BPB Reports

2019 - Vol. 2

2019 - Vol. 2

Regular Article
Protein Kinase C Regulates the Citrate Transport via Na+-Coupled Citrate Transporter NaCT in HepG2 Cells Vol.2, No.6, p.134-140
Maya Goto , Yusuke Kono , Ayako Yuki , Haruka Nishimura , Mizuki Ikawa , Kanta Ohno , Takuya Fujita
Received: November 01, 2019
Accepted: December 23, 2019
Released: December 31, 2019
Abstract Full Text PDF[1M]

Citrate, an intermediate of tricarboxylic acid cycle, plays a crucial role for the generation of biochemical energy and synthesis of fatty acids and cholesterol in liver. The cellular uptake of citrate is mediated by Na+-coupled di- and tricarboxylate transporters, particularly NaCT. Since NaCT expression level in liver is closely related to the pathogenesis of metabolic diseases, such as non-alcoholic fatty liver disease. Therefore, it is important to elucidate the regulation mechanism of NaCT function in liver. In this study, we focused on protein kinase C (PKC), and evaluated the influence of PKC activation on the citrate transport in human hepatocellular carcinoma HepG2 cells. The uptake of citrate in HepG2 cells depended on Na+, and it also occurred via a saturable process. Its Michaelis constant (Km) and maximal velocity (Vmax) was 5.12 mM and 106 nmol/mg protein/30 min, respectively. These results suggest that the citrate transport in HepG2 cells is primarily mediated by NaCT. In addition, we observed that the Na+-dependent citrate uptake in HepG2 cells was significantly decreased by the preincubation of the cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator. We also found that this decrease of citrate uptake by PMA was attributed to the reduction of Vmax, without affecting Km value. These results indicate that PKC regulates the transport activity of NaCT in HepG2 cells. The present findings contribute to the elucidation of the regulation mechanism of NaCT function in hepatic metabolic diseases.

Sensitization of Gastric Cancer Cells to Irinotecan by p53 Activation Vol.2, No.6, p.130-133
Shun Zhang , Yoshinori Kohira , Hajime Orita , Momoko Ishimine , Toshiyuki Kobayashi , Sharlyn Mae Buendia Chua , Hirofumi Nakaoka , Ituro Inoue , Okio Hino , Takehiko Yokomizo , Tetsu Fukunaga , Hyeon-Cheol Lee-Okada
Received: December 11, 2019
Accepted: December 17, 2019
Released: December 27, 2019
Abstract Full Text PDF[1M]

Irinotecan (camptothecin-11 [CPT-11]) is a topoisomerase I inhibitor that has been used in the treatment of a wide spectrum of cancers including gastric cancer. Recent reports suggest that the expression of CES2, a serine hydrolase that converts irinotecan to its active compound SN-38, is regulated by the tumor-suppressor p53. In this study, we investigated whether irinotecan acted synergistically with a p53 activator nutlin-3a in human gastric cancer cells. Nutlin-3a treatment enhanced the expression of CES2 in gastric cancer cell lines with wild-type p53. However, this effect was not observed in cells with non-functional p53. Irinotecan showed synergistic antitumor effects in combination with nutlin-3a in gastric cancer cells with wild-type p53, whereas the survival of cells with non-functional p53 was not significantly affected by the presence of nutlin-3a. These results provide evidence that p53 activation can enhance the antitumor effect of irinotecan or other anticancer prodrugs activated by CES2 in gastric cancer cells through upregulation of CES2 expression.

The Efficacy of a Mattress Type Sleep Measuring Device in Analyzing Sleep in Healthy University Students: Comparison with Actigraphy Vol.2, No.6, p.125-129
Hiroshi Kawai , Yutaro Togashi , Takuya Ishibashi , Reiko Iwadate , Atsushi Mitsumoto
Received: November 15, 2019
Accepted: December 10, 2019
Released: December 24, 2019
Abstract Full Text PDF[665K]

Sleep disturbance relates to various disorders and is a significant public health issue. Evaluation of sleep quality is necessary to analyze and improve sleep quality. Polysomnography (PSG) is an efficient method for sleep analysis. However, complicated systems are required for the analysis. Also, PSG can be stressful for participants and is, therefore, not suitable for long term sleep monitoring. Sleepscan is a non-invasive mattress type sleep measuring device developed by TANITA. Sleepscan measures the participant’s heart rate, respiration, and body movement during sleep, and evaluates sleep quality objectively. We measured the sleep quality of healthy university students with Sleepscan and a widely-used sleep measuring device, actigraph. We also discussed the efficacy of using Sleepscan daily. Sleepscan detected longer sleep latency and shorter awake episodes during sleep than actigraph. Although these devices showed quite different results for some sleep variables, the sleep score recorded by Sleepscan and sleep efficiency by actigraph correlates well. Since sleep efficiency is used as a representative index for comprehensive sleep quality in actigraphy, the sleep score by Sleepscan can be an alternative index used to evaluate sleep quality objectively. Sleepscan can also analyze the depth of sleep. The deep sleep variables recorded by Sleepscan did not correlate with the sleep variables by actigraph, suggesting that these variables may represent aspects of sleep quality that cannot be detected by actigraphy. Sleepscan may be useful in analyzing sleep quality objectively more comprehensively over a long period.

