BPB Reports

2020 - Vol. 3

2020 - Vol. 3

Regular Article
Lovastatin Suppresses the Transcriptional Regulation of CLDND1 in Human Hepatoma Cells Vol.3, No.4, p.113-118
Akiho Shima , Hiroshi Matsuoka , Kaoruko Miya , Akihiro Michihara
Received: April 11, 2020
Accepted: June 18, 2020
Released: July 03, 2020
Abstract Full Text PDF[1000K]

Claudin family proteins play an important role in the formation of tight junctions in several tissues. Individual claudins display organ- and tissue-specific expression. Claudin domain containing 1 (CLDND1), also known as claudin 25 (CLDN25), is a homolog of the claudin family, and its expression was reported to be downregulated in a mouse model of cerebellar hemorrhage. We have also reported that the retinoic acid receptor-related orphan receptor α (RORα) is involved in the transcriptional activation of CLDND1 by binding to the RORα responsive element (RORE) in the CLDND1 promoter region. Cholesterol and its derivative oxysterol reportedly serve as ligands for the nuclear receptor RORα. However, the effect of cholesterols on CLDND1 expression is unclear. The present study aimed to evaluate the effect of inhibiting steroid synthesis via lovastatin on RORα-mediated CLDND1 transcriptional regulation. Chromatin immunoprecipitation and luciferase reporter assays revealed that RORα-mediated transcriptional regulation of CLDND1 was suppressed upon lovastatin treatment of HepG2 cells; however, this inhibitory effect was attenuated by supplementation with cholesterol. Furthermore, quantitative reverse transcription-PCR and immunoblotting analyses revealed the downregulated expression of CLDND1 mRNA and protein in HepG2 cells upon lovastatin treatment with no parallel changes in RORα mRNA and protein levels. These results confirm that cholesterol serve as ligands for RORα and are, therefore, involved in the activation of CLDND1 transcriptional regulation by RORα.

Regular Article
Brain Regions with Reduced Amounts of Mevalonate Pyrophosphate Decarboxylase Correspond to Sites of Strokes in Stroke-Prone Spontaneously Hypertensive Rats Vol.3, No.3, p.106-112
Miyu Katayama , Hiroshi Matsuoka , Takahiro Hamashima , Akihiro Michihara
Received: December 07, 2019
Accepted: December 28, 2019
Released: June 11, 2020
Abstract Full Text PDF[1M]

Cholesterol deficiency is believed to result in fragile plasma membranes. It remains unclear whether a reduction in the amounts of both mevalonate pyrophosphate decarboxylase (MPD), which is involved in cholesterol biosynthesis, and cholesterol content occurs in the cerebrum and brain stem (diencephalon and midbrain) in or near the sites of strokes in stroke-prone spontaneously hypertensive rats (SHRSP). In this study, we investigated whether a reduction in both the amounts of MPD and cholesterol content corresponded to the sites of strokes in the SHRSP brain. The results obtained suggested that a reduction in the amount of MPD was involved in the decrease observed in cholesterol content, and was also important as a risk factor for stroke in SHRSP because the reductions in cholesterol content and MPD protein levels were associated with the sites of strokes. The mechanism responsible for reducing MPD protein levels in the brains of SHRSP differed with each region.

Different Correlation between Serum Levels of Indoxyl Sulfate and Estimated GFR in the Elderly with or without Dementia Vol.3, No.3, p.102-105
Hideyuki Suga , Yuichi Ichimura , Masako Oda , Hiroshi Saitoh
Received: December 22, 2019
Accepted: May 20, 2020
Released: June 01, 2020
Abstract Full Text PDF[837K]

A decrease in renal function leads to the accumulation of various uremic toxins (UTs) which exert unfavorable physiological effects on the body. Indoxyl sulfate (IS), a tryptophan-derived UT, is known to closely associate with the progression of cognitive disorders (CD) including dementia, in addition to chronic kidney disease (CKD) and cardiovascular events. It is, therefore, important to assess blood IS levels in CKD patients with CD. In this study, we assayed serum IS levels in 37 residents who had been admitted to a geriatric health services facility with stage G3b to G5 CKD and evaluated the correlation between serum IS levels and estimated glomerular filtration rate (eGFR). Eighteen out of 37 residents were considered to suffer dementia. When plotting all serum IS levels against eGFR, a weak but significant correlation was observed with a regression coefficient (r) of -0.420. In the non-dementia group, the correlation between serum IS levels and eGFR became stronger (r = -0.720). However, no correlation was observed in the dementia group. At CKD stage G3b, mean serum IS level was higher in the dementia group than in the non-dementia group. These results suggest that eGFR becomes a good marker to predict serum IS level in the case of CKD patients without dementia, but not in those with dementia. Therefore, direct monitoring of serum IS level is essential to assess the onset and/or progression of dementia in the elderly, irrespective of CKD stages.

