BPB Reports

2023 - Vol. 6

2023 - Vol. 6

Regulation of the ER-Resident Mannosidase EDEM2 in HEK293 Cells Vol.6, No.6, p.193-199
Ryoichi Murase , Genki Kato , Kentaro Oh-hashi
Received: October 06, 2023
Accepted: November 09, 2023
Released: December 05, 2023
Abstract Full Text PDF[1M]

EDEM2 plays an important role as the first enzyme that acts during mannose trimming of N-glycosylated proteins in the ERAD machinery. Although EDEM2 expression has been reported to be transcriptionally regulated by the IRE1-sXBP1 pathway, very little is known about how endogenous EDEM2 protein expression is regulated. In this work, three different ER stress inducers were used to treat HEK293 cells. Thapsigargan slightly increased both EDEM2 mRNA and protein levels in the cells. Treatment with MG132 did not increase the level of mature EDEM2 protein, and a truncated form of the protein appeared. In SEL1L-deficient cells, there was a slight increase in EDEM2 protein as well as in TXNDC11, a protein that has been reported to form disulfide bonds with EDEM2. On the other hand, EDEM2 protein level decreased in TXNDC11-deficient cells. DTT treatment decreased EDEM2 and TXNDC11 protein levels in a time-dependent manner. The decrease in EDEM2 protein after DTT treatment was attenuated by treatment of the cells with MG132 and by SEL1L deficiency. These findings demonstrate that endogenous EDEM2 protein is regulated posttranscriptionally and that it is in part an SEL1L-mediated ERAD substrate.

Effects of Xanthine Oxidoreductase Inhibitors on Reactive Oxygen Species Produced In Vitro from Xanthine Oxidase Vol.6, No.6, p.189-192
Masato Noda , Chigusa Kikuchi , Ryota Tarui , Takashi Nakamura , Takayo Murase , Tamihide Matsunaga
Received: August 11, 2023
Accepted: November 14, 2023
Released: November 30, 2023
Abstract Full Text PDF[982K]

Xanthine oxidase (XO) produces reactive oxygen species (ROS) and has been associated with vascular endothelial dysfunction. While the effects of xanthine oxidoreductase (XOR) inhibitors on inhibiting the generation of uric acid from xanthine have been reported, much less is known about their effects on XO-induced ROS. The mechanisms of action of each XOR inhibitor vary, but it is not known whether XOR inhibitors’ effects on oxidative stress also vary. The purpose of this study is to compare the effects of different XOR inhibitors on XO-induced ROS. We used an in vitro chemiluminescence assay with clinically relevant doses of XOR inhibitors (allopurinol, oxypurinol, febuxostat, and topiroxostat) to investigate their effects on circulating XO-derived ROS. All XOR inhibitors significantly inhibited ROS production, with febuxostat and topiroxostat showing strong effects. These results confirm differences in the effects at clinical did among XOR inhibitors on XO, with topiroxostat demonstrating a strong suppression of ROS production. This study should help guide clinical practice in using XOR inhibitors to improve patient care and management.

Improved Palatability of Gummy Drugs of Epinastine Hydrochloride Using Organoleptic Taste-Masking Methods Vol.6, No.6, p.184-188
Shimako Tanaka , Sayuri Kawamoto , Yasuharu Kashiwagura , Akio Hakamata , Keiichi Odagiri , Takashi Okura , Naoki Inui , Hiroshi Watanabe , Noriyuki Namiki , Shinya Uchida
Received: October 02, 2023
Accepted: November 13, 2023
Released: November 30, 2023
Abstract Full Text PDF[1M]

Gummy drugs are dried jelly formulations prepared by adding a gelling agent to saccharides, which are then cooled and solidified. Epinastine hydrochloride (Epi) is commonly used to treat allergic diseases as a prescription and over-the-counter drug. However, the extremely bitter taste of Epi would affect its acceptability among patients. In this study, we aimed to improve the palatability of a gummy drug containing Epi (Epi-G) via organoleptic masking. Epi-G (10 mg of Epi/3.5 g of gummy drug) with two different organoleptic masking formulations, namely aspartame, cocoa powder, and chocolate flavoring (C-Epi-G) or aspartame, L-menthol, and lemon flavoring (L-Epi-G). The gustatory sensation test included six healthy adult volunteers (23.3 ± 1.8 years). We used a visual analogue scale (VAS) to evaluate bitterness, sweetness, and the overall palatability of each Epi-G formulation during chewing and after spitting out the drugs. In the gustatory sensation test, the VAS scores for bitterness and sweetness were decreased and increased for C-Epi-G and L-Epi-G, respectively, compared with the values for Epi-G without organoleptic masking. The VAS scores for overall palatability during chewing for C-Epi-G and L-Epi-G were significantly increased by 2.3- and 2.0-fold, respectively, versus the value for Epi-G. The score after spitting out C-Epi-G remained higher than that of Epi-G. These data illustrated that Epi-G with organoleptic masking had good palatability, which could improve patient adherence to treatment. The gummy drugs could represent an alternative dosing formulation for pediatric and geriatric patients by allowing them to take the drugs more easily than other oral formulations.

Corrigendum: An in Vitro Short-Term Treatment with Black Tea-Derived Theaflavins Reduced Infectivity of SARS-CoV-2 in Saliva
[Notice] The Original Article was published on October 6, 2023
Vol.6, No.6, p.183-183
Motohiko Ogawa , Mana Murae , Shuetsu Fukushi , Kohji Noguchi , Hideki Ebihara , Masayoshi Fukasawa
Received: November 16, 2023
Accepted: November 16, 2023
Released: November 25, 2023
Abstract Full Text PDF[578K]

Regular Article
Vitamin K Converting Enzyme UBIAD1 Promotes the Proliferation and Differentiation of Mouse Chondrogenic ATDC5 Cells Vol.6, No.6, p.176-182
Shunsuke Hirashima , Shinichiro Kaetsu , Yukino Kiyooka , Kimie Nakagawa
Received: October 21, 2023
Accepted: October 25, 2023
Released: November 16, 2023
Abstract Full Text PDF[4M]

Background: Vitamin K derivatives have an important role in bone formation and blood clotting. Vitamin K2 [menaquinone-4 (MK-4)] is used for the treatment and prevention of osteoporosis. Recently, vitamin K was found to be effective for the prevention of osteoarthritis and may play a role in cartilage formation; however, the function of UBIAD1 (MK-4 biosynthesis enzyme) and MK-4 in cartilage is unclear. In this study, we examined the function of UBIAD1 and MK-4 in chondrogenesis and differentiation using chondrogenically differentiated cells. Methods: Mouse chondrocyte progenitor ATDC5 cells were used for siRNA knockdown of UBIAD1 to determine its effects on cell proliferation and chondrogenic differentiation. Proliferation and differentiation were assessed using the WST-8 assay and Alcian blue staining, respectively. The effects of MK-4 treatment and transfection of a human UBIAD1 expression plasmid on UBIAD1 knockdown cells were also examined. RESULTS: UBIAD1 knockdown significantly decreased the proliferation and chondrogenic differentiation of ATDC5 cells. MK-4 treatment suppressed ATDC5 cell proliferation and chondrogenic differentiation. It also affected UBIAD1 knockdown ATDC5 cells during chondrogenic differentiation. However, overexpression of human UBIAD1 promoted ATDC5 cell proliferation and chondrogenic differentiation and exhibited a similar effect on UBIAD1 knockdown ATDC5 cells. These results suggest that MK-4 has an inhibitory effect on the proliferation and differentiation of UBIAD1, which exhibits a promoting effect. These results suggest that UBIAD1 and MK-4 have a role in regulating chondrocyte proliferation and differentiation and are important regulators of chondrogenic differentiation.

