BPB Reports

2021 - Vol. 4

2021 - Vol. 4

Regular Article
Protective Effects of the Alga Fucoidan Against Amyloid-β-Induced Neurotoxicity in SH-SY5Y Cells Vol.4, No.6, p.206-213
Miki Nagata , Mayumi Tsuji , Tatsunori Oguchi , Yutaro Momma , Tetsuhito Nohara , Hideaki Ohashi , Naohito Ito , Ken Yamamoto , Yuko Udaka , Akiko Sasaki , Yuji Kiuchi , Satoshi Numazawa
Received: November 16, 2021
Accepted: December 13, 2021
Released: December 27, 2021
Abstract Full Text PDF[2M]

Dementia is expected to affect an increasing number of patients with global aging populations. About 70% of all dementia is related to Alzheimer's disease (AD). Overaccumulation of amyloid-β protein (Aβ) in the brain forms senile plaques, one of the main features of neurodegeneration in AD. However, there are few drugs available to specifically inhibit senile plaque formation. Fucoidan, a sulfated polysaccharide derived from brown algae, has various bioactivities, such as anti-tumoral and anti-obesity effects. This study aimed to clarify the mechanism underlying the protective effect of fucoidan against Aβ-induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Cell viability and Aβ-induced cytotoxicity were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, calcein AM, and ethidium homodimer-1. Aβ-induced oxidative stress was evaluated through reactive oxygen species (ROS), cell membrane phospholipid peroxidation, mitochondrial ROS, and Mn-SOD, a mitochondrial radical scavenger. In addition, mitochondrial membrane permeability transition, and ATP concentration were evaluated. Fucoidan significantly improved Aβ-reduced cell viability. With respect to oxidative stress, Aβ exposure increased ROS, lipid peroxidation, and mitochondrial ROS, while fucoidan significantly suppressed these changes. Fucoidan also suppressed the decline in mitochondrial permeability transition and ATP caused by Aβ. Therefore, through its numerous antioxidant activities, fucoidan might have a neuroprotective role in preventing Aβ-induced neurotoxicity.

Exposure to (–)-Xanthatin during the Haploid Formation of Mouse Spermatocyte GC-2spd(ts) Cells, an in vitro Male Germ Cell Model Vol.4, No.6, p.202-205
Masayo Hirao-Suzuki , Shuso Takeda , Mitsuru Shindo
Received: November 08, 2021
Accepted: December 05, 2021
Released: December 21, 2021
Abstract Full Text PDF[1013K]

We and others have previously reported the possible usefulness of (–)-xanthatin, one of the naturally occurring xanthanolides present in the Cocklebur plant, as an anticancer cytotoxic agent in both in vivo and in vitro settings. Anticancer agents generally act as a double-edged sword during cancer chemoprevention, as these drugs can cause infertility in males and females. Although (–)-xanthatin is known to be an anti-proliferator against cancer cells, the adverse effect on spermatogenesis has not yet been investigated, even in vitro. In this study, we utilized chemically synthesized pure (–)-xanthatin to analyze whether this molecule affects the appearance of haploid cells in the population of a GC-2spd(ts) cell line, an in vitro model of male germ cells. We did not observe any remarkable effects on the haploid formation of GC-2spd(ts) at a (–)-xanthatin concentration below 0.5 μM.

Indoxyl Sulfate Induces an Inflammatory Response in the Proximal Tubule via Macrophages Vol.4, No.6, p.198-201
Ryo Isaka , Nanan Ishida , Go Kitahara , Yuji Sakahashi , Yuya Haga , Hirofumi Tsujino , Kazuya Nagano , Kazuma Higashisaka , Yasuo Tsutsumi
Received: October 11, 2021
Accepted: November 30, 2021
Released: December 09, 2021
Abstract Full Text PDF[1013K]