Hepatic Expression of the Na+-Coupled Citrate Transporter (NaCT/Slc13a5) and Cellular Uptake of Citrate in a Mouse Model of Type 1 Diabetes Induced by Streptozotocin Vol.3, No.3, p.97-101
Maya Goto , Yusuke Kono , Kanta Ohno , Takuya Fujita
Received: April 23, 2020
Accepted: May 22, 2020
Released: June 01, 2020
Abstract Full Text PDF[955K]

Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic disorder, characterized by the fat accumulation in hepatocytes without significant alcohol assumption. In lipogenesis in hepatocytes, the tricarboxylic acid cycle citrate plays a crucial role as a carbon source. Citrate is transported into hepatocytes via Na+-coupled citrate transporter, NaCT. It has been demonstrated that knockdown of NaCT expression ameliorates diet-induced NAFLD in mice. In addition, NaCT expression in the liver has been reported to be induced in type 2 diabetic mice. Based on these findings, NaCT is considered to be involved in the high prevalence of NAFLD in patients with type 2 diabetes. On the other hand, it is still unclear for the expression level of NaCT under type 1 diabetic condition and its relationship to hepatic lipid accumulation. In this study, we investigated the gene and functional expression level of NaCT in streptozotocin (STZ)-induced type 1 diabetic mice. The mRNA and protein expression levels of NaCT in STZ-treated mice were gradually decreased after STZ treatment. On the other hand, the Na+-dependent citrate uptake activity in hepatocytes isolated from STZ-treated mice was not different from that isolated from non-treated mice. Nevertheless, the plasma triglyceride, cholesterol, and nonesterified fatty acid levels were much higher in STZ-treated mice. These results suggest that NaCT expression level is not closely related to the citrate uptake in hepatocytes under type 1 diabetic condition. In conclusion, unlike type 2 diabetes, NaCT may not be responsible for the pathogenesis of NAFLD in type 1 diabetes.

Regular Article
Tauroursodeoxycholic Acid Promotes Neuronal Survival and Proliferation of Tissue Resident Stem and Progenitor Cells in Retina of Adult Zebrafish Vol.3, No.3, p.92-96
Yuichi Saito , Hiroyuki Okuyoshi , Shinsuke Nakamura , Wataru Otsu , Akihiro Yamaguchi , Peter F. Hitchcock , Mikiko Nagashima , Masamitsu Shimazawa , Hideaki Hara
Received: March 26, 2020
Accepted: May 12, 2020
Released: May 26, 2020
Abstract Full Text PDF[1M]

Regenerative medicine aims to replenish damaged tissue. Boosting the capacity of intrinsic stem cells to proliferate is one key for successful regeneration. Adult zebrafish possess tissue resident stem and progenitor cells, which contribute to homeostatic growth and tissue regeneration. In the intact retina, Müller glia sporadically divide to generate fate restricted, proliferative precursors. Cell death reprograms Müller glia into stem cells that divide and produce multi-potent retinal progenitors. Using zebrafish, we evaluated the effect of taurine-conjugated bile acid, Tauroursodeoxycholic acid (TUDCA) on retinal regeneration. In the intact retina, treatment with TUDCA significantly promotes proliferation of the fate restricted precursors, but has no effect on Müller glia. Following constant light exposure, TUDCA attenuates photoreceptor death, indicating that TUDCA is neuroprotective. Following a stab wound, which initiates death of retinal neurons and reprogramming of Müller glia, treatment with TUDCA significantly increases the number of proliferating cells. In the intact retina, TUDCA-induced proliferation was accompanied by decreased expression of cell cycle inhibitors. These results suggest that TUDCA promotes proliferation of actively-cycling stem and progenitors, identifying TUDCA as a potential reagent to promote regeneration of retinal neurons.

Regular Article
A Safety Evaluation Study in Mice Revealed that Albumin Dimer is Safe for Medical and Pharmaceutical Applications Vol.3, No.2, p.87-91
Mai Hashimoto , Victor Tuan Giam Chuang , Yu Ishima , Mayumi Ikeda , Toru Maruyama , Keishi Yamasaki , Kazuaki Taguchi , Masaki Otagiri
Received: March 24, 2020
Accepted: April 06, 2020
Released: April 10, 2020
Abstract Full Text PDF[2M]