Corrigendum: Development of a Standard Test Method for Insecticides in Indoor Air by GC-MS with Solid-Phase Adsorption/Solvent Extraction
[Notice] The Original Article was published on May 12, 2023
Vol.6, No.5, p.175-175
Taichi Yoshitomi , Iwaki Nishi , Aya Onuki , Tokuko Tsunoda , Masahiro Chiba , Shiori Oizumi , Reiko Tanaka , Saori Muraki , Naohiro Oshima , Hitoshi Uemura , Maiko Tahara , Shinobu Sakai
Received: October 26, 2023
Accepted: October 26, 2023
Released: October 31, 2023
Abstract Full Text PDF[554K]

Thyroid Transcription Factor-1 as a Potential Hematologic Toxicity Indicator for the Three-Drug Combination Regimen of Carboplatin, Pemetrexed, and Pembrolizumab in Patients with Advanced Recurrent Non-Squamous Non-Small Cell Lung Cancer Vol.6, No.5, p.172-174
Shoma Mori , Takayoshi Maiguma , Keisuke Yoshii , Hikari Hashimoto , Atsushi Komoto , Yuto Haruki , Tetsuhiro Sugiyama , Kenichi Shimada
Received: July 27, 2023
Accepted: October 16, 2023
Released: October 26, 2023
Abstract Full Text PDF[830K]

Thyroid transcription factor-1 (TTF-1) expression in patients with non-squamous non-small cell lung cancer (NS-NSCLC) is reportedly useful in selecting treatment regimens and predicting life expectancy. However, only a few studies have reported the association between TTF-1 expression and the efficacy of the current first-line regimens containing immune checkpoint inhibitors. It is unclear whether TTF-1 can be a hematologic toxicity indicator in patients receiving these treatment regimens. Patients who received the three-drug combination regimen of carboplatin, pemetrexed, and pembrolizumab, i.e., KEYNOTE-189, between April 2019 and December 2021 at Tsuyama Chuo Hospital and who had known TTF-1 expression were retrospectively studied using electronic medical records. Among the seven patients included, four patients were TTF-1 positive, while three were TTF-1 negative. TTF-1-positive patients showed a trend toward improved progression-free survival and were more likely to experience thrombocytopenia than the TTF-1-negative patients. These results suggest that TTF-1 expression in patients with NS-NSCLC could play a role in determining both treatment efficiency and hematologic toxicity.

Regular Article
Mechanism of Blue Light-Induced Asthenopia and the Ameliorating Effect of Tranexamic Acid Vol.6, No.5, p.166-171
Keiichi Hiramoto , Sayaka Kubo , Keiko Tsuji , Daijiro Sugiyama , Yasutaka Iizuka , Tomohiko Yamaguchi
Received: July 21, 2023
Accepted: October 19, 2023
Released: October 26, 2023
Abstract Full Text PDF[1M]

Tranexamic acid exerts various effects on living bodies; however, its effects on asthenopia remain unknown. In this study, an asthenopia-like model was developed and used to investigate the effects of tranexamic acid on asthenopia. Mice were placed in special cages constructed for the test, and continuous irradiation with blue light was applied for 20 days. The tranexamic acid-treated group was orally administered tranexamic acid daily during the test period. Motor activity was measured for 10 days after irradiation, and reactive oxygen species (ROS), plasmin, and transforming growth factor (TGF)-β levels in the ciliary muscle of the mice were measured on the last day. Blue light irradiation induced asthenopia and increased ROS, plasmin, and TGF-β levels. In contrast, tranexamic acid administration improved asthenopia and significantly decreased plasmin and TGF-β levels compared to blue light irradiation alone; however, ROS levels remained unchanged. The study results indicate that blue light irradiation induces asthenopia by activating the ROS/plasmin/TGF-β pathways and that tranexamic acid improves asthenopia by suppressing plasmin production.

An in Vitro Short-Term Treatment with Black Tea-Derived Theaflavins Reduced Infectivity of SARS-CoV-2 in Saliva
[Notice] An Corrigendum to this article was published on 22 November 2023
Vol.6, No.5, p.163-165
Motohiko Ogawa , Mana Murae , Shuetsu Fukushi , Kohji Noguchi , Hideki Ebihara , Masayoshi Fukasawa
Received: September 15, 2023
Accepted: September 22, 2023
Released: October 06, 2023
Abstract Full Text PDF[896K]

The coronavirus disease 2019 is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is abundant in the saliva of an infected person; therefore, saliva is an important source of infection. The present study evaluated the efficacy of short-term treatment using tea-derived compounds against SARS-CoV-2 infectivity in the saliva. The antiviral efficacy of theaflavin 3,3’-gallate (TF3) and two black tea-derived theaflavin concentrates (TF35 and TF80) against a prototype Wuhan and a recent Omicron strain was evaluated using human saliva. TF3, TF35, and TF80 reduced the infectivity of both strains at high (1 mM or 1 mg/ml) and low (0.25 mM or 0.25 mg/ml) concentrations; however, antiviral efficacy against the Wuhan strain was stronger than that against the Omicron strain. Furthermore, the antiviral agents at high concentrations showed better efficacy against both strains than those at low concentrations. For example, treatment with 1 mM TF3 for 10 min decreased the infectivity of Wuhan and Omicron strains to approximately 0.05% and 3%, respectively; these reduction rates are attributable to the inactivation of large amounts of viruses (9.995 × 105 and 9.7 × 104 TCID50, respectively). Considering these facts, it was expected that the inclusion of the main components of black tea (TF3) and the black tea-derived theaflavin concentrates (TF35 and TF80) in the oral cavity for a short time might inactivate the virus in saliva and, thus, can be considered an effective suppressor of the spread of infection.

Regular Article
Neudesin, A Secretory Protein, Suppresses Cytokine Production in Bone Marrow-Derived Dendritic Cells Stimulated by Lipopolysaccharide Vol.6, No.5, p.155-162
Naoto Kondo , Yuki Masuda , Yoshiaki Nakayama , Ryohei Shimizu , Takumi Tanigaki , Yuri Yasui , Nobuyuki Itoh , Morichika Konishi
Received: July 14, 2023
Accepted: September 11, 2023
Released: September 25, 2023
Abstract Full Text PDF[1011K]

Neudesin was identified as a secretory factor expressed in the nervous system. On the other hand, neudesin is expressed in various organs and cells, suggesting that it plays roles in tissues other than neural tissues. We found that neudesin was expressed in dendritic cells (DCs) in the mouse spleen, which play a crucial role in the initiation of adaptive immune responses. Therefore, considering the possibility that neudesin may affect the acquired immune response, we first investigated whether neudesin has an effect on DCs using bone marrow-derived dendritic cells (BMDCs). Neudesin expression levels increased during the differentiation of bone marrow cells to BMDCs, and its expression level in BMDCs was reduced by lipopolysaccharide (LPS) treatment. BMDCs from neudesin knockout mice showed increased production of various cytokines, such as IL-12p70 and TNF-α, under LPS-stimulated conditions, compared with BMDCs from wild-type mice. In addition, treatment with recombinant neudesin suppressed the expression of cytokine genes in LPS-stimulated BMDCs from neudesin knockout mice. T cell proliferation was more strongly induced by co-culture with BMDCs from neudesin knockout mice than by those from wild-type mice. BMDCs from neudesin knockout mice showed increased lactate production, glucose consumption, and expression levels of glycolysis-related factors, suggesting that neudesin inhibits glycolysis, which promotes DC activation. The increased cytokine production in BMDCs from neudesin knockout mice was suppressed by the glycolytic inhibitor, 2-deoxyglucose. These results suggested that neudesin is a novel suppressor of DC function through the inhibition of glycolysis.