Indoxyl sulfate is a uremic toxin and is difficult to remove by hemodialysis owing to its tight binding to albumin in the blood. There is therefore concern that it could cause inflammatory responses in renal tissue, leading to worsening of renal failure. Recent reports suggest that indoxyl sulfate not only directly affects renal cells but also induces an inflammatory response via macrophages infiltrating renal tissues. However, the mechanism of the indoxyl-sulfate-induced inflammatory response mediated by macrophages and the effect of macrophages on renal cells have not yet been elucidated. Here, we evaluated the effect of indoxyl sulfate on the inflammatory response of macrophages and the effect of macrophages on renal cells. Indoxyl sulfate upregulated the expression of tumor necrosis factor-α (TNF-α) by THP-1 macrophages. Moreover, it activated the transcriptional regulator nuclear factor-kappa B (NF-κB) p65, and an inhibitor of NF-κB suppressed indoxyl-sulfate-induced TNF-α elevation. Supernatant of THP-1 macrophages treated with indoxyl sulfate increased the mRNA expression levels of inflammatory cytokines in HK-2 renal proximal tubular epithelial cells. These results suggest that indoxyl sulfate increases TNF-α expression through the activation of NF-κB in THP-1 macrophages and that macrophages stimulated with indoxyl sulfate induce an inflammatory response in the proximal tubular epithelial cells.

Regular Article
Characteristics of Japanese Medical and Healthcare University Students with Habitual Consumption of Energy Drinks: Relationship with Perceived Stress Vol.4, No.6, p.193-197
Tomoyuki Enokiya , Eiichi Azuma , Masahiko Hayashi
Received: October 21, 2021
Accepted: November 09, 2021
Released: December 01, 2021
Abstract Full Text PDF[1M]

This cross-sectional study examined the sociodemographic characteristics associated with energy drink consumption at a medical and welfare university in Japan. Data were collected and analyzed from 1249 students (first to fourth year) belonging to a single university. We examined 19 sociodemographic variables through a questionnaire. Furthermore, we identified sociodemographic characteristics associated with the consumption of energy drinks using multivariate logistic regression analysis. We characterized students who consumed energy drinks using principal component analysis and hierarchical cluster analysis. Significance was established at P < 0.05. The findings of our study revealed that perceived stress (P = 0.01) and experiencing strong sleepiness (P = 0.01) during the day in males and females, as well as the frequency of alcoholic drink consumption were related to the habitual consumption of energy drinks. The students who consumed energy drinks were categorized into three clusters: male students who perceived stress, female students who perceived stress and wanted to consume alcohol, and male students who perceived stress and experienced excessive daytime sleepiness. Thus, perceived stress may be strongly correlated with the consumption of energy drinks. Therefore, educational interventions to promote awareness of the health risks of excessive energy drink consumption among students are warranted.

Analysis of Volatile Organic Compounds Emitted from Bedding Products Vol.4, No.6, p.182-192
Naohiro Oshima , Maiko Tahara , Shinobu Sakai , Yoshiaki Ikarashi
Received: September 13, 2021
Accepted: November 06, 2021
Released: November 29, 2021
Abstract Full Text PDF[709K]

In this study, we analyzed the volatile organic compounds (VOCs) emitted from a sample of bedding products. These items are intended for long-term use indoors and therefore will be present for long periods of time in the breathing zone of household occupants. Forty bedding products (20 pillows and 20 mattresses) were obtained from the Japanese domestic market for analysis. We have pioneered the measurement of VOCs from bedding products using the sampling bag method, and our measurements showed that a variety of VOCs were emitted from the items. In the pillow sample, polyethylene pillows emitted the most aliphatic hydrocarbons, while buckwheat hull pillows emitted fewer chemicals overall. All pillows emitted tetradecane, toluene, and xylene. VOCs emissions from the mattresses tended to be higher than from the pillows. The mattresses emitted 2-ethyl-1-hexanoic acid frequently and at high concentrations. To further understand the effects of indoor air pollution, it is necessary to continue research into testing the emissions from bedding products and other household items.

Regular Article
Fibroblast Growth Factor-2 Upregulates Reactive Sulfur Species Production via ERK1/2 Signal-Mediated Cystathionine γ-Lyase Induction in Cultured Bovine Aortic Endothelial Cells Vol.4, No.6, p.175-181
Musubu Takahashi , Ayaka Kubota , Tomoya Fujie , Yasuhiro Shinkai , Yoshito Kumagai , Tsuyoshi Nakano , Takato Hara , Chika Yamamoto , Toshiyuki Kaji
Received: August 10, 2021
Accepted: October 13, 2021
Released: November 12, 2021
Abstract Full Text PDF[1M]