Human serum albumin (HSA) dimer, where two molecules of HSA are genetically fused with a linker of 10 amino acid, has superior blood retention property, compared with HSA monomer. Moreover, HSA dimer derivative, s-nitrosated HSA dimer, functions as an enhanced permeability and retention effects enhancer. HSA dimer has gained considerable attention as drug delivery system carrier based on its prominent function. However, for the HSA dimer to be used clinically, the safety profile of the HSA dimer is required in order to exclude any potential toxicity or unwanted effects. Thus, the present study was undertaken to investigate the occurance of tissue damage and serologic changes due to repeated administration of HSA dimer (66.5 mg/kg) to mice every 3 d for 56 d, as part of a basic consideration on safety evaluation. The evaluation on survival, behavior and body weight indicate that HSA dimer has no effect on physical growth and physiological functions. Hematological tests suggest that HSA dimer has no direct influence on hemocytes, such as hemolysis and platelet aggregation. Moreover, plasma clinical chemistry and histological examinations indicate that the HSA dimer has no deleterious effect on liver and renal functions. The results obtained here indicate HSA dimer is safe and should be useful for medical and pharmaceutical applications.

Regular Article
Calcined Ni–Al Complex Hydroxide and Its Use for the Removal of Phosphate Ion from Aqueous Solution Vol.3, No.2, p.80-86
Fumihiko Ogata , Chiharu Ito , Megumu Toda , Masashi Otani , Chalermpong Saenjum , Takehiro Nakamura , Naohito Kawasaki
Received: March 06, 2020
Accepted: March 27, 2020
Released: April 10, 2020
Abstract Full Text PDF[2M]

Calcined Ni–Al complex hydroxide (NA12) was produced through calcination at 400°C, and its capability on phosphate ion adsorption was examined. Initially, the physicochemical characteristics including specific surface area, the number of hydroxyl groups, pore volume, scanning electron microscope images, and X-ray diffraction patterns of calcined Ni–Al complex hydroxides were evaluated. The level of phosphate ion adsorbed onto NA12 in the value of 128.5 mg/g was higher than that of other compared adsorbents. This study indicated that the level of phosphate ion adsorbed using calcined Ni–Al complex hydroxide was correlated to the properties of an adsorbent surface. Moreover, the binding energy of the NA12 surface before and after the phosphate ion adsorption was also determined, and phosphorus energy (2p and 2s) could be detected after adsorption. The results demonstrated that the NA12 surface properties were important for phosphate ion removal from the aqueous solution. Additionally, the effects of pH, temperature, and contact time on the phosphate ion adsorption were also investigated. The results confirmed a potent recovery of the phosphate ion (over 90%) when using a NaOH solution at 1000 mmol/L in this experiment. Thus, NA12 is a promising adsorbent for the phosphate ion.

Anti-Metastatic Effects of Curcumin Analogues in a Mouse Breast Cancer Model Vol.3, No.2, p.76-79
Sisca Ucche , Retno Murwanti , Ritmaleni , Yoshihiro Hayakawa
Received: January 29, 2020
Accepted: April 02, 2020
Released: April 10, 2020
Abstract Full Text PDF[1M]

Curcumin is a polyphenol compound derived from the roots of Curcuma longa. Although the biological activities of curcumin, such as its anti-inflammatory, anti-oxidant, anti-microbial and anti-cancer effects, have been well applied, its poor chemical stability is a major problem. Pentagamavunon-0 (PGV-0) and Pentagamavunon- 1 (PGV-1) were developed as curcumin analogues with higher bioavailability; however, their anti-cancer activity has not yet been assessed. In this study, we evaluated the anti-metastatic activity of PGV-0 and PGV-1 in 4T1 breast cancer cells. Although both curcumin analogues demonstrated similar anti-proliferative effects to curcumin in 4T1 breast cancer cells, they did not inhibit nuclear factor kappa B (NF-ĸB) activity which is a well-defined molecular target of curcumin for its anti-cancer effects. As PGV-0 and PGV-1 exhibited stronger inhibition of the metastatic capacity in a 4T1 breast cancer model than curcumin, PGV-0 and PGV-1 may be promising curcumin analogues to target cancer metastasis having a distinct molecular mechanism from that of curcumin.

Regular Article
Activation of TRPV4 Channel Regulates Differentiation to and Function of Myeloid-Derived Suppressor Cells Vol.3, No.2, p.70-75
Moeka Yamamoto , Ichiro Horie , Yoichiro Isohama , Mitsutoshi Tsukimoto
Received: December 23, 2019
Accepted: March 16, 2020
Released: March 25, 2020
Abstract Full Text PDF[2M]