Regular Article
Effectiveness of Third-Generation Cephalosporins Against Enterobacter Cloacae Infection Vol.6, No.4, p.150-154
Yusuke Niinuma , Keisuke Kagami , Mitsuru Sugawara , Yoh Takekuma
Received: August 09, 2023
Accepted: August 12, 2023
Released: August 30, 2023
Abstract Full Text PDF[784K]

Enterobacter cloacae are thought to exhibit resistance to third-generation cephalosporins (3GCs) despite their sensitivity to these drugs; therefore, broad-spectrum antibiotics such as carbapenems are primarily used against them. Reports elucidating the treatment outcomes of 3GCs against E. cloacae are limited; therefore, we aimed to accumulate further evidence in this regard. Patients with isolated E. cloacae infections who were treated with antibiotics between April 2008 and April 2021 at Hokkaido University Hospital were included in the study. The primary endpoint was the difference in treatment efficacy between patients treated with the 3GCs after the detection of E. cloacae and those treated with other drugs. As a secondary endpoint, we compared the differences in treatment efficacy rates according to specimen type, severity, and type of antibiotics. Furthermore, multivariate analysis was performed to identify the factors influencing treatment failure. Among the 146 cases analyzed, 25 and 121 were categorized into the 3GCs and others groups, respectively. The treatment efficacy rate did not significantly differ between the two groups (3GC vs. others: 80.0% vs. 84.3%; P = 0.564). Only one patient in the 3GCs group developed resistance. The treatment efficacy rate did not differ according to the specimen type, severity, or antibiotic class between the groups. Multivariate analysis confirmed that the use of 3GCs did not affect treatment failure. 3GCs may be considered a potential therapeutic option for E. cloacae infections.

Regular Article
LATS Inhibitor Protects 6-OHDA Induced Neuronal Cell Death In Vitro and In Vivo Vol.6, No.4, p.144-149
Honoka Fujimori , Takuya Ohba , Shinsuke Nakamura , Yoshinobu Shimazawa , Daiki Takahashi , Takumi Iwawaki , Junji Kamon , Masamitsu Shimazawa , Hideaki Hara
Received: March 20, 2023
Accepted: July 27, 2023
Released: August 22, 2023
Abstract Full Text PDF[3M]

Parkinson's disease (PD) is a neurodegenerative disorder marked by progressive motor dysfunction. Dopaminergic neurons in the substantia nigra are likely the main cause of PD onset. The Hippo signaling pathway regulates organ size and tumor suppression. The Yes-associated protein (YAP) is a nuclear effector of the Hippo signaling pathway, and activation of YAP may be beneficial in several neurodegenerative diseases. In this study, we examined the effects of compound A [N-(tert-butyl)-2-(pyridin-4-yl)-1,7-naphthyridin-4-amine], a large tumor suppressor kinase (LATS) inhibitor, on 6-hydroxydopamine (6-OHDA)-induced cell damage in vitro and in vivo. In human neuroblastoma SH-SY5Y cells, compound A showed a protective effect against 6-OHDA-induced cell death without exhibiting any cytotoxicity. In order to investigate the effects of compound A on dopaminergic neurons, compound A was orally administrated to mice twice a day for 21 d. Next, mouse brains were harvested to assess the expression of (1) a dopaminergic neuron marker and (2) a YAP transcriptional target. Treatment of mice with 6-OHDA suppressed the expression of tyrosine hydroxylase (TH), a dopaminergic neuron marker, and compound A (3 mg/kg, per os) administration ameliorated the TH expression levels. In addition, compound A upregulated the mRNA expression levels of connective tissue growth factor (CTGF), a YAP transcriptional target. These results suggest that activation of the Hippo signaling pathway by LATS inhibition may be used as a novel therapeutic target for treating PD.

Overcoming Native Macrolide and Acquired Multidrug-Resistant Pseudomonas aeruginosa with Azithromycin and Polymyxin B Nonapeptide Vol.6, No.4, p.141-143
Aoi Kimishima , Hidehito Matsui , Kazunari Sakai , Masako Honsho , Sota Honma , Miho Sugamata , Naozumi Kondo , Serino Maruyama , Paul Wasuwanich , Kamrun Naher , Naoaki Arima , Kazutoyo Abe , Hideaki Hanaki , Yukihiro Asami
Received: June 21, 2023
Accepted: July 23, 2023
Released: August 04, 2023
Abstract Full Text PDF[697K]

Semi-synthetic antibiotic, azithromycin (AZM) does not show significant bactericidal activity against Pseudomonas aeruginosa (P. aeruginosa). We focused on potential for AZM as a multi targeting drug against P. aeruginosa and found combination of AZM and Polymyxin B nonapeptide (PMBN) to increase the permeability of the outer membrane. This combination is effective for P. aeruginosa including multidrug-resistance clinical isolates and shows 32-512-fold potentiation of the anti-P. aeruginosa activity of AZM. We found a great opportunity to create new anti-P. aeruginosa drug candidates based on AZM and PMBN.

Effects of Indomethacin Administration on the Small Intestines of 40-Week-Old Mice Vol.6, No.4, p.136-140
Kiyoko Maruyama , Shota Tanaka , Keiichi Hiramoto , Kazuya Ooi
Received: June 15, 2023
Accepted: July 19, 2023
Released: August 04, 2023
Abstract Full Text PDF[2M]

It is well known that administration of non-selective cyclooxygenase (COX) non-steroidal anti-inflammatory drugs (NSAIDs) increases the risk of developing gastric and duodenal ulcers. Previously, we reported that administration of indomethacin, one of these COX non-selective drugs, results in acute inflammation of the small intestine and that the subsequent increased release of cytokines, such as interleukin-6 and tumor necrosis factor-α, and histamines can lead to the development of dry skin. However, the effect of indomethacin administration in aging mice remains unknown. We aimed to investigate the effect of indomethacin administration on the small intestine of 40-week-old adult mice. As a result, both macroscopic and histologic abnormalities were observed in their small intestines. Expression of diamine oxidase, a histamine-degrading enzyme in the jejunum, was also decreased. In addition, expressions of mucin-2 and zonula occludens-1, both of which regulate the intestinal barrier function, were also decreased. Therefore, it was suggested that administration of indomethacin to aging mice may cause the release of histamines, which consequently increase in concentration in the small intestine, infiltrate into the blood, and circulate throughout the body as the intestinal barrier function declines.