Regular Article
Application of Activated Clay for Improvement of Water Quality in Wire Electric Discharge Machining Vol.2, No.6, p.119-124
Eri Nagahashi , Fumihiko Ogata , Takehiro Nakamura , Naohito Kawasaki
Received: November 25, 2019
Accepted: December 13, 2019
Released: December 23, 2019
Abstract Full Text PDF[1M]

In this study, the availability of activated clay treatment for decreasing the electrical conductivity in the real wire cut water was demonstrated. Activated clay (AC) was prepared, and its characteristics were investigated. The ability to adsorb Zn2+ and the removal ability through electrical conductivity were evaluated. It was found that the AC had a montmorillonite-like structure. Its specific surface area, micropore volume, mesopore volume, and macropore volume were 125.0 m2/g, 0.7 μL/g, 172.6 μL/g, and 13.7 μL/g, respectively. The cation exchange capacities at pH 5 and pH 10 were 56.2 and 67.2 cmol/kg, respectively. The effects of temperature, contact time, and pH of the solution on the adsorption of Zn2+ were measured. The amount of Zn2+ adsorbed by the AC increased with an increase in the adsorption temperature or in the pH. Adsorption isotherms data were fitted to the Freundlich equation compared to the Langmuir equation. Adsorption equilibrium was reached within 30 min, and kinetic data were fitted to the pseudo-second-order model compared to the pseudo-first-order model. Additionally, the AC was proven to effectively suppress the electrical conductivity. The suppression achieved by using washed AC was higher than that obtained by virgin AC. Washing AC with distilled water is useful for removing released ions (SO42-). Therefore, the column treatment packed with washed AC was evaluated in this paper. Finally, washed AC could remove Zn2+, resulting in the reduction of electrical conductivity (removal percentage is 47%). These findings provide significant information that can be useful for the removal of Zn2+ and reducing the electrical conductivity from wire cut water.

Adenovirus Fiber can Distribute Itself to the Cell Surface without Membrane Damage Vol.2, No.6, p.113-118
Anna Sato , Takamasa Hirai , Naoya Koizumi , Saya Hatakeyama , Aine Watanabe , Tetsuya Nomura , Fuminori Sakurai , Hiroyuki Mizuguchi , Naoki Utoguchi
Received: October 04, 2019
Accepted: November 22, 2019
Released: December 20, 2019
Abstract Full Text PDF[2M]

Fiber is an adenovirus (Ad) capsid protein that binds to coxsackievirus and Ad receptor. It is secreted by Ad-infected cells in the early infection stage, and it increases the permeability of the epithelial cells. Accordingly, fiber may facilitate the apical escape of Ad particles from the basolateral side in Ad-infected cells. However, its behavior in the Ad-infected cells remains unclear. Therefore, we investigated the behavior of fiber in the Adinfected cells by fluorescence microscope analysis. Results showed that a higher proportion of fiber molecules were present in the apical side compared with that in the basolateral side, and electrical resistance, which represents cell–cell adhesion, remained unaffected in the Ad-infected cells. Furthermore, the association between fiber secretion and membrane damage was analyzed using annexin V and propidium iodide staining. We observed that fiber was distributed to the membrane surface without membrane damage. In addition, fiber distribution occurred in Ad-infected cells as well as in fiber-expressing cells. Therefore, fiber can distribute itself to the cell surface, and it plays a novel role in Ad infection. Further investigation of fiber distribution would be useful to completely elucidate Ad infection mechanism and develop antiviral strategies for Ad.

Regular Article
Induction of Versican V0 Variant Synthesis by A Thrombin Receptor Agonist Peptide in Cultured Human Coronary Smooth Muscle Cells Vol.2, No.6, p.106-112
Takato Hara , Takako Wakata , Yasuyuki Fujiwara , Chika Yamamoto , Toshiyuki Kaji
Received: November 06, 2019
Accepted: November 25, 2019
Released: December 18, 2019
Abstract Full Text PDF[1M]

Versican is a large aggregating chondroitin sulfate proteoglycan that accumulates in vascular wall during atherosclerosis. This proteoglycan is particularly synthesized by arterial smooth muscle cells and contributes to atherosclerosis progression by enhancing the retention of low density lipoproteins and inducing the proliferation and migration of the cells in atherosclerotic plaques. There is a strong interrelationship between atherosclerosis and thrombosis, suggesting that thrombin—a key coagulation factor—may stimulate versican synthesis in arterial smooth muscle cells. To determine the regulation of proteoglycan synthesis by thrombin receptor agonist peptide (SFLLRN), cultured human coronary smooth muscle cells were stimulated by the peptide, and proteoglycans synthesized by the cells were characterized by biochemical techniques. The experiments indicate that SFLLRN enhances the synthesis of versican V0 variant without affecting the length and disaccharide composition of its chondroitin sulfate chains under high cell density condition. This suggests that the procoagulant state of blood may accelerate atherosclerosis progression through a high accumulation of versican V0 variant derived from arterial smooth muscle cells after the cell density becomes higher in atherosclerotic plaques.