Myeloid-derived suppressor cells (MDSCs), which are derived from immature bone marrow cell (BMC) populations that proliferate in the tumor microenvironment, suppress T cell immune responses. Transient receptor potential vanilloid (TRPV) 4, which is a Ca2+ channel, is involved in tumor growth, but the role of TRP channels in MDSC differentiation and function remains unclear. Here, we first investigated the involvement of TRP channels in the differentiation of MDSCs. The selective TRPV4 channel antagonist RN-1734 increased the population of MDSCs (CD11b+Gr-1+) at Day 3, while the TRPV4 agonist GSK1016790A decreased it, suggesting that stimulation of TRPV4 suppresses the differentiation of BMCs to MDSCs. GSK1016790A also increased the production of nitric oxide and reactive oxygen species, but suppressed the expression of Arg-1 mRNA, which encodes arginase-1, in MDSCs. Furthermore, GSK1016790A decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in MDSCs, thereby attenuating STAT3 signaling. Our results suggest that TRPV4 plays a role in regulating both the differentiation and function of MDSCs, and therefore could be a promising target for cancer immunotherapy.

Interaction Between Piperacillin/Tazobactam and Warfarin: a Single-Center Retrospective Single-Arm Cohort Study Vol.3, No.2, p.65-69
Shota Kadomura , Yoh Takekuma , Shungo Imai , Hitoshi Kashiwagi , Kotaro Kawamoto , Tatsuya Itoh , Mitsuru Sugawara
Received: January 26, 2020
Accepted: March 09, 2020
Released: March 16, 2020
Abstract Full Text PDF[1011K]

Antibiotics influence the anticoagulation effect of warfarin and increase the bleeding risk in patients who are receiving warfarin. Piperacillin/tazobactam (PIPC/TAZ) is commonly used to treat infections such as healthcare-associated infection. However, there have been few reports about the interaction between warfarin and PIPC/TAZ. In this study, we investigated the influence of PIPC/TAZ on the anticoagulation effect of warfarin in hospitalized patients. The primary outcome was elevation of prothrombin time international normalized ratio (PT-INR) after PIPC/TAZ administration. Secondary outcomes were the proportion of patients with supratherapeutic levels of PT-INR, discontinuation of administration or reduction in the dose of warfarin, bleeding, transfusion, and vitamin-K rescue. Fifteen patients were enrolled in this study. PT-INR elevation occurred in 11 (73.3%) of the 15 patients. The median value of PT-INR after administration of PIPC/TAZ was significantly higher than the value before administration: 2.22 (interquartile range (IQR), 2.05-2.76) and 1.90 (IQR, 1.36-2.45), respectively (p = 0.024). Three (20%) of the 15 patients had PT-INR over 4, and discontinuation of administration or reduction in the dose of warfarin was needed in 6 (40%) of the 15 patients. Bleeding occurred in one patient, transfusion was performed in one patient and vitamin-K rescue was performed in one patient. This study showed that PIPC/TAZ induced elevation of PT-INR in patients receiving warfarin and that discontinuation or reduction in the dose of warfarin was needed in 40% of the patients. Therefore, we recommend to close monitoring of PT-INR in patients treated with warfarin during PIPC/TAZ administration.

Urinary Bile Acid Shows Diurnal Fluctuation and Phase Shift with Daytime-Restricted Feeding in Rats Vol.3, No.2, p.60-64
Hiroshi Kawai , Ai Kurokawa , Takuya Ishibashi , Reiko Iwadate , Naomi Kudo , Yoichi Kawashima , Atsushi Mitsumoto
Received: January 09, 2020
Accepted: March 02, 2020
Released: March 16, 2020
Abstract Full Text PDF[804K]

Dysregulation of the biological clock disrupts the homeostasis of physiological functions, which may lead to the development of various disorders. To investigate the relationship between biological rhythms and disorders, an efficient monitoring method of the biological clock is necessary. In this study, we analyzed the circadian rhythmicity profile of bile acids in urine and investigated whether urinary bile acid (UBA) could reflect the circadian rhythm in liver physiology. Male Wistar-Hannover rats were maintained in metabolic cages under ad lib feeding and later subjected to restricted feeding (in which rats were fed only during light periods). Excreted urine was collected in each session, and bile acid contents were analyzed via an enzyme-based total bile acid assay. UBA content showed diurnal fluctuations under both ad lib and restricted feeding conditions and reached a peak during the dark period of ad lib feeding. In contrast, with restricted feeding, the peak was observed during light periods. Restricted feeding induced an 8-12 h phase shift. Diurnal fluctuation and phase shift by restricted feeding are distinctive features controlled by biological clocks in peripheral tissues. Since bile acids are synthesized in the liver, we propose that UBA might reflect the circadian fluctuation in liver physiology.