Report (Case Report)
Safe Co-Administration of Amenamevir with Calcineurin Inhibitors: Case Reports Vol.6, No.4, p.133-135
Toshinori Hirai , Tomohiro Murata , Akihiro Tanemura , Shugo Mizuno , Takuya Iwamoto
Received: June 23, 2023
Accepted: July 17, 2023
Released: July 26, 2023
Abstract Full Text PDF[770K]

Although co-administration of amenamevir (a helicase–primase inhibitor) reportedly decreases exposure to midazolam, a CYP3A4 substrate, it remains unclear whether amenamevir induces the metabolism of calcineurin inhibitors (tacrolimus and cyclosporine) metabolized by CYP3A4. Herein, we illustrated two cases of induction of metabolism for calcineurin inhibitors by amenamevir. The concentration/dose normalized by body weight was defined as exposure to calcineurin inhibitors. The first case is a 63-year-old female (CYP3A5*3/*3 in both the recipient and donor) who received sustained-release tacrolimus 1.0 mg × 1 after living-donor liver transplantation. Although she temporarily withdrew tacrolimus on days 1 and 2 (from the initiation of combination), the same dosage was restarted from day 3. There was no significant difference in the C/D ratio regardless of co-administration of amenamevir 400 mg × 1 for 4 days (day -16: 4.5 ng/mL, 277.2 ng/mL/mg/kg vs. day 5: 3.6 ng/mL, 221.8 ng/mL/mg/kg). The second case is a 71-year-old female who received an induction therapy of microemulsion cyclosporine 50 mg × 2 from day -2 for nephrotic syndrome. The genotype of CYP3A5 was unknown. Blood cyclosporine concentrations at 2 h post-dose were 153.5 ng/mL (80.6 ng/mL/mg/kg) on day 2 and 166.8 ng/mL (87.6 ng/mL/mg/kg) on day 4 when administered amenamevir 400 mg × 1 for 5 days from day 0. After the discontinuation of amenamevir, blood cyclosporine concentration at 2 h post-dose on day 19 remained unchanged (170.4 ng/mL, 89.5 ng/mL/mg/kg). In conclusion, amenamevir co-administered for ≤5 days had less impact on the pharmacokinetics of tacrolimus and cyclosporine at low concentrations.

Molecular Toxicology of Methylmercury and Phytoremediation of Toxic Metals for Human Health Vol.6, No.4, p.126-132
Masako Kiyono , Yuka Ohshiro , Ryosuke Nakamura , Shimpei Uraguchi , Yasukazu Takanezawa
Received: June 29, 2023
Accepted: July 14, 2023
Released: July 26, 2023
Abstract Full Text PDF[1M]

The history of methylmercury (MeHg) toxicity research goes back to Minamata disease. In Japan, histopathological examination of patients afflicted with Minamata disease, alongside meticulous investigations at both in vitro and in vivo, were robustly undertaken to elucidate the deleterious effects of MeHg. It is not an overstatement to assert that Japanese investigations on MeHg toxicity have spearheaded global advancements in this field. Nevertheless, more than half a century has passed since the onset of Minamata disease, yet the intricacies of MeHg toxicity remains enigmatic. Moreover, environmental pollution of mercury and toxic metals is a global problem that needs to be solved. Therefore, our research has focused on, "toxicology of MeHg" and "remediation of mercury and other toxic metals". This review discusses the molecular mechanisms underlying roles of autophagy in MeHg responses of mammalian cells, identification of an anti-MeHg natural product, analysis of bacterial mercury-resistant gene, and plant biotechnology using bacterial transporters for phytoremediation.

Regular Article
Bactericidal Effect of MA-T Against Escherichia coli in Davis Minimal Medium in the Presence of Organic Materials, Compared to Perchlorous Acid as a Control Vol.6, No.4, p.122-125
Takekatsu Shibata , Ryuta Urakawa , Takayoshi Sakai , Kiyoshi Konishi
Received: May 07, 2023
Accepted: June 23, 2023
Released: July 04, 2023
Abstract Full Text PDF[893K]

Effectiveness of disinfectant MA-T against Escherichia coli in various growth media was demonstrated by minimal inhibitory concentration (MIC). MIC of MA-T in (DM+1/100 volume of LBG [Luria-Bertani medium with 0.4% glucose]) at pH 6.5 and pH 8.5 was 2.5 μg/mL, and that in 100%LBG at pH 6.5 and pH 8.5 was 7.5 μg/mL and 10 μg/mL, respectively. MA-T is not markedly affected by organic materials or pH. MIC of HClO in DM+1/100LBG at pH 6.5 and pH 8.5 was 7.5 μg/mL and 12.5 μg/mL, respectively, but MIC in rich medium (100%LBG) at pH 6.5 and pH 8.5 was 300 μg/mL and 500 μg/mL, respectively, indicating that HClO does not affect bacteria because of preferential reaction with biological materials contained in LBG, with minimal difference at varying pH. Growth of E. coli as monitored in aerobic shaking culture in LBG was dramatically reduced by adding 25 μg/mL MA-T, but growth was not affected by 100 μg/mL HClO (pH 6.5) or NaClO (pH 8.5). The CFU/mL of E. coli in aerobic standing culture in LBG declined linearly with incubation time on a logarithm chart after addition of 25 μg/mL MA-T. Viability was not affected by the addition of 100 μg/mL HClO (pH 6.5) or NaClO (pH 8.5). In conclusion, bactericidal effect of MA-T against E. coli is minimally affected by biological substances at different pH values (pH 6.5 or 8.5), but bactericidal effect of both HClO (pH 6.5) and NaClO (pH 8.5) is completely abolished by biological substances.

Regular Article
Insulin Treatment Combined with Exercise Training Does Not Prevent the Exacerbation of 12-O-Tetradecanoylphorbol-13-Acetate-Induced Inflammation in Type 2 Diabetic db/db Mice Vol.6, No.3, p.115-121
Yoshinori Iba , Ryousuke Sugimoto , Kosuke Itoh , Mizuki Yamasaki , Shota En , Kiyokazu Ozaki , Tetsuro Matsuura
Received: March 23, 2023
Accepted: June 08, 2023
Released: June 21, 2023
Abstract Full Text PDF[1M]

We investigated whether 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear inflammation is exacerbated in type 2 diabetic db/db mice, and if it is prevented by the control of blood glucose levels. Ear inflammation was assessed by ear thickness and the mRNA expression of the pro-inflammatory cytokines IL-1beta, IL-6, and TNF-alpha. Six-week-old db/db mice received an insulin treatment combined with exercise training daily for three weeks to lower blood glucose to normal levels. The other db/db group and their control (db/m) mice received saline without exercise training. TPA-induced ear swelling peaked after 8 h in db/m mice and after 32 h in db/db mice. The gene expression levels of cytokines 24 and 32 h after the TPA treatment were higher in db/db mice than in db/m mice. The insulin treatment combined with exercise training significantly suppressed blood glucose and HbA1c levels in 9-week-old db/db mice, but did not prevent the TPA-induced inflammatory response. These results revealed that TPA-induced ear inflammation was exacerbated in db/db mice and was not prevented by the insulin treatment combined with exercise training.

Regular Article
Chloroform Fraction of Panax Ginseng Extract Enhances Zip4-Mediated Zinc Influx into the Cytosol Vol.6, No.3, p.108-114
Yoshito Ikeda , Mizuki Kawakami , Yasuyuki Yamada , Masayuki Munekane , Kohei Sano , Takahiro Mukai , Taiho Kambe , Nobukazu Shitan
Received: April 26, 2023
Accepted: June 05, 2023
Released: June 15, 2023
Abstract Full Text PDF[2M]

Zinc is an essential nutrient with important biological functions, and its deficiency can lead to several diseases. The zinc transporter families, ZIP and ZNT, play essential roles in regulating zinc homeostasis and dynamics in the body and cells. Specifically, ZIP4 is the primary zinc transporter responsible for zinc absorption in the small intestine. Previous studies have shown that Panax ginseng (P. ginseng) extract can promote mouse Zip4 expression, and ginsenosides, including Rc and Re, enhance zinc uptake. However, the effects of other metabolites present in P. ginseng extract remain unclear. Therefore, we fractionated P. ginseng extract using chloroform, ethyl acetate, and n-butyl alcohol, and evaluated the effect of each fraction on zinc uptake using mouse Hepa and Hepa/MRE-Luc cells that stably expressed luciferase under the promoter of metal-responsive elements. Luciferase activity assays demonstrated that the chloroform (F1), ethyl acetate (F2), and n-butyl alcohol (F3) fractions increased cellular zinc uptake. In particular, F1 fraction was found to induce Zip4 mRNA and protein expressions, which significantly enhanced zinc uptake. Ginsenosides were mainly present in the F2 and F3 fractions, indicating that metabolites other than ginsenosides in the F1 fraction would enhance zinc uptake by inducing Zip4 mRNA and protein expressions. Our study offers novel insights into the molecular mechanisms underlying zinc uptake by P. ginseng.

