Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
45 巻, 10 号
選択された号の論文の29件中1~29を表示しています
Current Topics - Rotational Catalysis and Diverse Functions of Proton-Pumping ATPases
Current Topics: Reviews
  • Mizuki Sekiya
    2022 年 45 巻 10 号 p. 1404-1411
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Proton pumping ATPases, both F-type and V/A-type ATPases, generate ATP using electrochemical energy or pump protons/sodium ions by hydrolyzing ATP. The enzymatic reaction and proton transport are coupled through subunit rotation, and this unique rotational mechanism (rotational catalysis) has been intensively studied. Single-molecule and thermodynamic analyses have revealed the detailed rotational mechanism, including the catalytically inhibited state and the roles of subunit interactions. In mammals, F- and V-ATPases are involved in ATP synthesis and organelle acidification, respectively. Most bacteria, including anaerobes, have F- and/or A-ATPases in the inner membrane. However, these ATPases are not believed to be essential in anaerobic bacteria since anaerobes generate sufficient ATP without oxidative phosphorylation. Recent studies suggest that F- and A-ATPases perform indispensable functions beyond ATP synthesis in oral pathogenic anaerobes; F-ATPase is involved in acid tolerance in Streptococcus mutans, and A-ATPase mediates nutrient import in Porphyromonas gingivalis. Consistently, inhibitors of oral bacterial F- and A-ATPases, such as phytopolyphenols and bedaquiline, strongly diminish growth and survival. Herein, we discuss rotational catalysis of bacterial F- and A-ATPases, and discuss their physiological roles, focusing on oral bacteria. We also review the effects of ATPase inhibitors on the growth and survival of oral pathogenic bacteria. The features of the catalytic mechanism and unique physiological roles in oral bacteria highlight the potential for proton pumping ATPases to serve as targets for oral antimicrobial agents.

  • Atsuko Iwamoto-Kihara
    2022 年 45 巻 10 号 p. 1412-1418
    発行日: 2022/10/01
    公開日: 2022/10/01
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    The F-type ATPase family of enzymes, including ATP synthases, are found ubiquitously in biological membranes. ATP synthesis from ADP and inorganic phosphate is driven by an electrochemical H+ gradient or H+ motive force, in which intramolecular rotation of F-type ATPase is generated with H+ transport across the membranes. Because this rotation is essential for energy coupling between catalysis and H+-transport, regulation of the rotation is important to adapt to environmental changes and maintain ATP concentration. Recently, a series of cryo-electron microscopy images provided detailed insights into the structure of the H+ pathway and the multiple subunit arrangement. However, the regulatory mechanism of the rotation has not been clarified. This review describes the inhibition mechanism of ATP hydrolysis in bacterial enzymes. In addition, properties of the F-type ATPase of Streptococcus mutans, which acts as a H+-pump in an acidic environment, are described. These findings may help in the development of novel antimicrobial agents.

  • Ge-Hong Sun-Wada, Yoh Wada
    2022 年 45 巻 10 号 p. 1419-1425
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Vacuolar-type H+-ATPase (V-ATPase) was first identified as an electrogenic proton pump that acidifies the lumen of intracellular organelles. Subsequently, it was observed that the proton pump also participates in the acidification of extracellular compartments. V-ATPase plays important roles in a wide range of cell biological processes and physiological functions by generating an acidic pH; therefore, it has attracted much attention not only in basic research but also in pathological and clinical aspects. Emerging evidence indicates that the luminal acidic endocytic organelles and their trafficking may function as important hubs that connect and coordinate various signaling pathways. Various pharmacological analyses have suggested that acidic endocytic organelles are important for the maintenance of cell polarity. Recently, several studies using genetic approaches have revealed the involvement of V-ATPase in the establishment and maintenance of apico-basal polarity. This review provides a brief overview of the relationship between the polarity of epithelial cells and V-ATPase as well as V-ATPase driven luminal acidification.

  • Mayumi Nakanishi-Matsui, Naomi Matsumoto
    2022 年 45 巻 10 号 p. 1426-1431
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Vacuolar-type ATPase (V-ATPase) shares its structure and rotational catalysis with F-type ATPase (F-ATPase, ATP synthase). However, unlike subunits of F-ATPase, those of V-ATPase have tissue- and/or organelle-specific isoforms. Structural diversity of V-ATPase generated by different combinations of subunit isoforms enables it to play diverse physiological roles in mammalian cells. Among these various roles, this review focuses on the functions of lysosome-specific V-ATPase in bone resorption by osteoclasts. Lysosomes remain in the cytoplasm in most cell types, but in osteoclasts, secretory lysosomes move toward and fuse with the plasma membrane to secrete lysosomal enzymes, which is essential for bone resorption. Through this process, lysosomal V-ATPase harboring the a3 isoform of the a subunit is relocated to the plasma membrane, where it transports protons from the cytosol to the cell exterior to generate the acidic extracellular conditions required for secreted lysosomal enzymes. In addition to this role as a proton pump, we recently found that the lysosomal a3 subunit of V-ATPase is essential for anterograde trafficking of secretory lysosomes. Specifically, a3 interacts with Rab7, a member of the Rab guanosine 5ʹ-triphosphatase (GTPase) family that regulates organelle trafficking, and recruits it to the lysosomal membrane. These findings revealed the multifunctionality of lysosomal V-ATPase in osteoclasts; V-ATPase is responsible not only for the formation of the acidic environment by transporting protons, but also for intracellular trafficking of secretory lysosomes by recruiting organelle trafficking factors. Herein, we summarize the molecular mechanism underlying secretory lysosome trafficking in osteoclasts, and discuss the possible regulatory role of V-ATPase in organelle trafficking.