(–)-Isostemonamine Can Enhance the Anti-Proliferative Activity of Trichostatin A Against Human Breast Cancer MDA-MB-231 Cells Vol.3, No.2, p.56-59
Masayo Hirao-Suzuki , Shuso Takeda , Takayuki Iwata , Satoshi Fujita , Taishi Tomiyama , Masufumi Takiguchi , Akihisa Toda , Mitsuru Shindo
Received: January 06, 2020
Accepted: February 13, 2020
Released: March 12, 2020
Abstract Full Text PDF[1M]

Recent findings established (–)-isostemonamine as an anti-proliferator for estrogen receptor α-negative human breast cancer cells, MDA-MB-231, known to grow/divide at an aggressive rate. However, ST-4, a thioamide derivative of (–)-isostemonamine, is believed to not affect the viability of these cells. Epigenetic changes, such as DNA methylation and histone modification, are involved in the progression of many cancers, including breast cancer. In the present study, we investigated whether ST-4 and its related compounds (ST-3 and ST-5) can potentiate the anti-proliferative activity of the established epigenetic modifiers, 5-aza-2’-deoxycytidine (5-aza-dC; a DNA methyltransferase 1 inhibitor), trichostatin A (TSA; a class I/II histone deacetylase inhibitor), and etoposide (a DNA topoisomerase IIα inhibitor). Data obtained from this study demonstrate that, among the studied compounds, ST-4 displays the strongest enhancement of the anti-proliferative activity of TSA, against MDA-MB-231 cells (IC20 of TSA = 14 ± 3.4 nM versus that of TSA/ST-4 combination = 7.8 ± 1.1 nM). However, this effect was not observed at higher concentrations of above 25 nM of TSA, at which the IC50 values of TSA with or without ST-4 were not significantly different (30 ± 4.4 nM versus 28 ± 1.4 nM, respectively). Results from the study suggest that combining ST-4 with established anti-cancer agents could potentiate the latter’s anti-proliferative activity, thereby potentially minimizing the concentration of these agents needed for optimal clinical efficacy and safety.

Regular Article
Involvement of TRPV1 and TRPV4 Channels in Enhancement of Metastatic Ability Induced by γ-Irradiation in Human Lung Cancer A549 Cells Vol.3, No.1, p.50-55
Kurumi Washiya , Rie Sasaki , Yuto Nakanishi , Kazuki Kitabatake , Keisuke Nishino , Sei-ichi Tanuma , Shuji Kojima , Mitsutoshi Tsukimoto
Received: January 17, 2020
Accepted: February 15, 2020
Released: February 27, 2020
Abstract Full Text PDF[4M]

Radiation therapy is an important local treatment for malignant tumors, but ionizing radiation may also facilitate tumor invasion and metastasis. The transient receptor potential (TRP) superfamily, which is a diverse group of ion channels activated by various stimuli, has a variety of pathophysiological functions, including a role in malignancy. However, it is not clear whether TRP channels influence radiation-induced biological effects. Here, we show that TRPV1 and TRPV4 channels contribute to the γ-irradiation-induced enhancement of migration of human lung cancer A549 and mouse melanoma B16 cells. We found that γ-irradiation induced both cell migration and actin stress fiber formation of A549 cells, but both effects were suppressed by the TRPV1 inhibitors capsazepine AMG9810, SB366791, and BCTC, and by the TRPV4 inhibitors RN-1734 and GSK2193874. γ-Irradiation induced migration was also suppressed by knockdown of TRPV1 and TRPV4 channels. Furthermore, γ-irradiation of B16 mouse melanoma cells increased the number of lung metastases in C57BL/6 mice, compared to non-irradiated B16 cells, and TRPV1 and TRPV4 channel inhibitors suppressed this increase. These results suggest that TRPV1 and TRPV4 channels are potential targets for intervention to block the acquisition of motility by cancer cells during radiotherapy in order to reduce the risk of metastasis.

Effect of Remifentanil-Based Anesthesia on Perioperative Phagocytic Function of Human Monocytes Vol.3, No.1, p.45-49
Manzo Suzuki , Yoshinori Abe , Yusuke Taguchi , Hiroyasu Bito
Received: December 29, 2019
Accepted: February 10, 2020
Released: February 19, 2020
Abstract Full Text PDF[1M]

Although morphine-induced modulation of immune cells has been well studied, modulation of immune cells by fentanyl and remifentanil, the latter of which has been used in recent anesthesia procedures, has not been well-studied. Our aim was to identify the effects of fentanyl and remifentanil on phagocytosis and respiratory burst in leucocytes in in vivo and in vitro studies. In the in vivo study, twelve patients were assigned to receive fentanyl-based anesthesia (fentanyl group, n=6) or remifentanil-based anesthesia (remifentanil group, n=6). Blood samples were obtained from before anesthesia to 30 min after anesthesia in each group. In the in vitro study, blood samples were obtained from three healthy volunteers and incubated with various blood concentrations of fentanyl or remifentanil (from 0.3 ng/mL to 9 ng/mL). Phagocytic activity (percentage of phagocyting cells) and respiratory burst activity (percentage of cells producing oxygen radicals) were analyzed. In the in vivo study, phagocytosis was suppressed only before incision in the fentanyl group whereas suppression of phagocytosis was continued until emergence from anesthesia in the remifentanil group. In the in vitro study, incubation with fentanyl or remifentanil tended to enhance phagocytic function of monocytes and had no dose-dependent effect over various concentrations of fentanyl or remifentanil, respectively. Remifentanil-based anesthesia suppressed the phagocytic function of monocytes during its administration.