Cell Morphology-Based Screening Identified Vitetrifolin D from Vitex Rotundifolia as an Inhibitor of Phorbol Ester–Induced Downregulation of E-Cadherin in HHUA Endometrial Cells Vol.6, No.3, p.103-107
Yusuke Hanaki , Nichika Iwase , Yasunori Sugiyama , Sena Miyoshi , Ryo C. Yanagita
Received: April 28, 2023
Accepted: June 02, 2023
Released: June 13, 2023
Abstract Full Text PDF[1M]

The epithelial–mesenchymal transition (EMT) of endometrial cells contributes to the development of endometrial cancer, endometriosis, and adenomyosis. We have recently reported that 12-O-Tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, induces EMT in HHUA endometrial cells cultured on collagen type I gels. HHUA cells showed an obvious morphological change from spheroids to scattered spindle cells during the TPA-induced EMT. In this study, we searched for inhibitors of the TPA-induced morphological change, and isolated vitetrifolin D from Vitex rotundifolia leaves. Vitetrifolin D suppressed the TPA-induced downregulation of E-cadherin, a major component of the epithelial adherens junction. Since vitetrifolin D showed a modest cytotoxicity at the concentration required to inhibit E-cadherin downregulation, it would not be suitable as a drug to treat endometrial disorders. However, our cell morphology-based screening could contribute to the discovery of new compounds that inhibit the loss of epithelial cell junctions.

Report (Case Report)
Warfarin Drug-Drug Interactions with Amiodarone and Tramadol in a Patient with Paroxysmal Atrial Fibrillation: A Case Report Vol.6, No.3, p.98-102
Marina Higa , Eisuke Katsuren , Jose Carlos S. Tayag , Masashi Iwabuchi , Yusuke Ohya , Hideo Shiohira , Katsunori Nakamura
Received: March 14, 2023
Accepted: May 08, 2023
Released: May 26, 2023
Abstract Full Text PDF[868K]

Amiodarone and tramadol are known to have drug-drug interactions that potentiate the effects of the anticoagulant warfarin. Herein we report a case which suggests increasing prothrombin time-international normalized ratio (PT-INR) and PT-INR/dose ratio due to the concomitant administration of warfarin, amiodarone (a cytochrome P450 2C9 inhibitor) and tramadol (an enhancer of anticoagulant effects of warfarin). A 72-year-old woman underwent emergency surgery following a suspected non-obstructive mesenteric ischemia, and diagnosis was confirmed. After surgery, the patient was given 2 mg/day of WF, and tramadol/acetaminophen, followed by amiodarone. After the concomitant dose of the triple combination, the PT-INR increased to over 7.02 (above the upper limit of the laboratory). The physician consulted the pharmacist for dose adjustment, and the pharmacist recommended a reduction in the dose to 0.5 mg. After restarting warfarin, PT-INR was controlled to 1.66–2.02. However, the PT-INR/dose ratio increased from 1.22 to 3.32–4.04 compared to the initial dose. The results suggest that these enhanced anticoagulant effects may be due to the inhibition of WF metabolism. Although the patient underwent resection of the small intestine, the effects of oral vitamin K1 were observed one day after administration. In conclusion, frequent PT-INR monitoring, and pharmacist intervention such as the assessment and dose adjustment in this report should be beneficial during anticoagulation therapy when multiple concomitant medications with suspected drug-drug interactions are present.

Regular Article
Blueberry Stem Extract Suppresses Blue Light-Emitting Diode Light-Induced Endoplasmic Reticulum Stress on Retinal Photoreceptor Cells Vol.6, No.3, p.87-97
Kenjirou Ogawa , Takara Karitani , Wataru Otsu , Kazuo Nishiyama , Hisato Kunitake , Yo Goto , Shota Nomiyama , Hideaki Hara , Masao Yamasaki
Received: March 16, 2023
Accepted: April 20, 2023
Released: May 26, 2023
Abstract Full Text PDF[4M]

Background: Blue light causes retinal photoreceptor damage via oxidative and endoplasmic reticulum (ER) stress. A previous study showed that blueberry stem extract (BStEx) and its active components have cytoprotective effects against blue-light-induced photoreceptor cell damage by suppressing oxidative stress. This study demonstrated the inhibitory effect of BStEx against blue light-induced ER stress in photoreceptor cells. Methods: The photoreceptor cells treated with BStEx or the antioxidant N-Acetyl-L-cysteine (NAC) as a positive control were used and then exposed to blue light. The cytoprotective effects of BStEx and NAC were evaluated using CCK-8. The ER stress-related protein expression changes over time, and its levels were measured after each exposure time to blue light in photoreceptor cells treated with BStEx or NAC. Results: BStEx and NAC showed protective effects against blue-light-induced photoreceptor morphological abnormalities and cell damage. Although blue light triggered ER stress factors such as BiP, PERK, ATF6, eIF2α, ATF4, and CHOP, which in turn stimulated cell cycle arrest factors p53 and p21 and upregulation of apoptosis-inducing factors caspase-3. However, BStEx suppressed the increase in expression of BiP, ATF4, ATF6, CHOP, p53, p21, and caspase-3, but not mitochondrial apoptotic factors Bax and cytochrome c. Furthermore, the antioxidant NAC showed similar suppressive effects on BStEx. Conclusion: Our findings suggest that blue light-induced ER stress is primarily caused by oxidative stress, and BStEx might suppress ER stress via an antioxidant effect. The antioxidant NAC contributes to the cell proliferative capacity and suppression of apoptosis in photoreceptor cells.

Regular Article
Bactericidal Effect of 405-nm LED Light against Aggregatibacter Actinomycetemcomitans Is Due Primarily to Disruption of Respiratory Chain Terminal Members Cytochrome bd Oxidase and Quinol Peroxidase Vol.6, No.3, p.81-86
Tomoyuki Nakada , Kiyoshi Konishi
Received: March 19, 2023
Accepted: April 27, 2023
Released: May 12, 2023
Abstract Full Text PDF[1M]

Irradiation with 405-nm visible violet LED light without additional photosensitizers decreased the viability of the aggressive periodontopathic bacterium Aggregatibacter actinomycetemcomitans. The number of CFU/mL decreased linearly on a logarithm chart versus irradiation time, with a 1-log reduction time of 1.32 min. The antimicrobial photodynamic effect of 405-nm LED light involved inhibition of the activity of membrane-bound cytochrome bd, a terminal quinone: oxygen oxidoreductase, and quinol peroxidase, a terminal quinone:H2O2 oxidoreductase. The 405-nm LED irradiation reduced minus oxidized difference spectrum showed that the 640-nm peak (α-peak of heme d) completely disappeared, and the height of the 556-nm (α-peak of hemes b and c) and Soret band (425 nm; γ-peak of hemes b, c, and d) was reduced to approximately half of the peak heights of non-irradiated controls. Survival of bacteria-injected silkworm larvae was also examined. Fifth-instar silkworm larvae were almost completely killed by approximately 40 h after bacterial injection, but almost all silkworm larvae irradiated with 405-nm LED light (20 mW/cm2 for 5 min, energy density: 6 J/cm2) survived, similar to controls not injected with bacteria, indicating that 405-nm LED light killed the injected bacteria. The bactericidal effect of 405-nm blue-light on A. actinomycetemcomitans is primarily due to disruption of cytochrome bd oxidase and quinol peroxidase of the respiratory chain.