Regular Article
DC-Based Immunotherapy Using Vascular Endothelial Cells Cultured in Conditioned Medium as a Vaccine Antigen Exerts an Anti-Tumor Effect by Inhibiting Angiogenesis Vol.2, No.6, p.99-105
Makie Yamakawa , Tetsuya Nomura , Mariko Yamagata , Takamasa Hirai , Naoya Koizumi , Kazuo Maruyama , Naoki Utoguchi
Received: November 06, 2019
Accepted: December 02, 2019
Released: December 18, 2019
Abstract Full Text PDF[2M]

Blood vessels are essential for the maintenance and growth of tumor tissues. Furthermore, tumor angiogenesis promotes metastasis, which greatly affects prognosis. Therefore, tumor blood vessels are considered an important target in cancer therapy. Cancer immunotherapy has been developed recently as a new cancer therapeutic. Notably, vaccine therapy with dendritic cells (DCs), which possess potent antigen-presenting capacities, is expected to induce tumor-associated, antigen-specific immunity. In this regard, as tumor endothelial cells (TECs) constituting tumor blood vessels are derived from endothelial cells (ECs) in the host, DC vaccine therapy targeting tumor blood vessels may have applicability in several cancer types. Thus, we attempted to develop DC vaccine therapy that targeted TEC. In our previous studies, in vitro TEC models were created by culturing normal ECs in the culture supernatants of tumor cells. Moreover, we demonstrated that the molecules’ permeability is enhanced in an in vitro TEC model compared with normal ECs. In this study, we examined whether immunotherapy using TEC-extracted proteins as vaccine antigens would be an effective cancer therapy. The results showed that DC vaccine therapy targeting TECs induced anti-tumor effects in a murine Colon-26 solid tumor model and in a lung metastases model using murine B16 melanoma cells. Moreover, anti-angiogenic effects of immunization with TECs were demonstrated. Thus, immunotherapy using the in vitro TEC model as an antigen may be an effective cancer therapeutic. In the future, identifying specific TEC antigens will help generate promising new strategies to inhibit angiogenesis.

Regular Article
Distribution of 58 Semi-Volatile Organic Chemicals in the Gas Phase and Three Particle Sizes in Indoor Air and House Dust in Residential Buildings During the Hot Season in Japan Vol.2, No.6, p.91-98
Shinji Takeuchi , Toshiko Tanaka-Kagawa , Ikue Saito , Hiroyuki Kojima , Hideto Jinno
Received: November 14, 2019
Accepted: November 20, 2019
Released: December 04, 2019
Abstract Full Text PDF[1M]

A variety of semi-volatile organic chemicals (SVOCs), such as plasticizers and flame retardants, are released into indoor air and dust from building materials, furniture, and housekeeping products in residential housing. In this study, we measured 58 SVOCs in indoor air and dust from 50 and 48 dwellings, respectively, from 19 prefectures across Japan during the hot season (from July to September). In order to reveal the current situation regarding these compounds in indoor air, we obtained indoor air samples using a newly designed four-stage multi-nozzle cascade impactor and measured the concentrations of the chemicals in the indoor air in three different particle size ranges (<2.5, 2.5-10, and >10μm), as well as a gas phase. From the results obtained using the multi-nozzle cascade impactor, smaller compounds were mainly detected in the gas phase and larger compounds were found in the particle phases. However, the three cyclic polysiloxanes, including decamethylcyclooctasiloxane (D5), with large molecules were detected in the gas phase in all of the houses. Among the 58 chemicals, D5 showed the highest median concentration (1.1 μg/m3) in the range from 0.2 to 36 μg/m3 in the indoor air samples. Our analysis of house dust revealed that di-2-ethylhexyl phthalate (DEHP) was present in all samples at the highest median concentration (590 μg/g) in the range from 200 to 6,200 μg/g. These results suggest that the residential indoor environment in Japan is mainly polluted with siloxanes in the gas phase of indoor air and by DEHP in house dust.

Regular Article
Method Validation for the Determination of Phthalates in Indoor Air by GC-MS with Solid-Phase Adsorption/Solvent Extraction using Octadecyl Silica Filter and Styrene–Divinylbenzene Copolymer Cartridge Vol.2, No.5, p.86-90
Toshiko Tanaka-Kagawa , Ikue Saito , Aya Onuki , Maiko Tahara , Tsuyoshi Kawakami , Shinobu Sakai , Yoshiaki Ikarashi , Shiori Oizumi , Masahiro Chiba , Hitoshi Uemura , Nobuhiko Miura , Ikuo Kawamura , Nobumitsu Hanioka , Hideto Jinno
Received: October 02, 2019
Accepted: October 09, 2019
Released: November 01, 2019
Abstract Full Text PDF[715K]