Regular Article
Persistence, Effects, and Adverse Events Associated with Real-World Daily Teriparatide Use in Japanese Patients with Osteoporosis Vol.3, No.1, p.39-44
Tomoko Matsumoto , Kazuyuki Niki , Asuka Hatabu , Takuya Oshima , Kimihiko Sato , Mikiko Ueda , Tetsuya Sato , Etsuko Uejima
Received: November 06, 2019
Accepted: January 06, 2020
Released: February 14, 2020
Abstract Full Text PDF[812K]

Few studies have addressed the effects and adverse events associated with daily teriparatide use, as well as the adherence and causes for non-adherence, among Japanese patients with osteoporosis, particularly those older than 80 years. In this study, we aimed to clarify various parameters associated with daily teriparatide use in Japanese patients with osteoporosis in a real-world clinical setting. This retrospective study compared the persistence of daily teriparatide use and the associated effects and adverse events in older (≥80 years, n=52) and younger patients (<80 years, n=106) treated with teriparatide between May 2013 and May 2018 at a single orthopedic clinic. We observed a significantly higher treatment completion rate among younger patients compared to their older counterparts (59.6% vs. 40.6%, p=0.036). Of the 74 patients (both patient groups) who completed a 24-month treatment course, only one (1.35%) developed new vertebral fractures. Our findings suggest that older patients would benefit from consistent osteoporosis treatment, particularly with a generally safe and effective agent, such as teriparatide. However, Log-rank test also shows the older patients exhibits a greater tendency to drop out than the younger patients (p=0.0238). The older patients tended to continue to drop out from the beginning. Accordingly, our results emphasize the importance of interventions, especially continuous encouraging from the first self-injection of teriparatide.

Regular Article
Cadmium Inhibits All-Trans-Retinoic Acid-Induced Increase of Nitroblue Tetrazolium Reduction Activity and Induces Metallothionein 1G Expression in Human Acute Myelocytic Leukemia HL-60 Cells Vol.3, No.1, p.34-38
Shoko Ogushi , Shuji Ikemoto , Nobuhiko Miura , Tsuyoshi Nakanishi , Tomoki Kimura
Received: January 07, 2020
Accepted: January 30, 2020
Released: February 10, 2020
Abstract Full Text PDF[2M]

Cadmium is an environmental pollutant. Metallothioneins are cysteine-rich, low-molecular-weight proteins that are induced by cadmium, which they chelate for detoxification. In humans, the functional metallothionein isoforms are MT1A, 1B, 1E, 1F, 1G, 1H, 1M, 1X, 2A, 3, and 4. It has been reported that overexpression of MT1G inhibits all-trans-retinoic acid (ATRA)–induced hematopoietic differentiation. Here, we found that cadmium inhibited ATRA-induced nitroblue tetrazolium reduction activity, a marker of hematopoietic differentiation, in human acute myelocytic leukemia HL-60 cells. Reverse transcription – quantitative polymerase chain reaction analysis of HL-60 cells revealed the expression of the metallothionein isoforms MT1G, MT1X, and MT2A and showed that MT1G expression increased significantly after cadmium treatment. ATRA treatment significantly attenuated this cadmium-induced increase in MT1G expression; however, MT1G expression remained significantly higher than that in untreated cells. No significant changes in MT1X and MT2A expression were observed. We also found that the cadmium-induced increase in MT1G expression was independent of CpG demethylation. ATRA is a standard chemotherapy treatment for acute promyelocytic leukemia; our findings suggest that cadmium may inhibit the effect of this chemotherapy.