Communication to the Editor
  • Yuki Kiguchi, Izumi Morita, Akari Tsuruno, Norihiro Kobayashi
    2022 年 45 巻 10 号 p. 1432-1437
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Previously, we generated high-affinity antibody mutants that enabled sensitive immunoassays by exploring diverse libraries of single-chain Fv fragments (scFvs) displayed on bacteriophage. To isolate rarely-occurring desirable clones, “panning” has commonly been performed but is often unsuccessful. Therefore, we previously developed a clonal array profiling (CAP) method, wherein scFv-displaying phage (scFv-Ph) clones in a library were examined individually regarding their ability to target antigens immobilized on microwells. Clones that showed strong reactivity were recovered via dissociation using an acidic treatment. The CAP successfully discovered cortisol-specific scFvs showing 17–31-fold improved Ka from libraries generated via site-directed insertions in a prototype anti-cortisol scFv (wt-scFv; Ka, 3.6 × 108 M−1), but their Ka did not exceed 1.1 × 1010 M−1. In this study, to break this possible affinity ceiling, we devised a new system employing a dissociation-independent recovery. scFv-Phs were individually reacted to target antigen (cortisol) immobilized on microwells via a linker containing a disulfide bond. Following acidic and basic treatments to eliminate scFv-Phs with “ordinary affinities,” dissociation-resistant scFv-Phs remaining on the microwells were retrieved via reductive cleavage of the disulfide bonds. This system allowed for a straightforward and efficient discovery of scFv mutants with 33–56-fold increased Ka (1.2–2.0 × 1010 M−1), exceeding the previous affinity ceiling. These scFvs enabled an enzyme-linked immunosorbent assay for cortisol with 18–51-fold higher sensitivity than the assay performed using wt-scFv.

Regular Articles
  • Azliza Mad Anuar, Akira Minami, Hiroshi Matsushita, Kanako Ogino, Kose ...
    2022 年 45 巻 10 号 p. 1438-1443
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Estrogen deficiency during menopause causes a variety of neurological symptoms, including depression. The edible Lion’s Mane mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (HE), is a medicinal mushroom that has the potential for a neuroprotective effect and ameliorating neurological diseases, such as depression, anxiety, and neurodegenerative diseases. HE contains phytoestrogens, including daidzein and genistein. However, the ameliorating effect of HE on menopausal symptoms is not well understood. Here we investigated the impact of methanol extract of the HE fruiting body on depressive-like behavior in postmenopausal model rats. The activation of estrogen receptor alpha (ERα) causes body weight loss and uterine weight gain. Body weight gain and uterine weight loss by estrogen deficiency in ovariectomized (OVX) rats were reversed with 17β-estradiol (E2) but not with HE. Thus, the phytoestrogens in HE may hardly activate ERα. Estrogen receptor beta (ERβ) is expressed in the brain, and activation of ERβ ameliorates menopausal depressive symptoms. Notably, depressive-like behavior in OVX rats evaluated in forced swim test was reduced by administration of not only E2 but also HE for 92 d. Long-term activation of ERα increases the risk of breast and uterine cancers. HE, therefore, may be effective in treating menopausal depression without the risk of carcinogenesis caused by ERα activation.

  • Bingjiang Dong, Xinhui Liu, Jiwei Li, Bin Wang, Jian Yin, Hailong Zhan ...
    2022 年 45 巻 10 号 p. 1444-1451
    発行日: 2022/10/01
    公開日: 2022/10/01
    [早期公開] 公開日: 2022/07/20
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    Cartilage regenerative medicine, wherein the stem cells from adults exert a crucial role, has high potential in the treatment of defective articular cartilage. Recently, Bone marrow mesenchymal stem cells (BMSCs) are being increasingly recognized as an alternative source of adult stem cells, which are capable of differentiating into several cell types (e.g., adipocytes, chondrocytes, and osteoblasts). However, their proliferative properties and tendency to dedifferentiate restrict their use in clinical settings. Recently, a possible bioactive material PRP-exos (exosomes derived from platelet-rich plasma), has emerged, which can effectively facilitate the differentiation and proliferation of cells. Recent studies have reported that berberine (Ber), known to have anti-inflammatory properties, plays a role in osteogenesis. Since biological molecules are used in combinations, we attempted to assess the effect of Exos-Ber (PRP-exos in combination with Ber) on the chondrogenic differentiation of BMSCs in vitro. In this study, Exos-Ber was observed to promote the proliferation of BMSCs and cause their chondrogenic differentiation in vitro. Additionally, Exos-Ber could promote the migration of BMSCs and increase the protein expression of the chondrogenic genes (Collagen II, SOX9, Aggrecan). After treatment with Exos-Ber, significant induction of β-catenin expression was observed, which could be repressed successfully by adding β-catenin inhibitor XAV-939. Interestingly, the repression of the Wnt/β-catenin axis also resulted in reduced gene expression levels of Collagen II, SOX9, and Aggrecan. These observations indicated that Exos-Ber facilitated the differentiation of chondrogenic BMSCs by modulating the Wnt/β-catenin axis, which offers innovative insights into the reconstruction of cartilage.