This study proposes and evaluates a precise and labor-saving method for quantifying phthalic-acid esters (PAEs) in indoor air based on solid-phase extraction. Three different adsorbents were evaluated; i.e., two types of octadecyl silica (ODS) filter and a styrene–divinylbenzene (SDB) copolymer cartridge. Calibration curves for five PAEs [diethyl phthalate (DEP), diisobutyl phthalate, di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), and di(2-ethylhexyl) phthalate (DEHP)] were created using an internal standard (DBP-d4). Values of the coefficient of determination (R2) indicated good linearity of the calibration curves (R2 > 0.9953). Among the three adsorbents, the SDB cartridge was easiest to handle because it can be used without cleaning and has the lowest blank value. The recovery of deuterated PAEs (DEP-d4, DBP-d4, BBP-d4, and DEHP-d4) did not differ significantly among the three adsorbents; values were consistently > 89.7% for an air volume of 2.88 m3. During simultaneous indoor air sampling, PAE concentrations were very similar for the three adsorbents. Interlaboratory validation studies were conducted in five laboratories to validate the proposed method for two PAEs (DBP and DEHP). The mean recoveries of the two PAEs added to two types of adsorbent were 91.3–99.9%, the reproducibility relative standard deviations (RSDR) were 5.1–13.1%, and the Horwitz ratio (HorRat) values were 0.31–0.79. The proposed method using solid-phase extraction with two types of adsorbents provides accurate estimates of PAEs in ambient air.

Comparison of Predictive Performance of Drug Dose Settings Using Renal Function Estimation Equations Based on the Japanese Population: A Preliminary Retrospective Study Using Vancomycin Dosing Data Vol.2, No.5, p.80-85
Shungo Imai , Soyoko Kaburaki , Takayuki Miyai , Hitoshi Kashiwagi , Mitsuru Sugawara , Yoh Takekuma
Received: September 18, 2019
Accepted: October 18, 2019
Released: October 30, 2019
Abstract Full Text PDF[1M]

The Cockcroft–Gault (C–G) equation is widely used for drug dose settings in Japan. However, several reports have questioned its accuracy. In previous decades, estimation equations of creatinine clearance (CCr), such as the Orita–Horio equation, have been established based on the Japanese population. We previously built the fitted C–G and fitted Orita–Horio equations by fitting the coefficients of the estimation equations to the study population. However, the usefulness of these equations for drug dose settings remains unclear. Our preliminary study verifies the accuracy of these equations by comparing the predictive performance of the initial vancomycin (VCM) trough value between four equations: the conventional C–G (as control), conventional Orita– Horio, fitted C–G, and fitted Orita–Horio equations. Patients receiving VCM intravenously between January 2015 and March 2019 at Hokkaido University Hospital were enrolled. Overall, 308 patients were included. As initial dose setting methods, we selected two therapeutic drug monitoring (TDM) analysis software: SHIONOGI-VCM-TDM ver.2009 (VCM-TDM) and Vancomycin MEEK TDM analysis software Ver2.0 (MEEK). Predictive performances were evaluated by calculating mean prediction error and mean absolute prediction error (MAE). The lowest MAE was obtained with the conventional C–G equation using VCM-TDM, indicating high predictive performance. However, contrasting result was obtained with MEEK, where the highest MAE was obtained using conventional C–G equation. Moreover, no significant differences were observed in MAE between the other three equations, suggesting that accurate dose settings are not always achieved, despite using accurate CCr equations based on the Japanese population.

Regular Article
A PRNP-Disrupted Human Neuroblastoma Cell Line and Its Stable Transformants Expressing Prion Protein Variants Vol.2, No.5, p.73-79
Yuko Okemoto-Nakamura , Isei Tanida , Toshiyuki Yamaji , Kentaro Hanada , Ken’ichi Hagiwara
Received: July 25, 2019
Accepted: September 11, 2019
Released: October 16, 2019
Abstract Full Text PDF[4M]

A key event in prion diseases is the conformational conversion of a cellular normal form of the prion protein (PrPC) to disease-associated conformers (PrPSc). Polymorphisms or mutations in the human prion protein gene (PRNP) are associated with acquired or hereditary human prion diseases. When human prion protein (PrP) variants are characterized by cDNA expression systems in cultured cells, the endogenous prion protein may affect the behavior of ectopically expressed prion variants. To eliminate this issue, we herein created a PRNP-disrupted mutant cell line from human neuroblastoma BE(2)-M17 cells. No morphological differences were observed between PRNP-null and parental cells. We stably introduced cDNAs encoding E196K-PrP and E200K-PrP (both of which are Creutzfeldt-Jakob disease (CJD)-related mutants), a non-pathogenic E219K-PrP, and wild-type control PrP into PRNP-null mutant cells using the PiggyBac system, and found that the glycosylation pattern of E196K-PrP differed from that of other PrPs. We also found that non-glycosylated PrPs for E196K- and E200K-PrPs had distinct electrophoretic mobilities from that of WT-PrP, and E219K-PrPs exhibited slightly different mobilities in polyacrylamide gel electrophoresis under endogenous PrP-null conditions, demonstrating that the PRNP-disrupted human neuroblastoma cell line serves as a useful tool for examining PrPs with mutations or polymorphisms.