Regular Article
Reconstitution of Bacterial Tyrosine Kinase-Modulator Interaction in a Human Cell Line Vol.3, No.1, p.28-33
Hidesuke Fukazawa , Mari Fukuyama , Yoshitsugu Miyazaki
Received: October 21, 2019
Accepted: January 30, 2020
Released: February 07, 2020
Abstract Full Text PDF[4M]

Many bacterial species express tyrosine kinases termed BY-kinases that share no homology with eukaryotic enzymes. We have previously reported that the Staphylococcus aureus BY-kinase CapB2 when fused with the C-terminal activation domain of its modulator CapA1, can translate into an active tyrosine kinase in HEK293T cells. In the present study, full-length CapA1 and CapB2 tagged with different fluorescent proteins were transfected into HEK293T cells. When expressed individually, the modulator CapA1, a membrane protein in bacteria, also appeared to localize to the cell membrane in HEK293T cells. In contrast, the catalytic subunit, CapB2, was found to be cytosolic. Coexpression of the two proteins resulted in apparent translocation of CapB2 to the membrane with concomitant activation of tyrosine kinase activity. This translocation and activation of CapB2 did not occur when the cytoplasmic C-terminal tail of CapA1 was deleted. Conversely, the CapA1 cytoplasmic C-terminal tail alone, when attached to a membrane localization sequence, was sufficient for CapB2 translocation and kinase activation. Our results indicate that the kinase activity of CapB2 is stimulated by direct interaction with the C-terminal cytoplasmic domain of CapA1 and that the process can be reconstituted and visualized in a human cell line. We created various mutants of CapA and CapB, and present data that demonstrate the correlation between CapA-CapB interaction and kinase activation.

Regular Article
Role of Metallothionein in Transcriptional Regulation by Metal-Responsive Element-Binding Transcription Factor 1 Vol.3, No.1, p.22-27
Yu-ki Tanaka , Yoshiaki Futami , Yasunori Fukumoto , Noriyuki Suzuki , Yasumitsu Ogra
Received: November 15, 2019
Accepted: December 16, 2019
Released: February 05, 2020
Abstract Full Text PDF[1M]

Owing to the high metal binding affinity of metallothionein (MT), newly synthesized MT was speculated to attenuate the activity of metal-responsive element (MRE)-binding transcriptional factor 1 (MTF-1) by removing Zn from the activated MTF-1. To investigate the potential role of MT in the inactivation of MTF-1, we examined the transcriptional levels of reporter and endogenous MRE-dependent genes using mouse embryonic fibroblasts (MEFs) established from MT-knockout (KO) and wild-type (WT) mice. The activation of MTF-1 by the Cd exposure of MT-KO MEFs was sustained for 12 h, whereas that of MT-WT MEFs showed the rapid attenuation. Consequently, MT was found to negatively regulate MTF-1 activity, which can control the expression of MT itself.

Regular Article
The Antibiotic Cefotaxime Works as Both an Activator of Nrf2 and an Inducer of HSP70 in Mammalian Cells Vol.3, No.1, p.16-21
Mayuka Yamada , Midori Suzuki , Takuya Noguchi , Takumi Yokosawa , Yuto Sekiguchi , Natsumi Mutoh , Takashi Toyama , Yusuke Hirata , Gi-Wook Hwang , Atsushi Matsuzawa
Received: November 27, 2019
Accepted: January 23, 2020
Released: January 31, 2020
Abstract Full Text PDF[1M]

Both NF-E2-related factor 2 (Nrf2) and heat shock protein 70 (HSP70) contribute to cellular defense to various stresses, and have emerged as candidates of therapeutic targets to improve or prevent tissue damage. Cefotaxime (CTX), a third-generation cephalosporin antibiotic, is conceived as a safe drug largely free from side effects. CTX exhibits broad-spectrum antimicrobial activity, and thereby, is most commonly prescribed for the treatment of infectious diseases induced by Gram-positive or Gram-negative bacteria. In this study, we unexpectedly found the beneficial properties of CTX that upregulate both Nrf2 and HSP70 to the extent that stress-induced damage is ameliorated. Non-toxic levels of reactive oxygen species (ROS) induced by CTX activated the Nrf2 pathway without cytotoxicity, which in turn upregulated HSP70. Interestingly, the cytotoxicity of Fas/CD95 ligand (FasL), a cytotoxic cytokine that strongly induces apoptosis, was significantly ameliorated by pre-treatment with CTX, most likely because of the upregulation of Nrf2 and HSP70. Our results therefore show novel properties of CTX, which raise the possibility that CTX works as a non-toxic therapeutic agent for preventing and repairing tissue damage.