  • Makoto Kataoka, Sakiho Ikkou, Yuki Kato, Keiko Minami, Haruki Higashin ...
    2022 年 45 巻 10 号 p. 1452-1457
    発行日: 2022/10/01
    公開日: 2022/10/01
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    In this study, we investigated the effects of ingested water volume on the oral absorption of fenofibrate (FEN) with several formulations to confirm the applicability of rats for oral formulation screening. Oral absorption of suspended crystalline FEN was significantly improved by increasing ingested water volume (from 0.5 to 2 mL). FEN absorption improvement by particle size reduction and the linearity in oral absorption by dose escalation suggested that the rate-limiting step of FEN absorption in rats was the dissolution rate, consistent with that in humans. When FEN, as an amorphous solid dispersion (ASD) formulation, was suspended in water followed by immediate administration, oral FEN absorption was significantly higher than when administered in crystalline form and was not influenced by the differences in ingested water volume. Oral absorption of FEN from encapsulated ASD formulation in 1 or 2 mL of water was comparable with that of the suspension form. However, 0.5 mL of water significantly reduced the oral absorption of the solid ASD FEN formulation. These results indicate that to improve the oral absorption of poorly water-soluble drugs when performing a preclinical study with rats, 1 mL of water is the minimum preferable ingested volume to evaluate in vivo formulation performance.

  • Yasuhito Nobushi, Taira Wada, Yuzuki Koike, Hikari Kaneko, Shigeki Shi ...
    2022 年 45 巻 10 号 p. 1458-1465
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Obesity is currently the most common cause of metabolic diseases including type 2 diabetes and hyperlipidemia. Obesity results from excess lipid accumulation in adipose tissue. Several studies have investigated the inhibitory effects of natural plant-derived products on adipocyte differentiation and lipid accumulation. In this study, we examined the effect of hydrolysable tannins composed of gallic acid and glucose on adipocyte differentiation in 3T3-L1 cells. 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) (1), a representative gallotannin, inhibited lipid accumulation in 3T3-L1 cells, whereas ellagitannins (tellimagrandin I, eugeniin and casuarictin) did not. The expression of adipocyte differentiation-related genes, including peroxisome proliferator activator γ2 (Pparγ2), CCAAT/enhancer binding protein α (C/EBPα) and adipocyte fatty acid binding protein (aP2), was significantly suppressed in PGG (1)-treated 3T3-L1 cells beginning at day 2 after induction of differentiation. While PGG (1) did not directly reduce Pparγ2 expression, it reduced the expression of its target genes in mature adipocytes. In addition, PGG (1) treatment inhibited mitotic clonal expansion, one of earliest events of adipocyte differentiation. These findings indicate that PGG (1) has an inhibitory effect on adipocyte differentiation through the suppression of mitotic clonal expansion.

  • Huiwen Guo, Lixiang Zheng, Yepin Guo, Lu Han, Jing Yu, Fuchong Lai
    2022 年 45 巻 10 号 p. 1466-1475
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Curculigoside (Cur) is a natural component from Curculigo orchioides Gaertn, with various bioactivities. The function of Cur in the nervous system and osteoarthritis has been reported. However, its role in osteosarcoma (OS) needs to be investigated. Hence, we focus on probing the impact of Cur on OS. In vitro, cell counting kit 8 (CCK-8), flow cytometry and Transwell assay were used to investigate the effects of Cur on OS cell proliferation, apoptosis, migration and invasion. In vivo, we developed a xenograft model to figure out the effect of Cur on tumor growth in nude mice. Western blotting (WB) was conducted to compare the levels of Cur on apoptosis-related proteins (C-caspase-3, Bax, and Bcl-2), epithelial–mesenchymal transition (EMT)-related proteins (N-cadherin, Snail, and E-cadherin) and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and nuclear factor-κB (NF-κB) pathways in vitro and in vivo. In-vitro data testified that Cur treatment markedly hampered OS cells’ growth, migration and invasion and intensified their apoptosis compared to that of the control group. In vivo, Cur treatment notably hampered the growth of OS tumors in mice. In addition, both in vitro and in vivo experiments demonstrated that the phosphorylation of JAK2, STAT3, and NF-κB were inhibited through Cur treatment. Furthermore, the inhibition of Cur in OS cells was demonstrated by up-regulating the expression of JAK/STAT and NF-κB pathways protein levels. In summary, the data suggest that Cur curbs OS growth by down-regulating the JAK/STAT and NF-κB pathways, which is an underlying therapeutic option for OS treatment.