Regular Article
Development of a Standard Test Method for Insecticides in Indoor Air by GC-MS with Solid-Phase Adsorption/Solvent Extraction
[Notice] An Corrigendum to this article was published on 31 October 2023
Vol.6, No.3, p.76-80
Taichi Yoshitomi , Iwaki Nishi , Aya Onuki , Tokuko Tsunoda , Masahiro Chiba , Shiori Oizumi , Reiko Tanaka , Saori Muraki , Naohiro Oshima , Hitoshi Uemura , Maiko Tahara , Shinobu Sakai
Received: November 16, 2022
Accepted: April 22, 2023
Released: May 12, 2023
Abstract Full Text PDF[1M]

Semi-volatile organic compounds (SVOCs), which can cause indoor air pollution, include plasticizers, insecticides, and flame retardants. In Japan, the Ministry of Health, Labour and Welfare has set guidelines for indoor air concentrations of di-n-butyl phthalate and di-2-ethylhexyl phthalate in plasticizers and fenobucarb, diazinon, and chlorpyrifos in insecticides. However, this analytical method has only been tentatively proposed for more than 20 years. In this study, we attempted to construct an analytical method for insecticides for which guideline values have been established based on recently standardized sampling and extraction methods for phthalates in indoor air. The results of the recovery tests for the insecticides were excellent, with recovery rates and relative standard deviations in the ranges 88%–104% and 1.4%–7.5%, respectively. Furthermore, the limits of detection and quantification were less than 1/50 of the current guideline values. Additionally, inter-laboratory validation was conducted at five research institutions. By excluding outliers with the Grubbs test, the accuracies were in the ranges of 81.9%–126.3%, 76.8%–121.7%, and 76.7%–112.8% for chlorpyrifos, diazinon, and fenobucarb, respectively. The target ranges for repeatability (RSDr) and reproducibility (RSDR) were 30% and 35%, respectively, and the validation results met these criteria. Based on these results, we propose the developed method as the standard test method for insecticide-originated pollutants in indoor air in Japan.

A Demonstration of the Validation Process for Alternative Endotoxin Testing Methods Using PyroSmart NextGen® Recombinant Cascade Reagent Vol.6, No.2, p.68-75
Madeline Kelley , Ingrid Stevens , Toshio Oda , Junko Akiyoshi , Edwin G. Jahngen
Received: April 04, 2023
Accepted: April 14, 2023
Released: April 28, 2023
Abstract Full Text PDF[1M]

The Bacterial Endotoxins Test (BET) uses Limulus amoebocyte lysate (LAL) reagents derived from the blood of horseshoe crabs for detection and quantification of endotoxins from Gram-negative bacteria in parenteral drug products and medical devices. Two types of recombinant reagents using genes cloned from the horseshoe crab genome have become available from several suppliers. One is a recombinant Factor C reagent (rFC), containing only recombinant Factor C, and the second is a recombinant Cascade Reagent (rCR) containing recombinant factor C, recombinant factor B and recombinant proclotting enzyme. Implementation of these recombinant reagents for BET requires validation that demonstrates results equal to or better than those determined by LAL reagents. Previous studies have shown that the PyroSmart NextGen® rCR meets the analytical performance requirements for both the plate and tube reader testing methods and provides equivalent results when testing samples containing autochthonous endotoxin. This study directly compares PyroSmart NextGen® to LAL reagent performance when testing a parenteral drug, which is a critical step for end-user implementation of alternative methods. It is the first published demonstration of an approach to the validation of alternative reagents that includes testing of a specific parenteral drug sample, and the data indicates that PyroSmart NextGen® is more precise when compared to LAL reagents. Relative recovery, linear regression, and Bland-Altman plot analyses also illustrate that PyroSmart NextGen® results are equal to or better than those determined by naturally sourced LAL reagents. This indicates that PyroSmart NextGen® is a useful alternative method for quantifying bacterial endotoxins in parenteral drugs.

Survey on Headache During COVID-19 Infection in People with Chronic Headache Vol.6, No.2, p.62-67
Masakazu Ishii, , Ikumi Ito , Hirotaka Katoh
Received: February 16, 2023
Accepted: March 27, 2023
Released: April 07, 2023
Abstract Full Text PDF[793K]

A questionnaire survey was conducted to clarify the headache situation in people with chronic headache during the new coronavirus disease 2019 (COVID-19) infection. A questionnaire survey was conducted on the internet. The subjects were 600 women in their 20s to 40s who were infected with the 7th wave of COVID-19 infection from July to October 2022. Subjects (55.7%) had headaches at the time of infection, and most of the infected people were recuperating at home. Other headaches (excluding migraine) accounted for about 60% of existing headaches in both the headache group and the headache-free group, but people with migraine accounted for 30.5% of the headache group, and those without headache accounted for 23.3% of the headache-free group. In the headache group, 40.3% had headaches even at the time of vaccination against COVID-19. In both the migraine group and other headache groups, compared to regular headaches, headaches during COVID-19 infection had a greater impact on daily life. Migraine headaches may have worsened in migraine carriers, as accompanying symptoms of migraine were observed at the time of infection. It is therefore important to note that chronic headache patients may develop severe headaches during COVID-19 infection.

PPARγ Protein Expression is Regulated by Cited1 and Cidec in Mouse 3T3-L1 Adipocytes Treated with Troglitazone Vol.6, No.2, p.55-61
Kohei Moriyama , Ayato Kokabu , Atsuko Masumi
Received: December 13, 2022
Accepted: February 07, 2023
Released: April 07, 2023
Abstract Full Text PDF[1M]

We previously reported that treatment with thiazolidinediones (TZDs), such as troglitazone (Tro), downregulates the protein levels of peroxisome proliferator-activated receptor gamma (PPARγ), with enhanced lipid accumulation during 3T3-L1 murine adipocyte differentiation in the presence of 3-isobutyl methylxanthine, dexamethasone, and insulin (MDI). In this study, we performed DNA microarray analysis to compare the gene expression profiles of MDI-induced and MDI/Tro-induced 3T3-L1 adipocytes to elucidate the mechanism underlying the reduction in PPARγ protein expression by Tro treatment. Apoptotic process genes of Gene Ontology were selected from the upregulated genes in MDI/Tro-induced cells and analyzed using real-time RT-PCR and western blotting. For several proteins, higher expression was detected in MDI/Tro-treated 3T3-L1 cells than in MDI-treated cells. Plasmid expression analysis using 293T cells revealed that the expression of cell death-inducing DFFA-like effector C (Cidec) or Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (Cited1) reduced PPARγ protein expression compared with the vector control. When 3T3-L1 preadipocytes transfected with small interfering RNA targeting Cidec or Cited1 were differentiated in response to MDI or MDI/Tro treatment, the reduction in PPARγ expression in MDI/Tro-treated 3T3-L1 adipocytes was partially suppressed. Our findings indicate that the expression of PPARγ protein is regulated in part by the induction of Cidec and Cited1 in MDI/Tro-treated 3T3-L1 adipocytes.