Regular Article
Targeting of Wild-Type and Mutated Forms of Lysosome-Associated Membrane Protein-1 (LAMP-1) to Late Endosomes/Lysosomes Depends on Affinities of Their Cytoplasmic Tail Peptides with a Medium Subunit of Adaptor Protein Complex-3 (AP-3) Vol.2, No.5, p.67-72
Kenji Akasaki , Hiroshi Sakane , Yusuke Ohdoi , Kenta Okamoto , Kazutaka Nushida , Toshiyuki Hata
Received: September 02, 2019
Accepted: September 24, 2019
Released: October 11, 2019
Abstract Full Text PDF[3M]

Lysosome-associated membrane protein-1 (LAMP-1) is a type I membrane glycoprotein with a COOH-terminal cytoplasmic tail (CT) containing a lysosome-targeting signal of GYQTI382-COOH. This sequence is categorized as a tyrosine-based motif of GYXXΦ where Φ is a bulky hydrophobic amino acid residue. Lysosomal localization of LAMP-1 varies by changing the COOH-terminal amino acid residues. Adaptor protein (AP) complexes are reported to recognize the tyrosine-based signal peptide for efficient lysosomal transport of LAMP-1. In order to better understand the role of APs in lysosomal transport of LAMP-1, we have studied interactions of wild-type (WT) and mutated CTs of LAMP-1 with medium (μ) subunits of the four APs by a yeast two-hybrid (Y2H) system and subsequent computer-based molecular modeling. Among the μ subunits of AP-1, AP-2, AP-3 and AP-4, called μ1, μ2, μ3A, and μ4, respectively, the WT-CT significantly interacted with μ3Α in the Y2H system. The degree of interaction of the WT and mutated CTs with μ3A from the Y2H analyses correlated with that of their dissociation constants determined by computer-based molecular modeling, and also with that of the late endosomal and lysosomal amount of WT and the similar mutants of LAMP-1. These results reinforce the notion that AP-3 makes a major contribution to the vesicular transport of LAMP-1 to late endosomes and lysosomes via a direct intracellular route.

Regular Article
Advantage of a Co-expression System for Estimating Physiological Effects of Functional Interaction Between Cytochrome P450 3A4 and Uridine 5’-Diphospho-Glucuronosyltransferase 2B7 Vol.2, No.5, p.61-66
Yuu Miyauchi , Hideyuki Yamada , Yuji Ishii
Received: July 19, 2019
Accepted: September 15, 2019
Released: October 10, 2019
Abstract Full Text PDF[1M]

Cytochrome P450 (CYP, P450) and uridine 5’-diphospho-glucuronosyltransferase (UGT) play crucial roles in drug metabolism phase I and II, respectively. Our previous studies suggest that there are functional interactions between P450 and UGT. We previously established a co-expression system featuring CYP3A4 and UGT2B7 using baculovirus-infected insect cells. Commercial microsomes are available that individually express CYP3A4/NADPH P450 reductase (CPR) or UGT2B7. It would be much easier if we could evaluate the functional interaction of CYP3A4 and UGT2B7 by simply mixing the microsomes. To address this issue, we presently compared our established co-expression system with a simple microsome mixing system. Co-expressed UGT2B7 suppressed CYP3A4 activity. On the contrary, adding UGT2B7 microsomes to CYP3A4/CPR microsomes significantly enhanced CYP3A4 activity. The enhancement was systematic and strongly dependent on UGT2B7 microsomes, and was abrogated by detergent treatment. The collective results suggested that the enhancement of CYP3A4 activity resulted from a non-physiological interaction between CYP3A4 and UGT2B7, which were both expressed on different membranes. The phenomenon was distinguishable and hardly ever reflected the physiological interaction. This pitfall can be avoided by not using simple mixing procedures. In selecting experimental materials and methods depending on the subject of the study, a co-expression system should be applied in the analysis of functional P450-UGT interaction.

Oleanolic Acid-3-(1′2′Orthoacetate-Glucoside)-28-Glucoside Alleviates Methylmercury Toxicity in Vitro and in Vivo Vol.2, No.4, p.56-60
Ryosuke Nakamura , Tatsuya Shirahata , Tatsuya Katsumi , Naruki Konishi , Yasukazu Takanezawa , Yuka Ohshiro , Shimpei Uraguchi , Yoshinori Kobayashi , Masako Kiyono
Received: July 27, 2019
Accepted: August 14, 2019
Released: August 29, 2019
Abstract Full Text PDF[1M]