  • Hitomi Nakatsukasa, Masaya Takahashi, Katsuyuki Takahashi, Tsutomu Tak ...
    2022 年 45 巻 10 号 p. 1476-1481
    発行日: 2022/10/01
    公開日: 2022/10/01
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    The cyclin-dependent kinase (CDK) 4/6 inhibitors, palbociclib and abemaciclib, have been approved in Japan. However, the selection criteria for these drugs have not been established. Hence, we aimed to identify the risk factors for CDK4/6 inhibitor-induced intolerable adverse events requiring dose reduction or therapy cessation and to establish useful markers for choosing the appropriate CDK4/6 inhibitor, based on the incidence of the intolerable adverse events. This retrospective cohort analysis included patients with advanced breast cancer who received 125 mg/d palbociclib or 300 mg/d abemaciclib. We defined significant adverse events (SAEs) as side effects requiring dose reduction or therapy cessation. Thirty-six percent of the patients who received palbociclib (9/25) and 27.3% of those who received abemaciclib (9/33) experienced SAEs. In palbociclib and abemaciclib groups, baseline white blood cell (WBC) counts and serum albumin (ALB) levels, respectively, were significantly lower in patients who experienced SAEs than in those who did not (palbociclib: p = 0.007; abemaciclib: p = 0.004). According to the receiver operating characteristic curve analysis, the optimal cutoff values for baseline WBC count and ALB level were 5700/µL and 4.0 g/dL, respectively. Among patients with ALB levels >4.0 g/dL, the incidence of abemaciclib-induced SAEs was significantly lower than that of the palbociclib-induced SAEs (1/17 (5.9%) vs. 6/14 (42.9%), odds ratio: 11.0, 95% confidence interval: 1.07–583, p = 0.0281). Thus, a baseline WBC count ≤5700/µL and ALB level ≤4.0 g/dL may be risk factors for palbociclib and abemaciclib-induced SAEs, respectively. Also, high ALB levels can serve as a useful marker for choosing abemaciclib.

  • Yuki Koshino, Hiroaki Tanaka, Takakiyo Tatsumichi, Yuuri Houchi, Jun N ...
    2022 年 45 巻 10 号 p. 1482-1488
    発行日: 2022/10/01
    公開日: 2022/10/01
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    This study aimed to evaluate the effects on the medical economy of the use of tracing reports by pharmacy-based pharmacists for pharmaceutical interventions, including to reduce leftover medicines. These effects were estimated by analyzing 267 tracing reports issued by pharmacy pharmacists over a period of 1 year, 2020–2021. We estimate that these interventions created cost savings of USD108170.02/year (USD104800 via pharmaceutical interventions, USD3370.02 via interventions to reduce leftover medicines). The cost savings from pharmaceutical interventions prompted by patient follow-up was estimated to be USD47650. The medical economic effect per tracing report was estimated to be USD392.51 from pharmaceutical interventions, USD12.62 from reducing leftover medicines, and USD445.33 from pharmaceutical intervention prompted by patient follow-up. Overall, therefore, pharmaceutical interventions by pharmacy pharmacists using tracing reports, including those designed to reduce leftover medicines, may benefit the medical economy.

  • Kenji Momo, Takeo Yasu, Seiichiro Kuroda, Sonoe Higashino, Eiko Mitsug ...
    2022 年 45 巻 10 号 p. 1489-1494
    発行日: 2022/10/01
    公開日: 2022/10/01
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    The aim of this study was to determine the proportion of near-miss dispensing errors in hospital pharmacies in Japan. A prospective multi-center observational study was conducted between December 2018 and March 2019. The primary objective was to determine the proportion of near-miss dispensing errors in hospital pharmacy departments. The secondary objective was to determine the predictive factors for near-miss dispensing errors using multiple logistic regression analysis. The study was approved by the ethical committee at The Institute of Medical Sciences, University of Tokyo, Japan. A multi-center prospective observational study was conducted in 20 hospitals comprising 8862 beds. Across the 20 hospitals, we assessed data from 553 pharmacists and 53039 prescriptions. A near-miss dispensing error proportion of 0.87% (n = 461) was observed in the study. We found predictive factors for dispensing errors in day-time shifts: a higher number of drugs in a prescription, higher number of quantified drugs, such as liquid or powder formula, in a prescription, and higher number of topical agents in a prescription; but we did not observe for career experience level for clinical pharmacists. For night-time and weekend shifts, we observed a negative correlation of near-miss dispensing errors with clinical pharmacist experience level. We found an overall incidence of near-miss dispensing errors of 0.87%. Predictive factors for errors in night-time and weekend shifts was inexperienced pharmacists. We recommended that pharmacy managers should consider education or improved work flow to avoid near-miss dispensing errors by younger pharmacists, especially those working night or weekend shifts.

  • Toshiyuki Shizuya, Seiko Miyazaki
    2022 年 45 巻 10 号 p. 1495-1502
    発行日: 2022/10/01
    公開日: 2022/10/01
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    In Japan, there have been many requests for off-label drugs from academic societies or patient groups to the “Evaluation Committee on Unapproved or Off-labeled Drugs with High Medical Needs.” Thus, drug indications in Japan may be limited compared to those in other countries. To clarify whether drug indications in Japan are limited, the drugs containing new active ingredients approved in Japan, the United States, and Europe from 2001 to 2020 and sold in these three regions as of the end of 2020 were identified and their indications were compared. The indications of antineoplastic agents, psycholeptics, drugs from non-Japanese companies, and drugs approved in Japan from 2011 to 2020 were limited in Japan and Europe compared to the United States. These trends were more notable among antineoplastic agents. Thirty-seven indications for 19 antineoplastic agents were approved in the United States but not in Japan, and the most common indications were urothelial carcinoma (4), hepatocellular carcinoma (3), and thyroid cancer (3). The numbers of indications and drugs with different indications in Japan and Europe were generally comparable, and no specific imbalance was observed. The same indications of antineoplastic agents should be made promptly available in Japan and Europe as in the United States, as malignancy is one of the leading causes of death.

