involved in the anesthetic effects of phenobarbital. When phenobarbital was infused in the ventricle, anesthesia was induced 2.2-times faster in ARF rats than in sham rats, and there was no detectable increase in the cerebral expression of the GABAA receptor in ARF rats. It was additionally noted that the cerebral expression of KCC2 decreased, whereas that of NKCC1 was unaltered in ARF rats. These findings indicated that the anesthetic effects of phenobarbital are potentiated in ARF rats, probably due to imbalanced cerebral expression of KCC2 and NKCC1, suggesting that altered cation-chloride handling in nerve cells is associated.

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  • Toshiyuki Takasu, Masanori Yokono, Atsuo Tahara, Shoji Takakura
    2019 Volume 42 Issue 3 Pages 507-511
    Published: March 01, 2019
    Released: March 01, 2019
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    Ipragliflozin, a selective sodium glucose cotransporter 2 (SGLT2) inhibitor, is used for the treatment of type 2 diabetes mellitus. To date, the only known in vitro pharmacological characteristic of ipragliflozin is its selectivity for SGLT2 over SGLT1, which was previously reported by our group. Therefore, in this study, we investigated other in vitro pharmacological characteristics of ipragliflozin and compared them with those of phlorizin, a naturally occurring SGLT inhibitor. Selectivity of ipragliflozin and phlorizin for human (h) SGLT2 over hSGLT3, hSGLT4, hSGLT5, hSGLT6 and hSodium/myo-inositol (MI) cotransporter 1 (hSMIT1) was examined in Chinese hamster ovary (CHO) cells overexpressing each transporter using specific radio-ligands. Ipragliflozin had higher selectivity for hSGLT2 than other hSGLTs. Phlorizin showed lower selectivity for hSGLT2 compared to ipragliflozin. Studies using CHO cells overexpressing hSGLT2 demonstrated that both ipragliflozin and phlorizin competitively inhibited SGLT2-mediated methyl-α-D-glucopyranoside (AMG) uptake with an inhibitory constant (Ki) of 2.28 and 20.2 nM, respectively. Ipragliflozin, but not phlorizin, inhibited hSGLT2 in a wash-resistant manner, suggesting that binding of ipragliflozin to hSGLT2 was persistent. These data demonstrate that ipragliflozin is a competitive inhibitor of SGLT2, has high selectivity for SGLT2 over not only SGLT1 but also other SGLT family members, and binds persistently to hSGLT2.

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  • Shoji Seyama, Hisae Nishioka, Hidemasa Nakaminami, Keisuke Nakase, Tak ...
    2019 Volume 42 Issue 3 Pages 512-515
    Published: March 01, 2019
    Released: March 01, 2019
    [Advance publication] Released: December 20, 2018
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    Recently, 1.5% olanexidine gluconate, a biguanide compounds, was launched as a new antiseptic agent in Japan. However, the comprehensive bactericidal spectrum of olanexidine gluconate is still unknown. In this study, we evaluated in vitro bactericidal activity of olanexidine gluconate using time-kill assay against various bacteria, mycobacteria, and fungi. With the exception of Burkholderia cepacia and Mycobacterium spp., 1.5% olanexidine gluconate exhibited fast-acting (≤60 s) bactericidal activity against all tested Gram-positive and Gram-negative bacteria, including vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, extended spectrum β-lactamase producing Klebsiella pneumoniae, and multidrug-resistant Pseudomonas aeruginosa. Furthermore, 1.5% olanexidine gluconate eradicated Candida albicans, Microsporum canis, and Malassezia furfur within 3 min. Our findings indicate that olanexidine gluconate has broad spectrum bactericidal activity; therefore, it may be useful for the prevention of a wide range of infectious diseases.

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