Methylmercury (MeHg) is one of the most toxic environmental pollutants and causes serious health hazards worldwide. Recently, we demonstrated that oleanolic acid (OA) 3-glucoside (OA3Glu), a saponin derivative in which glucose is bound to the C3 position of OA, has anti-MeHg activity by suppressing Hg accumulation in organs of mice. In this study, we examined the anti-MeHg activity of OA-3-(1′2′orthoacetate-Glu)-28-Glu in which glucose is bound to the C3 position of OA in a different binding form from that in OA3Glu. We found that OA-3-(1′2′orthoacetate-Glu)-28-Glu suppressed cellular MeHg uptake and improved cell viability upon exposure to MeHg in Caco-2 cells. To verify the in vivo anti-MeHg activities of OA-3-(1′2′orthoacetate-Glu)-28-Glu, mice were orally administered MeHg (0.02, 1.0, or 5.0 mg·kg−1·d−1), with or without OA-3-(1′2′orthoacetate-Glu)-28-Glu. The mice cotreated with 0.02 mg·kg−1·d−1 MeHg and OA-3-(1′2′orthoacetate-Glu)-28-Glu showed significantly lower Hg content in the liver and kidney than those treated with MeHg alone. In addition, interleukin (IL)-1β and IL-6 levels in the brain of mice cotreated with 5.0 mg·kg−1·d−1 MeHg and OA-3-(1′2′orthoacetate-Glu)-28-Glu were significantly lower than those of mice treated with MeHg alone. These results suggested that OA-3-(1′2′orthoacetate-Glu)-28-Glu had potential as an anti-MeHg accumulation compound, owing to its ability to suppress MeHg distribution into organs especially under low-level MeHg exposure condition. Taken together, it was suggested that glucose binding to the C3 position of OA is important for anti-MeHg activity of OA saponin derivatives.

Regular Article
Two Modes of Toxicity of Lipid Nanoparticles Containing a pH-Sensitive Cationic Lipid on Human A375 and A375-SM Melanoma Cell Lines Vol.2, No.4, p.48-55
Ahmed Y. AlBaloul , Yusuke Sato , Nako Maishi , Kyoko Hida , Hideyoshi Harashima
Received: May 10, 2019
Accepted: August 02, 2019
Released: August 28, 2019
Abstract Full Text PDF[1M]

Melanomas are one of the most aggressive form of skin cancer and are resistant to many cancer therapies. Lipid nanoparticles (LNPs) containing a pH-sensitive cationic lipid, YSK05 (YSK05-LNPs), for delivering short interfering RNA (siRNA) were found to strongly triggers in vitro toxicity in human A375 and A375-SM melanoma cell lines regardless of gene silencing. Assessing the localization of the toxicity was done by controlling the cellular uptake of the YSK05-LNPs that contained different polyethyleneglycol (PEG)-lipid. The YSK05-LNPs exhibited consistent dose- and time-dependent toxicity, independent of their cellular uptake, indicating that the toxicity is triggered by an interaction between the YSK05-LNPs and the cell surface. Treatment with free YSK05 resulted in only time-dependent toxicity. These results suggest that the YSK05-LNPs trigger two modes of action; a fast-acting component that is related to the LNP formulation and a slow-acting mode, which is related to the YSK05 lipid itself. Necrosis was determined to be the cause of cell death, as evidenced by the results of Annexin V assays, which are specific for confirming lipid-based toxicity. These findings indicate that these YSK05-LNPs have substantial potential for use as an antimelanoma agent as both an RNA interference-based drug and as a chemotherapeutic drug.

Regular Article
Prostaglandin-Modulated Interaction of Thymic Progenitor Cells with Blood Vessels during Estradiol-Induced Thymic Involution Vol.2, No.4, p.39-47
Nurhanani Razali , Io Horikawa , Hirofumi Hohjoh , Chihiro Yoshikawa , Hiroshi Hasegawa
Received: July 10, 2019
Accepted: July 13, 2019
Released: August 01, 2019
Abstract Full Text PDF[6M]

Thymic involution-associated disfunction of thymus is implicated in aging, microbial infection, pregnancy, improper nutrition, and etc., therefore it is clinically important especially in aged societies. Excess administration of estradiol to male mice is known to induce thymic involution and used as a mouse model of thymic involution, whereas the mechanisms of which have not been well understood. Here we examined the role of prostanoids in the estradiol-induced thymic involution in mice. The administration of 17β-estradiol for 7 consecutive days induced thymic involution. In the involute thymus, the expression of mRNAs for some synthetic enzymes of prostanoids, including Ptgs1, Ptgs2, Ptgds, Hpgds, Ptges1, and Tbxas, are upregulated. In order to examine the roles of prostanoids in the thymic involution, we treated the mice with an NSAID, etodolac, following 17β-estradiol-administration. The etodolac-treatment partially inhibited the estradiol-induced reduction of thymic size and disorganization of the boundary between thymic cortex and medulla, as indicated by keratin 5 expression as well as by localization of innate immune cells. CD34-positive thymic progenitor cells localized near the blood vessels in the estradiol-administered thymus, although they were more dispersed by the etodolac-treatment. The association of CD34-positive cells with blood vessels is known to be mediated by E- and P-selectins, whose expressions were also regulated by estradiol-administration in an etodolac-sensitive manner. These results indicated the role of prostanoids in the histological change of thymus during estradiol-induced thymic involution.