Regular Article
Intake of Lactobacillus Pentosus TJ515 Prevents the Formation of Retinal Edema in Retinal Vein Occlusion Model Mice Vol.6, No.2, p.47-54
Yae Hidaka , Aika Kuriyama , Anri Nishinaka , Kei Takahashi , Seigo Nakaya , Takeshi Kubota , Shinsuke Nakamura , Masamitsu Shimazawa , Hideaki Hara
Received: January 13, 2023
Accepted: March 19, 2023
Released: April 03, 2023
Abstract Full Text PDF[2M]

Macular edema is a sight-threatening symptom in patients with retinal vein occlusion (RVO) and diabetic retinopathy. It is treated by the intravitreal injection of anti-vascular endothelial growth factor agents, but this has a physical burden on patients. Thus, it is important to develop a treatment that can be administered orally. The probiotic Lactobacillus pentosus TJ515 can reduce inflammation by regulating host immunity via its induction of interleukin-10 (IL-10). However, its action on inflammatory diseases of the retina remains unclear. This study aimed to investigate the preventive effects of L. pentosus TJ515 intake on retinal edema using RVO model mice. Occlusion of the retinal vein led to an increase in the thickness of all retinal layers and the inner nuclear layer. Intake of L. pentosus TJ515 for three weeks suppressed the formation of retinal edema versus vehicle and the control strain (L. pentosus JCM1558). The expression of MCP-1 and MCP-3 was increased in RVO model mice but suppressed by the intake of L. pentosus TJ515. F4/80-positive cells (i.e., activated macrophages) were increased in the retinas of RVO model mice but decreased by the intake of L. pentosus TJ515. Therefore, L. pentosus TJ515 suppressed macrophage migration in the retinas. Treatment of mouse bone marrow-derived macrophages (BMDM) with plasma from mice that had ingested L. pentosus TJ515 decreased the mRNA expression of IL-6 and increased the mRNA expression of IL-10. Thus, oral intake of L. pentosus TJ515 can prevent retinal edema through its anti-inflammatory effects on macrophages and inhibiting effects on macrophage migration.

Promoted Papilloma Formation in a Two-Stage Carcinogenesis Model in Rassf6 Knockout Mice Vol.6, No.2, p.43-46
Mayu Morishita
Received: January 27, 2023
Accepted: March 15, 2023
Released: March 27, 2023
Abstract Full Text PDF[1M]

We have previously reported the generation of Rassf6 knockout mice using CRISPR/Cas technology. Furthermore, we have reported that RASSF6 is implicated in the regulation of the canonical nuclear factor (NF)-κB signaling pathway and that 7,12-dimethylbenz(a)anthracene induced skin inflammation in Rassf6 knockout mice more remarkably than in wild-type mice. In this study, we investigated the role of RASSF6 in skin tumorigenesis using a two-stage carcinogenesis model in Rassf6 knockout mice. Rassf6 knockout mice initially developed significantly more papillomas than did the control wild-type mice. Additionally, macrophages were detected and the canonical NF-κB signal was elevated in papillomas of Rassf6 knockout mice. These results indicated that canonical NF-κB signaling may be involved in papilloma formation. The suppression of NF-κB signaling may have implications for preventing cancer initiation and progression. The findings of this study indicate a tumor suppressive role of RASSF6.

The Structure-Activity Relationship of MPBD and Dictyoquinone Analogs in Dictyostelium discoideum Cell Aggregation Vol.6, No.2, p.37-42
Salma Zulqaida , Tamao Saito , Chihiro Ito , Yoshiaki Takaya
Received: January 05, 2023
Accepted: February 07, 2023
Released: March 07, 2023
Abstract Full Text PDF[1M]

4-Methyl-5-pentylbenzene-1,3-diol (MPBD) (1) and dictyoquinone (DQ or 2-hydroxy-5-methyl-6-pentyl-1,4-benzoquinone) (2) are two polyketides involved in the cell development of Dictyostelium discoideum. We synthesized several MPBD and DQ analogs and tested their ability to recover the cell aggregation delay in the stlA null strain. According to the findings, the alkyl side chain of both compounds facilitates their activity. The parent n-pentyl side chain is required for the cell aggregation.

Cerium (IV) Oxide Nanoparticles Enhance Hepatotoxic and Nephrotoxic Effects of Paraquat, Cisplatin, or Acetaminophen in Mice Vol.6, No.2, p.33-36
Yoshihiro Takahashi , Isao Ishida , Katsuhiro Isoda
Received: February 01, 2023
Accepted: February 23, 2023
Released: March 06, 2023
Abstract Full Text PDF[777K]

Nanomaterials are central to nanotechnology and have many useful properties. Cerium(IV) oxide nanoparticles (nCeO) have catalytic and antioxidant activity, and hold promise in industrial and medical products. However, the potential biological hazards of nCeO have not been addressed. We therefore investigated the in vivo effects and drug interactions of nCeO. Tail vein administration of nCeO did not induce liver and kidney injury in mice but induced liver or kidney damage when co-administered with paraquat, cisplatin or acetaminophen. Therefore, our findings indicate that nCeO are potentially hepatotoxic and nephrotoxic due to drug interactions.

Adenosine Acts as an Active Antiplatelet Constituent in Strawberries (Fragaria × ananassa) Vol.6, No.1, p.27-32
Natsuki Ichihara , Shiho Maekawa , Nanako Ogawa , Aiko Yamada , Tomoka Nagasato , Ikuro Maruyama , Kazuyoshi Sone , Masaharu Yasuda , Kenji Matsushita , Chihiro Ito , Yoshiaki Takaya
Received: January 20, 2023
Accepted: February 04, 2023
Released: March 01, 2023
Abstract Full Text PDF[1M]

Many foods have been reported to be effective against thrombosis, but most of them may be difficult to ingest in effective doses to prevent the disease. In this study, we focused on strawberries as one of the foods reported to have antithrombotic activities because they are highly palatable to many people and have several intake methods (e.g., raw fruits, juices, jams, etc.). In this study, strawberry ‘Minomusume’ was selected as a material to explore antiplatelet active compounds. The activity-guided fractionation of the strawberry extract resulted in the active compound being adenosine. Furthermore, we investigated the correlation between adenosine content and the antiplatelet activity of strawberries. As a result, as for the cultivars whose adenosine was detected, significant positive correlation was observed.

Regular Article
Elucidation of the Usefulness of Glypican-3, a Hepatocellular Carcinoma Biomarker, with the Fully Automated LUMIPULSE® System Vol.6, No.1, p.21-26
Hisashi Nojima , Tomonori Nishii , Kumiko Iida , Paul Franck Adeyissimi Adjou Moumouni , Katsumi Aoyagi , Shintaro Yagi
Received: November 15, 2022
Accepted: January 24, 2023
Released: February 15, 2023
Abstract Full Text PDF[1M]

Background: Glypican-3 (GPC3) is expressed in most of hepatocellular carcinoma (HCC) and GPC3 immunohistochemical staining is widely used in the clinical setting, but it has not been recommended as a blood biomarker, mainly due to its heterogeneous nature and the lack of established assay system. Here, we developed and evaluated the basic performance of fully automated GPC3 immunoassay kits which detect the full-length or the N-terminal fragments. We analyzed the molecular forms of GPC3 in HCC serum and evaluated the diagnostic performance of GPC3 and other biomarkers. Methods: We examined the analytical performance of the GPC3 kits. Then, the automated GPC3 assays were compared with an established ELISA kit. Afterwards, we determined the clinical cutoff of GPC3 and compared its diagnostic performance to alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) using 180 serum samples from clinically diagnosed patients. Results: GPC3 assays showed good analytical performance. The level of GPC3 in HCC was higher than recorded in healthy or other liver diseases’ sera. The AUC of GPC3 was 0.90, whereas the AUCs of AFP and PIVKA-II were 0.89 and 0.76, respectively. Conclusion: Automated GPC3 assays with stable performance against GPC3 in screening HCC have been established and the diagnostic accuracy of GPC3 was as good as AFP.