  • Shiro Watanabe, Zhuoer Chen, Kyosuke Fujita, Masashi Nishikawa, Hirosh ...
    2022 年 45 巻 10 号 p. 1503-1509
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Secondary bile acids (SBAs) with high hydrophobicity are abundant in the colonic lumen. However, both aggravating and protective roles of SBAs have been proposed in the pathogenesis of inflammatory bowel diseases (IBDs). We observed that oral administration of hyodeoxycholic acid (HDCA), a hydrophilic bile acid, prevented the development of dextran sulfate sodium (DSS)-induced colitis in mice. We then examined the individual effects of DSS and HDCA as well as their combined effects on fecal bile acid profile in mice. HDCA treatment increased the levels of most of fecal bile acids, whereas DSS treatment had limited effects on the levels of fecal bile acids. The combined treatment with DSS and HDCA synergistically increased the levels of fecal chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) in feces, which are potent activators of the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5). The overall hydrophobicity of fecal bile acids was not modified by any treatments. Our data suggest that the preventive effect of HDCA on DSS-induced colitis in mice is due to the synergism between DSS and HDCA in increasing the levels of the fecal bile acids with potencies to activate FXR and TGR5.

  • Keishi Ishida, Daisuke Matsumaru, Shinya Shimizu, Youhei Hiromori, His ...
    2022 年 45 巻 10 号 p. 1510-1517
    発行日: 2022/10/01
    公開日: 2022/10/01
    [早期公開] 公開日: 2022/08/02
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    Royal jelly (RJ) has beneficial effects on human health, and some of these effects are reported to be the result of its estrogenic activity; however, chemicals with estrogenic activities may disrupt physiological estrogen signaling leading to adverse effects on human health. Thus, clarification of the mode of action of RJ is needed. Here, we investigated whether the estrogen-like actions of RJ are induced via estrogen receptors (ERs)–mediated genomic actions by using an in vitro reporter assay in human choriocarcinoma JEG3 cells and an estrogen-responsive reporter (E-Rep) mouse line that can be used to sensitively detect transactivation of ERs in multiple organs simultaneously. In the in vitro reporter assay, ERs–dependent transcriptional activity was significantly increased by 17β-estradiol (E2) treatment at concentrations of 1 nM and above, confirming that the assay was highly responsive to estrogen; however, RJ did not exhibit any agonist activity via either the α or β form of ER. Similarly, in E-Rep mice, E2 showed significant ERs–dependent genomic action in 17 tissue types including uterus and mammary gland, whereas RJ did not. Thus, unlike endocrine-disrupting chemicals, the estrogen-like activity of RJ is unlikely to be due to genomic actions via ERs.

  • Yu Ishima, Nio Yamazaki, Victor T. G. Chuang, Taro Shimizu, Hidenori A ...
    2022 年 45 巻 10 号 p. 1518-1524
    発行日: 2022/10/01
    公開日: 2022/10/01
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    電子付録

    PEGylated liposomes (PL) lose their long-circulating characteristic when administered repeatedly, called the accelerated blood clearance (ABC) phenomenon. The ABC phenomenon is generally thought to occur when the anti-polyethylene glycol (PEG) antibody (anti-PEG immunoglobulin M (IgM)) expressed in the spleen B cells triggered by the first dose of PL binds to the second and subsequent doses of PL, leading to activation of the complement system. MAL-PEG-DSPE, a PEG lipid with a maleimide (MAL) group at the PEG terminal, is used in various studies as a linker for ligand-bound liposomes such as antibody-modified liposomes. However, most ABC phenomenon research used PL with a terminal methoxy group (PL-OCH3). In this study, we prepared MAL-PEG-DSPE liposomes (PL-MAL) to evaluate the effect of PL-MAL on the ABC phenomenon induction compared to PL-OCH3. Pharmacokinetic, anti-PEG IgM secretion and complement activation analyses of these liposomes were conducted in mice. Interestingly, despite C3 bound to the surface of the initially administered PL-MAL, the administered PL-MAL showed high blood retention, demonstrating the same results as PL-OCH3. On the other hand, although the secretion of anti-PEG IgM induced by PL-MAL was lower than PL-OCH3, the second dose of PL-MAL rapidly disappeared from the blood. These results suggest that the antibody produced from the first dose of PL-MAL binds to the second dose of PL-MAL, thereby activating C3 to act as an opsonin which promotes phagocytic uptake. In conclusion, PL-MAL induced the ABC phenomenon independent of the production of IgM antibodies against PEG. This study provides valuable findings for further studies using ligand-bound liposomes.

    Editor's pick

    PEGylated liposomes (PL) lose their long-circulating characteristic when administered repeatedly, called the accelerated blood clearance (ABC) phenomenon. A PEG lipid with a maleimide (MAL) group at the PEG terminal, MAL-PEG-DSPE, is used in various studies as a linker for ligand-bound liposomes such as antibody-modified liposomes. However, most ABC phenomenon research used PL with a terminal methoxy group. In this study, authors prepared MAL-PEG-DSPE liposomes (PL-MAL) to evaluate the effect of PL-MAL on the ABC phenomenon induction. These findings indicate PL-MAL induced the ABC phenomenon independent of the production of IgM antibodies against PEG. This study provides valuable findings for further studies using ligand-bound liposomes.