Decreased Risk of Fragility Fractures associated with Statin Use in the Older Japanese Population: a Nationwide Case–crossover Study Vol.2, No.3, p.35-38
Naomi Iihara , Eri Ohara , Kanako Baba , Soichiro Nagao , Yoshinori Bando , Tomoji Yoshida , Masaki Ohara , Yutaka Kirino
Received: June 08, 2019
Accepted: June 19, 2019
Released: July 02, 2019
Abstract Full Text PDF[679K]

The association between statin use and fractures has been investigated by several cohort and case–control studies, with inconsistent results, but no case–crossover study has been conducted. This case–crossover study aimed to analyze the association between statin use and fragility fractures in a large older cohort using the National Database of Health Insurance Claims and Specific Health Checkups of Japan. In this study, 446,101 patients aged ≥65 years in Japan who sustained fragility fractures from May 2013 to September 2014 were evaluated. Statin use was compared between the case window (3 days just before the date of the fragility fracture) and three control windows (31–33, 34–36, and 37–39 days before the fragility fracture), and the association between statin use and the occurrence of fragility fractures was estimated using a conditional logistic regression model with 1:3 matching of cases to controls. The adjusted odds ratio for the association between statin use and fragility fractures was 0.86 (95% confidence interval 0.83–0.89). Stratified analyses showed a tendency for a decreased risk of fractures with statin use; females (versus males), very old (versus old) individuals, and those with fractures in the vertebrae (versus proximal humerus, distal radius, or femoral neck) had substantially decreased risks of fractures. The results suggest a decreased risk of fragility fractures associated with statin use in older Japanese individuals, but further studies are needed.

Regular Article
Changes in Bile Acid Concentrations in Chimeric Mice Transplanted with Different Replacement Indexes of Human Hepatocytes Vol.2, No.2, p.29-34
Chieri Fujino , Seigo Sanoh , Yuka Tamura , Yuji Ishida , Chise Tateno , Shigeru Ohta , Yaichiro Kotake
Received: March 28, 2019
Accepted: April 17, 2019
Released: April 27, 2019
Abstract Full Text PDF[1M]

Chimeric mice with humanized liver that are repopulated with human hepatocytes are useful to study hepatitis B and C viruses, predict drug metabolism and pharmacokinetics (PK), and evaluate hepatotoxicity. Understanding the characteristics of chimeric mice is important for making predictions in humans. In general, chimeric mice with more than 70% of replacement indexes (RIs), a value representing the occupancy ratio of the region of the human liver to that of the mouse liver, are used. However, chimeric mice with RIs less than 70% are also useful in understanding the species differences between mice and humans. In this study, to elucidate the effects of proliferating human hepatocytes and remaining mouse hepatocytes on bile acid concentrations in detail, we investigated the differences in the total concentrations of bile acids and their compositions in chimeric mice with different RIs. The total concentrations of bile acids in their sera increased as the RIs increased. The ratios of primary to secondary bile acids, percentages of glycine conjugates, and hydrophobicity indexes, obtained upon classifying bile acids based on their compositions in the serum and comparing them with those in normal mice and humans, were found to approach the values observed in humans as the RIs increased. The percentages of taurine conjugates were high in chimeric mice with high RIs, although their values were close to those in humans. These results could be fundamental in providing knowledge to accurately predict human PK and toxicity in chimeric mice with humanized liver.

Effect of GATA Transcription Factors on Cadmium Toxicity in Human Proximal Tubular Cells Vol.2, No.2, p.25-28
Jin-Yong Lee , Maki Tokumoto , Gi-Wook Hwang , Masahiko Satoh
Received: March 09, 2019
Accepted: April 11, 2019
Released: April 26, 2019
Abstract Full Text PDF[1M]

Cadmium (Cd) is an environmental toxic heavy metal that causes renal dysfunction. Cd triggers renal dysfunction through proximal tubular cell toxicity. Our previous study demonstrated that Cd changed the activities of various transcription factors in human proximal tubular HK-2 cells. Interestingly, several GATA family members were included in the transcription factors whose activities were decreased by Cd treatment. The GATA family has diverse roles in cell proliferation, tissue development, disease regulation, and carcinogenesis. In this study, we examined whether knockdown of GATA family members affected the viability of HK-2 cells. The single knockdown of GATA1, GATA3 or GATA6 using siRNA significantly decreased HK-2 cell viability. In particular, GATA6 knockdown led to the greatest effect on HK-2 cells viability. Cd increased mRNA levels of GATA3 and GATA6 but did not affect that of GATA1. The GATA family may regulate the expression of downstream factors involved in common pathway. Therefore, the effect of combined knockdown of GATA1/3/6 on the viability of HK-2 cells was examined. Our results indicated that the effect on HK-2 cell viability following knockdown of multiple GATA family members was comparable to the single knockdown of GATA6. These results suggest, therefore, that multiple GATA family members may be involved in modulating Cd renal toxicity through a common pathway.