Dose-Related Difference and Involvement of Tyrosine Phosphorylation in Methylmercury-Induced Morphological Change in Cultured Astrocytes Obtained from Rat Cerebral Hemisphere Vol.6, No.1, p.16-20
Tatsumi Adachi , Hideaki Shimizu , Kohei Kawasumi , Yuka Suzuki , Tomoharu Hayashi , Ryota Tsurusaki , Yosuke Watanabe , Mitsunori Iwasaki , Yasuhide Yanagisawa , Ichiro Yasuda
Received: January 10, 2023
Accepted: January 17, 2023
Released: February 01, 2023
Abstract Full Text PDF[2M]

Astrocytes obtained from the cerebral hemisphere were maintained in serum-free defined medium containing dibutyryl cyclic AMP (dbcAMP) for 4 h, at which time almost all astrocytes showed a process-bearing stellate shape. They were then exposed to methylmercury (MeHg) at 1–3 μM or solvent alone (control) for up to 24 h. Astrocytes showed a flat polygonal shape after MeHg exposure at 3 µM for 1 h or at 2 µM for 3 h, whereas the shape was not affected after exposure at less than 1 µM for 24 h. Mercury accumulation levels were similar after MeHg exposure at 3 µM for 1 h and at 2 µM for 3 h, while the level after exposure at 1 µM did not reach those levels. The shape of astrocytes exposed to MeHg at 2 µM returned to stellate from polygonal after at least 24 h, although it remained polygonal in astrocytes exposed to MeHg at 3 µM. The viable cell number was significantly lower than in the control culture only in the culture exposed to MeHg at 3 µM for 24 h. In addition, genistein, a tyrosine kinase inhibitor, inhibited the morphological changes (spreading) induced by MeHg at 2–3 µM. These results suggest that a dose-dependent difference is observed in shape changes and cell death caused by MeHg in astrocytes, and that the responses to MeHg correlate to its accumulation levels, especially for the first few hours. They also suggest that tyrosine phosphorylation may play an important role in MeHg-induced spreading in astrocytes.

Evaluation of Recombinant Cascade Reagent PyroSmart NextGen® and Limulus Amebocyte Lysate Equivalency in a Plate and Tube Reader for Bacterial Endotoxins Testing Vol.6, No.1, p.11-15
Madeline Kelley , Ingrid Stevens , Nathaniel Marchessault , Junko Akiyoshi , Edwin G. Jahngen
Received: November 19, 2022
Accepted: December 08, 2022
Released: January 18, 2023
Abstract Full Text PDF[1M]

PyroSmart NextGen® is a recombinant cascade reagent (rCR) for the detection and quantification of bacterial endotoxins developed using cloned genes derived from the Limulus polyphemus horseshoe crab genome. Requirements for use of this alternative reagent include analysis of analytical performance, method suitability, and test result equivalency to Limulus amoebocyte lysate (LAL) reagents used in the compendial Bacterial Endotoxins Test (BET). The plate reader evaluation has been expanded to address two long-standing user preferences, with the inclusion of the tube reader method increasing the sensitivity of endotoxin detection from 0.005 EU/mL to 0.001 EU/mL. The utilization of PyroSmart NextGen® with the two different instrument types also allows for a more comprehensive equivalency analysis. Furthermore, the comparison results demonstrate that PyroSmart NextGen® detects equivalent levels of autochthonous endotoxin in water samples when compared to LAL reagents. Overall, this study provides the first large-scale example of equivalency analysis utilizing a robust rCR and has verified that PyroSmart NextGen® meets the expectations for alternative reagents.

Inhibitory Effects of Olive-Derived Phytochemicals on SARS-CoV-2 3C-Like Protease (3CLpro) Vol.6, No.1, p.8-10
Yoshimitsu Kiriyama , Hiromi Nochi
Received: November 22, 2022
Accepted: December 22, 2022
Released: January 12, 2023
Abstract Full Text PDF[761K]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that caused the global epidemic of COVID-19. 3C-like protease (3CLpro), called the main protease (Mpro), cleaves 11 sites in the long-stranded protein synthesized from viral genomic RNA. After the cleavage of the long-stranded protein, proteins such as RNA-dependent RNA polymerase are cut off and activated. Therefore, 3CLpro is crucial for SARS-CoV-2 replication and is the target of drugs against SARS-CoV-2. Polyphenols and triterpenoids in olives can bind to SARS-CoV-2 3CLpro in in silico and computational studies. However, not all phytochemicals have been shown to inhibit SARS-CoV-2 3CLpro. Here, we examined the inhibitory effects of olive-derived phytochemicals on SARS-CoV-2 3CLpro. Among these phytochemicals, luteolin and oleanolic acid inhibited the 3CLpro activity by about 40%. The actual enzyme inhibitory action of oleanolic acid has not been examined. Thus, we examined the dose response in the inhibition of 3CLpro by oleanolic acid. Oleanolic acid significantly inhibited the enzymatic activity of 3CLpro at concentrations above 50 μM, and it may be a potent molecule that inhibit SARS-CoV-2 replication via 3CLpro inhibition.

Regular Article
Effects of Hochuekkito on Lenvatinib-Induced Fatigue in Mice Vol.6, No.1, p.1-7
Jinyang Xu , Ikuo Nakamura , Makoto Sudo , Satoshi Noda , Naoki Fujitsuka , Sachiko Mogami , Keiko Mitani , Masaharu Tada , Yasuhiro Fujimoto , Tomohiro Terada , Seiko Hirono , Etsuro Hatano
Received: November 23, 2022
Accepted: December 08, 2022
Released: January 10, 2023
Abstract Full Text PDF[1M]

Lenvatinib has been approved for treating various cancers; however, it exerts numerous adverse effects. Hochuekkito, a Japanese Kampo medicine, can alleviate these adverse effects. Here, we aimed to evaluate the effect of Hochuekkito on lenvatinib-induced chronic fatigue in a murine model. BALB/C mice were fed a control diet or a diet supplemented with 1.5% Hochuekkito for six weeks. On days 15–42, the mice were intraperitoneally injected with dimethyl sulfoxide or lenvatinib. Accordingly, the mice were divided into control/dimethyl sulfoxide, control/lenvatinib, 1.5% Hochuekkito/dimethyl sulfoxide, and 1.5% Hochuekkito/lenvatinib groups. Body weight and food intake were recorded daily. Nesting tests were performed once a week, and the serum interleukin-6 (IL-6) concentration was measured. Liver drug-metabolizing enzyme, CYP3A4, breast cancer resistance protein (BCRP), and P-glycoprotein (P-gp) levels were determined. The serum lenvatinib concentration and CYP3A4, BCRP, and P-gp levels did not differ significantly between the control/lenvatinib and 1.5% Hochuekkito/lenvatinib groups. The usage rate of nesting material on day 42 was higher in the 1.5% Hochuekkito/lenvatinib group than in the control/lenvatinib group (p < 0.05). The serum IL-6 level was lower in the control/dimethyl sulfoxide and 1.5% Hochuekkito/lenvatinib groups than in the control/lenvatinib group (p < 0.05). Overall, Hochuekkito may alleviate lenvatinib-induced fatigue through IL-6 inhibition.