  • Tomoko Yamaguchi, Misae Nishijima, Kenji Kawabata
    2022 年 45 巻 10 号 p. 1525-1530
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Brain microvascular endothelial cells (BMECs) are essential component of the blood–brain barrier (BBB). BMECs strictly regulate the entry of various molecules into the central nervous system from the peripheral circulation by forming tight junctions and expressing various influx/efflux transporters and receptors. In vitro BBB models have been widely reported with primary BMECs isolated from animals, although it is known that the expression patterns and levels of transporters and receptors in BMECs differ between humans and animals. Recently, several methods to differentiate BMECs from human induced pluripotent stem (hiPS) cell have been developed. However, the expression of P-glycoprotein (P-gp), which is a key efflux transporter, in hiPS cell-derived BMECs was detected at a relatively low level compared with primary human BMECs. In this study, we examined the involvement of the canonical Wnt signaling pathway, which contributes to the development of BBB formation, in the regulation of P-gp expression in hiPS cell-derived BMECs. We found that the barrier integrity was significantly enhanced in hiPS cell-derived BMECs treated with glycogen synthase kinase-3ß (GSK-3ß) inhibitors, which are known to positively regulate the canonical Wnt signaling pathway. In addition, our data also showed P-gp expression level was increased by treatment with GSK-3ß inhibitors. In conclusion, physiological barrier function and P-gp expression in BMECs can be enhanced by the canonical Wnt signaling pathway. Our results may be useful for promoting the development of drugs for central nervous system diseases using in vitro BBB model.

  • Rei Tanaka, Hiroshi Ishikawa, Junya Sato, Takao Aoyama, Yoshiaki Shika ...
    2022 年 45 巻 10 号 p. 1531-1536
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Acne-like eruption caused by anti-epidermal growth factor receptor (EGFR) antibodies such as panitumumab reduces treatment adherence and patient QOL; an alternative therapy is desired. Meanwhile, the usefulness of oral Non-steroidal Anti-inflammatory Drugs (NSAIDs) for acne-like eruptions caused by low-molecular-weight EGFR inhibitors such as erlotinib has been reported in the treatment of lung cancer. This study aimed to investigate whether the combined use of oral NSAIDs and panitumumab for colorectal cancer patients helps prevent acne-like eruption. We retrospectively investigated 167 colorectal cancer patients who had been treated with panitumumab for three cycles or more. The observation period was set from the start of panitumumab treatment to the end of three cycles. Within this period, the incidence and severity of acne-like eruptions were compared. A total of 59 and 108 patients were in the NSAIDs use and non-use groups, respectively, showing differences in the incidence of acne-like eruption rates (78.0 vs. 90.7%, respectively; p = 0.033). In the use group, eruption severity grades 0, 1, 2, and 3 were observed in 13, 33, 13, and 0 patients, respectively; the corresponding values in the non-use group were 10, 60, 36, and 2, respectively (p = 0.007). Oral NSAIDs may help prevent acne-like eruptions caused by panitumumab.

    Editor's pick

    Acne-like eruption caused by anti-epidermal growth factor receptor (EGFR) antibodies such as panitumumab reduces treatment adherence and patient quality of life; an alternative therapy is desired. Although the mechanism underlying acne-like eruption associated with anti-EGFR antibody remains unclear, previous studies have suggested that the inhibition of cyclooxygenase (COX)-2 is effective to acne-like eruption caused by low-molecular-weight EGFR inhibitors such as erlotinib. Authors aimed to investigate whether the concurrent use of oral Non-steroidal Anti-inflammatory Drugs (NSAIDs) and EGFR antibodies and presented preliminary evidence that oral NSAIDs may help prevent acne-like eruptions caused by panitumumab.

  • Yuji Yamada, Toru Onda, Keisuke Hamada, Yamato Kikkawa, Motoyoshi Nomi ...
    2022 年 45 巻 10 号 p. 1537-1543
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Octa-arginine (R8) is a cell-permeable peptide with excellent cell adhesion properties. Surface-immobilized R8 mediates cell attachment via cell surface receptors, such as heparan sulfate proteoglycans and integrin β1, and promotes cell spreading and proliferation. However, it is not clear how these properties are affected by specific peptide composition and if they could be improved. Here, we synthesized XR8 peptides, in which half of the original R8 arginine residues were replaced with another amino acid (X). We then aimed to investigate the effect of the substitution on cell adhesion and proliferation on XR8-conjugated agarose matrices. The XR8-matrix showed slightly better cell attachment when X was a hydrophobic or aromatic amino acid. However, hydrophobic XR8-matrices tended to promote cell proliferation to a less extent. Eventually, YR8-matrix most efficiently promoted cell adhesion, spreading, and proliferation among the XR8-matrices tested. Collectively, these observations indicate that the properties of residue X play a major role in the biological activity of XR8-matrices and shed light on the interaction between small peptides and the cell membrane. Further, YR8 is a promising cell-adhesive peptide for the development of cell culture substrates and biomaterials.

