BPB Reports

2019 - Vol. 2

2019 - Vol. 2

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.