Regular Article
Improvement in the Quality of Deteriorated Blended Oil by Calcium Silicate Treatment Vol.2, No.2, p.19-24
Fumihiko OGATA , Haruka AMI , Eri NAGAHASHI , Takehiro NAKAMURA , Naohito KAWASAKI
Received: January 18, 2019
Accepted: February 13, 2019
Released: March 13, 2019
Abstract Full Text PDF[1M]

We prepared blended oil using soybean oil (SR10) and rapeseed oil (SR01), and evaluated the quality of deteriorated pure and blended oils (with weight/volume ratio of soybean oil to rapeseed oil of 2:1, 1:1, and 1:2) by heat treatment and aeration. The ratio of the acid value following deterioration (AV) to its initial value (AV0) i.e., the value of AV/AV0 was in the order SR10 (3.29) << SR01 (7.50). On the other hand, the ratio of the carbonyl value after deterioration (CV) to its initial value (CV0) i.e., the value of CV/CV0 decreased with increase in the proportion of rapeseed oil in the blended oil (SR01 (3.12) << SR10 (4.15)). We thus establish that the fatty acid component of the blended oil is a very important factor in the deterioration of edible oil by heat treatment and aeration. Next, we evaluated the improvement in the quality of deteriorated oil by calcium silicate adsorbent (Ca:Si = 1:3, CAS30) treatment i.e., adsorption. It is established that CAS30 exhibited the ability to remove AV and CV from deteriorated oil (treatment temperature: 80 °C < 100 °C). Moreover, we investigated the removal mechanism of AV and CV, and determined good positively or negatively linear relationship between AV or CV removal by CAS30 and the proportion of unsaturated fatty acid in deteriorated pure and blended oils. In summary, the results obtained from treatment with CAS30 provided useful information on improvement in the quality of deteriorated blended oil.

Regular Article
Calcineurin Binds to a Unique C-Terminal Region of NBCe1-C, the Brain Isoform of NBCe1 and Enhances its Surface Expression Vol.2, No.1, p.7-18
Naomi Hasegawa , Naoya Hatano , Suguru Tohyama , Sayaka Kita , Katsuhiko Mikoshiba , Akihiro Mizutani
Received: February 13, 2019
Accepted: February 13, 2019
Released: March 06, 2019
Abstract Full Text PDF[3M]

NBCe1 (electrogenic Na+/HCO3 – cotransporter 1) is a product of gene SLC4A4 and has five splice variants, NBCe1-A through NBCe1-E. In agreement with an essential role of NBCe1 in cellular pH regulation, human families carrying missense mutations of gene SLC4A4 show proximal renal tubular acidosis. Some of them exhibit brain function-related symptoms, such as migraine and mental retardation, but physiological roles of NBCe1 in brain function remain unclear. To gain insights into NBCe1-specific functions in the brain, we herein identified proteins that specifically bind to a unique C-terminal region of NBCe1-C, a brain-specific NBCe1 isoform. We found that a catalytic subunit of calcineurin binds to the C terminus of NBCe1-C in the mouse cerebellum. Heterologous-coexpression experiments revealed that calcineurin binds to NBCe1-C via a “PQIRIE” motif at its C terminus. The interaction enhanced cell surface expression of NBCe1-C, resulting in an increase of its transporter activity, for which the phosphatase activity of calcineurin was essential. When NBCe1-C was stably expressed in HeLa cells, its cell surface expression was enhanced by an intracellular Ca2+ concentration increase and was suppressed by FK506, a specific inhibitor of calcineurin. These mechanisms of surface expression and transport activity of NBCe1-C regulated by the Ca2+–calcineurin axis indicate specialized functions of NBCe1-C in the brain.

Regular Article
Structure-Activity Relationship of Anthocyanidins as an Inhibitory Effect on Osteoclast Differentiation Vol.2, No.1, p.1-6
Narumi Hirata , Tsukasa Tominari , Ryota Ichimaru , Keita Taniguchi , Chiho Matsumoto , Kenta Watanabe , Michiko Hirata , Sihui Ma , Katsuhiko Suzuki , Florian M.W. Grundler , Chisato Miyaura , Masaki Inada
Received: December 26, 2018
Accepted: January 17, 2019
Released: March 06, 2019
Abstract Full Text PDF[3M]

Anthocyanins are plant-derived pigments, and their aglycons are called anthocyanidin. Anthocyanidins have shown to exhibit various biological functions, such as anti-oxidant effects. However, their structure-activity relationship in bone tissue is not known. In this study, we examined the effects of three anthocyanidins, delphinidin, cyanidin and pelargonidin, on osteoclast differentiation and bone resorption to elucidate the structure-activity relationship. Anthocyanidins suppressed both IL-1 and LPS induced osteoclast differentiation in cocultures of bone marrow cells and primary osteoblasts, and bone resorbing activity in calvarial organ cultures. In osteoblasts, anthocyanidins inhibited prostaglandin (PG) E2 production via the downregulation of membrane-bound PGE synthase (mPGES)-1, leading to the suppression of PGE2-mediated receptor-activator of nuclear factorkappa B (NF-κB) ligand (RANKL) expression. In osteoclasts, anthocyanidins inhibited RANKL-induced osteoclast differentiation through the downregulation of osteoclast differentiation marker genes, nuclear factor of activated T-cells 1 (NFATc1), cathepsin K and tartrate-resistant acid phosphatase (TRAP). We further found that anthocyanidins suppressed the inhibitor of NF-κB kinase (IKK) activity in vitro assay, a signal component of NF-κB pathway, suggesting IKK was a novel target molecule of anthocyanidins. We found that delphinidin exerted the most potent inhibitory activity in these experiments, compared with cyanidin and pelargonidin. Anthocyanidins exhibits inhibitory activity in bone resorption, which may depend on the number of hydroxide residues.