  • Teruko Imai, Mizuki Isozaki, Kayoko Ohura
    2022 年 45 巻 10 号 p. 1544-1552
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Esmolol is indicated for the acute and temporary control of ventricular rate due to its rapid onset of action and elimination at a rate greater than cardiac output. This rapid elimination is achieved by the hydrolysis of esmolol to esmolol acid. It has previously been reported that esmolol is hydrolyzed in the cytosol of red blood cells (RBCs). In order to elucidate the metabolic tissues and enzymes involved in the rapid elimination of esmolol, a hydrolysis study was performed using different fractions of human blood and liver. Esmolol was slightly hydrolyzed by washed RBCs and plasma proteins while it was extensively hydrolyzed in plasma containing white blood cells and platelets. The negligible hydrolysis of esmolol in RBCs is supported by its poor hydrolysis by esterase D, the sole cytosolic esterase in RBCs. In human liver microsomes, esmolol was rapidly hydrolyzed according to Michaelis–Menten kinetics, and its hepatic clearance, calculated by the well-stirred model, was limited by hepatic blood flow. An inhibition study and a hydrolysis study using individual recombinant esterases showed that human carboxylesterase 1 isozyme (hCE1) is the main metabolic enzyme of esmolol in both white blood cells and human liver. These studies also showed that acyl protein thioesterase 1 (APT1) is involved in the cytosolic hydrolysis of esmolol in the liver. The hydrolysis of esmolol by hCE1 and APT1 also results in its pulmonary metabolism, which might be a reason for its high total clearance (170–285 mL/min/kg bodyweight), 3.5-fold greater than cardiac output (80.0 mL/min/kg bodyweight).

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    Esmolol, an antedrug, is clinically used for long period due to its rapid onset of action and elimination at a rate greater than cardiac output. The authors found the esterases involved in the hydrolysis of esmolol. Esmolol was hydrolyzed by human carboxylesterase 1 (hCE1) in leukocytes and platelets. The human hepatic clearance limited by hepatic blood flow was obtained by microsomal hydrolysis by hCE1 and cytosolic hydrolysis by acyl protein thioesterase 1 (APT1). The expression of hCE1 and APT1 in human lung might cause the high total clearance of esmolol, which is 3.5-fold greater than cardiac output.

  • Nao Yamagishi, Jun-ichiro Takahashi, Yue Zhou, Satoru Yokoyama, Teruhi ...
    2022 年 45 巻 10 号 p. 1553-1558
    発行日: 2022/10/01
    公開日: 2022/10/01
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    9,10-Phenanthrenequinone (9,10-PQ), a polycyclic aromatic hydrocarbon that is present in air pollutants, such as diesel exhaust gas and PM2.5, causes the production of excess reactive oxygen species. 9,10-PQ was recently shown to induce the activation of epidermal growth factor receptor (EGFR) by inhibiting protein tyrosine phosphatase 1B. In the present study, we focused on the non-canonical regulation of EGFR, including negative feedback and internalization. In contrast to previous findings, 9,10-PQ inhibited the constitutive tyrosine phosphorylation of EGFR via the mitogen-activated protein extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK)-mediated phosphorylation of Thr-669 in EGFR-overexpressing A431 and MDA-MB-468 cells. In addition, 9,10-PQ induced the clathrin-mediated endocytosis of EGFR via the p38 phosphorylation of Ser-1015 in HeLa and A549 cells. These results revealed that 9,10-PQ strongly induced the non-canonical regulation of EGFR by activating mitogen-activated protein kinase (MAPK).

  • Yasuhiro Hayashi, Kouki Matsuda, Kazunari Tanigawa, Takashi Tanikawa, ...
    2022 年 45 巻 10 号 p. 1559-1563
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity is inhibited with N-(4-hydroxyphenyl)-retinamide (4-HPR). We reported previously that 4-HPR suppresses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry through a DEGS1-independent mechanism. However, it remains unclear whether DEGS1 is involved in other SARS-CoV-2 infection processes, such as virus replication and release. Here we established DEGS1 knockout (KO) in VeroE6TMPRSS2 cells. No significant difference was observed in virus production in the culture supernatant between wild-type (WT) cells and DEGS1-KO cells, although the levels of dihydroceramide (DHCer), a DEGS1 substrate, were significantly higher in DEGS1-KO cells than WT cells. Furthermore, the virus-induced cytopathic effect was also observed in DEGS1-KO cells. Importantly, the EC50 value of 4-HPR in DEGS1-KO cells was almost identical to the value reported previously in WT cells. Our results indicated the lack of involvement of DEGS1 in SARS-CoV-2 infection.

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    Hayashi Y, et al. have already reported that N-(4-Hydroxyphenyl)-retinamide (4-HPR) inhibits dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity, and suppresses SARS-CoV-2 infection. Here, the authors describe the generation of DEGS1 knockout VeroE6/TMPRSS2 cells and their use to evaluate the infectivity and the effect of 4-HPR on SARS-CoV-2 infection. This study reports that DEGS1 is not involved in SARS-CoV-2 infection, including viral replication and the release of viral progeny. Further investigation is needed to elucidate the exact mechanism underlying the inhibition in SARS-CoV-2 infection by 4-HPR. 4-HPR has been extensively studied for cancer treatment. Therefore, the results of this study, along with accumulated clinical data on the safety of 4-HPR, are potential candidates for the treatment of COVID-19.