Regular Article
In Vitro Evaluation of the Interaction Between Activated Charcoal and N-Acetylcysteine after Acetaminophen Adsorption Vol.3, No.1, p.11-15
Yoshinori Tomoda , Mariko Fukumoto
Received: October 17, 2019
Accepted: January 11, 2020
Released: January 29, 2020
Abstract Full Text PDF[1M]

Gastrointestinal decontamination by activated charcoal (AC) is the most important treatment for acetaminophen (APAP) overdose. Because AC adsorbs a wide variety of toxins, it may also adsorb the oral antidote, N-acetylcysteine (NAC). NAC is a specific antidote for APAP overdose and administered as a 72-h oral regimen. We evaluated AC adsorption of NAC after APAP adsorption in vitro. Different concentrations of NAC solution diluted with simulated gastric fluids (SGF) and simulated intestinal fluids (SIF) were added to AC and incubated at 37°C for 1 h. The AC was then removed by filtration, and the NAC concentration was determined. This revealed that NAC was not only adsorbed onto the AC but also converted to N,N’-diacetyl-L-cystine (DAC), which is oxidized NAC. We then calculated the maximum adsorption capacity per gram of AC (Qm). The apparent Qm based on the amount of decreased NAC in the SGF was 400 mg/g, and that in the SIF was 714 mg/g. The actual Qm based on only the amount of adsorption in the SGF was 294 mg/g, and that in the SIF was 59 mg/g. We also determined whether or not AC could adsorb the loading and maintenance doses of NAC after APAP adsorption. The residual rate in the SGF was 2.1%, and that in the SIF was 0.3%. The rate of conversion to DAC was higher in the SIF than that in the SGF. By both the actions of adsorption and oxidation, AC may reduce the effect of loading and maintenance doses of NAC.

Regular Article
Construction of a PPARα Reporter Assay System with Drug-Metabolizing Capability Vol.3, No.1, p.7-10
Takuomi Hosaka , Ayano Wakatsuki , Takamitsu Sasaki , Ryota Shizu , Kouichi Yoshinari
Received: November 14, 2019
Accepted: December 29, 2019
Released: January 17, 2020
Abstract Full Text PDF[1M]

Peroxisome proliferator-activated receptor α (PPARα) belongs to the nuclear receptor superfamily and exerts hypolipidemic and anti-inflammatory functions when activated by ligand-binding. To screen its ligands, cell-based reporter assays have been widely used, but it is difficult to investigate the effects of the metabolites of test compounds on PPARα due to very low drug-metabolizing capability of cell lines generally used in those assays. The aim of this study was to construct a convenient PPARα reporter assay system with drug-metabolizing capability by using 9,000 x g supernatant (S9) of rat liver homogenate, which abundantly includes various drug-metabolizing enzymes. We used clofibrate as a model compound since it requires hydrolysis to clofibric acid to activate PPARα. In cell-based reporter assays using a PPARα-responsive luciferase reporter plasmid and a rat PPARα expression plasmid, reporter activity was increased by treatment with bezafibrate and clofibric acid, which directly activate PPARα as ligands, but not with clofibrate. The addition of S9 to culture media increased reporter activity of the cells treated with clofibrate, as expected. When heat-denatured S9 was used or a carboxylesterase inhibitor was included in the system, clofibrate-induced PPARα activation was not observed, suggesting that carboxylesterases are responsible for the hydrolysis of clofibrate to clofibric acid. Taken together, we have established a convenient PPARα reporter assay system with drug-metabolizing capability to assess PPARα-activating potency of both test compounds and their metabolites.

Regular Article
Factorial Analysis of Clostridioides Difficile Colitis and Pseudomembranous Colitis Using JADER Vol.3, No.1, p.1-6
Risako Takaya , Kana Misawa , Sho Tashiro , Yuki Enoki , Kazuaki Taguchi , Kazuaki Matsumoto
Received: October 25, 2019
Accepted: January 07, 2020
Released: January 15, 2020
Abstract Full Text PDF[1M]

Clostridioides difficile (C. difficile) colitis and pseudomembranous colitis are known as healthcare-associated intestinal infections. In this study, the incidence of C. difficile colitis and pseudomembranous colitis was investigated using the Japanese Adverse Drug Event Report (JADER). Using JADER data between April 2004 and September 2017, the patient who developed C. difficile colitis and pseudomembranous colitis were investigated. During the study period, 375 cases of C. difficile colitis and 903 cases of pseudomembranous colitis were reported. The numbers of reported cases of both C. difficile colitis and pseudomembranous colitis were largest in those in their 70s, accounting for 24.7% and 25.6%, respectively. Patients in their 60s-90s comprised the majority of all patients with both C. difficile colitis and pseudomembranous colitis. Both C. difficile colitis and pseudomembranous colitis were caused by antibiotics in many patients, and signals of all antibiotics were detected. In C. difficile colitis, signals of immunosuppressants, corticosteroids, and alkylating drugs were also detected among drugs other than antibiotics. For pseudomembranous colitis, the use of molecularly targeted drugs, antimetabolic drugs, and corticosteroids was reported other than antibiotics. Using JADER, we revealed risk factors for the development of C. difficile colitis and pseudomembranous colitis, and firstly revealed that molecularly targeted drugs other than antibiotics could also be potential risk factors. Our findings may be useful for the early detection of drug-induced C. difficile colitis and pseudomembranous colitis.