  • Rahma Tharwat Sabra, Amira A. Abdellatef, Essam Abdel-Sattar, Moustafa ...
    2022 年 45 巻 10 号 p. 1564-1571
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a potential target for inflammatory-breast cancer treatment as it participates in its pathogenesis, such as tumor initiation, progression, survival, metastasis, and recurrence. In this study, we aimed to discover a novel anti-cancer treatment from natural products by targeting NF-κB activity. Using the 4T1-NFκB-luciferase reporter cell line, we tested three pregnane glycosides extracted from the herb Caralluma tuberculata and discovered that Russelioside A markedly suppressed NF-κB activity in breast cancer. Russelioside A inhibited NF-κB (p65) transcriptional activity and its phosphorylation. Following NF-κB inhibition, Russelioside A exerted anti-proliferative and anti-metastatic effects in breast cancer cells in vitro. Moreover, it inhibited the NF-κB constitutive expression of downstream pathways, such as VEGF-b, MMP-9, and IL-6 in 4T1 cells. In addition, it reduced the metastatic capacity in a 4T1 breast cancer model in vivo. Collectively, our conclusions reveal that Russelioside A is an attractive natural compound for treating triple-negative breast cancer growth and metastasis through regulating NF-κB activation.

  • Fumie Mitani, Ryosuke Hayasaka, Akiyoshi Hirayama, Chitose Oneyama
    2022 年 45 巻 10 号 p. 1572-1580
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Extracellular vesicles (EVs) originating from intraluminal vesicles (ILVs) formed within multivesicular bodies (MVBs), often referred to as small EV (sEV) or exosomes, are aberrantly produced by cancer cells and regulate the tumor microenvironment. The tyrosine kinase c-Src is upregulated in a wide variety of human cancers and is involved in promoting sEV secretion, suggesting its role in malignant progression. In this study, we found that activated Src liberated synaptosomal-associated protein 23 (SNAP23), a SNARE molecule, from lipid rafts to non-rafts on cellular membrane. We also demonstrated that SNAP23 localized in non-rafts induced cholesterol downregulation and ILV formation, resulting in the upregulation of sEV production in c-Src-transformed cells. Furthermore, the contribution of the SNAP23-cholesterol axis on sEV upregulation was confirmed in pancreatic cancer cells. High SNAP23 expression is associated with poor prognosis in patients with pancreatic cancer. These findings suggest a unique mechanism for the upregulation of sEV production via SNAP23-mediated cholesterol downregulation in Src-activated cancer cells.

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    Cancer cells secrete large numbers of small extracellular vesicles (sEVs). These vesicles are derived from intraluminal vesicles (ILVs) and contribute to cancer progression by forming the tumor microenvironment. In this study, Mitani et al. demonstrated a novel role for SNAP23 in Src-dependent EV secretion: in Src-transfected cells, SNAP23 translocates to non-rafts and causes cholesterol depletion, resulting in the upregulation of ILV. They also found that pancreatic cancer patients with high SNAP23 expression have a poor prognosis. These findings suggest that the Src-SNAP23-sEV axis contributes to cancer progression.

Notes
  • Akira Maeda, Takashi Fujimura, Noriko Hirakawa, Kenji Baba, Seiji Kawa ...
    2022 年 45 巻 10 号 p. 1581-1584
    発行日: 2022/10/01
    公開日: 2022/10/01
    [早期公開] 公開日: 2022/08/18
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    Cellular senescence is an inherent tumor suppressive process, and cancer-targeted senescence induction represents an attractive anti-tumor strategy. Here, we show that a methoxyflavanone derivative (Perilla-derived methoxyflavanone, PDMF) from the Asian medicinal herb, Perilla frutescens, induces cellular senescence in A549 human adenocarcinoma cells but not in normal human bronchial epithelial (NHBE) cells. We also provide evidence that PDMF preferentially activates the p53–p21 pathway in A549 cells, and that p53 is essential for its pro-senescent activity.

  • Atsushi Kitamura, Kei Higuchi, Toshiki Kurosawa, Takashi Okura, Yoshiy ...
    2022 年 45 巻 10 号 p. 1585-1589
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood–brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

  • Kazuki Nagayasu, Chihiro Andoh, Hisashi Shirakawa, Shuji Kaneko
    2022 年 45 巻 10 号 p. 1590-1595
    発行日: 2022/10/01
    公開日: 2022/10/01
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    Transcriptional regulatory elements, including promoters and enhancers, play a key role in the cell-type specific regulation of the transcriptome. Application of rapidly evolving genetic tools, such as optogenetic/chemogenetic actuators and fluorescent reporters to elucidate the function of cell subtypes in vivo necessitates cell-type specific promoters or enhancers. In this context, methods for genome-wide functional screening of cis-regulatory elements, including enhancers, are of utmost importance. In this study, we describe a novel method for genome-wide functional screening of enhancer activity in vivo with minimal handling. Application of the method to cells from different brain structures and subsequent differential analysis allow identification of active enhancers in the target tissue or brain structures. To demonstrate proof of concept, we applied this method to samples from the dorsal raphe nucleus (DRN) and the medial prefrontal cortex of the mouse brain and successfully identified six enhancers with highly biased activity towards the dorsal raphe nucleus. Considering that these two structures consist of largely similar cell types whereas serotonin and dopamine neurons exist only in the DRN, our results confirm the validity of this method in identifying cell-type specific and brain-structure specific enhancers. Overall, this method will be helpful in identifying cis-regulatory elements suitable for cell-type specific manipulations.

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