Fibroblast growth factor-2 (FGF-2) regulates several vascular endothelial cell functions, including proliferation. It has been suggested that the regulation may be modulated by reactive sulfur species (RSS), which are hydrogen sulfide and biomolecules containing persulfide/polysulfide groups. Since RSS promote vascular endothelial cell proliferation, we hypothesized that FGF-2 regulates the levels of RSS-producing enzymes in the cells. Bovine aortic endothelial cells were cultured and treated with FGF-2, and intracellular RSS levels were determined. The expression of RSS-producing enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase, 3-mercaptopyruvate sulfurtransferase, and cysteinyl-tRNA synthetase 2, was evaluated, and the intracellular signaling pathway that mediates FGF-2 regulation of RSS accumulation was investigated. We revealed that FGF-2 upregulates the expression of RSS by selectively inducing CSE via the ERK1/2 signaling pathway in vascular endothelial cells. The effect of FGF-2 on the function of vascular endothelial cells may be modulated by intracellular RSS, especially higher-molecular-mass RSS such as protein persulfide, the levels of which are increased by the growth factor.

Drug-Drug Interaction between Azathioprine and Allopurinol in Patients with Chronic Kidney Disease: A Case Series Study Vol.4, No.5, p.170-174
Toshinori Hirai , Chihiro Shiraishi , Tomohiro Murata , Takuya Iwamoto
Received: August 09, 2021
Accepted: October 05, 2021
Released: October 28, 2021
Abstract Full Text PDF[809K]

The impact of renal impairment on the drug-drug interaction between azathioprine and allopurinol that causes myelosuppression and hepatotoxicity remains unclear. This case series study investigated adverse effects caused by azathioprine owing to drug-drug interaction considering renal impairment. Patients who started the combination therapy of azathioprine and allopurinol at Mie University Hospital between January 2013 and February 2021 were enrolled. The outcome of adverse events associated with azathioprine was assessed according to Common Terminology Criteria for Adverse Events version 5.0. The Drug Interaction Probability Scale was used to determine the probability of drug-drug interaction. Of the three patients, two were identified as exhibiting drug-drug interaction with the Drug Interaction Probability Scale > 5 points. They experienced grade 3 myelosuppression or hepatotoxicity with fatigue, after initiation of azathioprine (1.28 and 0.44 mg/kg once daily) and allopurinol (50 mg once daily). They received appropriate dose-adjusted allopurinol according to renal function. Additionally, both patients had the estimated glomerular filtration rate < 60 mL/min/1.73 m2. Thus, renal impairment might reduce the excretion of oxypurinol, an active metabolite of allopurinol, which certainly enhances the side effects of azathioprine.

Effect of Acylated Sugar Chain Terminals on the Intestinal Absorption of Large Molecular, Heavenly Blue Anthocyanin in Rats Vol.4, No.5, p.166-169
Takashi Ichiyanagi , Masayuki Nashimoto , Norihiko Terahara
Received: September 06, 2021
Accepted: October 11, 2021
Released: October 26, 2021
Abstract Full Text PDF[871K]

The gastrointestinal absorption of heavenly blue anthocyanin derived from morning glory (Pharbitis nil L.) was examined in rats. Ingested heavenly blue anthocyanin was directly absorbed from the gastrointestinal tract and detected in its original polyacylated form in rat blood plasma. The maximum plasma concentration and area under the plasma concentration curve during 8-h post-administration after a single oral dose of 0.0569 mmol/kg heavenly blue anthocyanin were 0.143 ± 0.023 μM and 23.30 ± 3.76 μM·min, respectively. Heavenly blue anthocyanin, which contains asymmetrical branched chains with glucosyl terminals, was absorbed at a similar level to that of total ternatin. By contrast, the plasma amount of heavenly blue anthocyanin was approximately 6-fold higher than that in a previous report of peonidin 3-O-β-(6''-O-caffeoyl)-sophoroside-5-O-β-D-glucopyranoside with an attached caffeoyl terminal on 6''-position.

Sperm Morphology is Different in Two Common Mouse Strains Vol.4, No.5, p.162-165
Nao Sekine , Satoshi Yokota , Shigeru Oshio
Received: June 03, 2021
Accepted: August 27, 2021
Released: September 30, 2021
Abstract Full Text PDF[723K]

ICR and C57BL/6J mice have been widely used in several research fields. The reproductive toxicology parameters, such as fertilization rate, which may differ between the two strains, are well known. However, the details of the sperm quality parameters are not well known. To reveal these, we compared the sperm morphology of the two strains. Eosin-stained sperm smears from adult ICR and C57BL/6J mice were analyzed. We observed that 79.6 ± 1.2 and 49.5 ± 1.7% of ICR and C57BL/6J mice sperm, respectively, showed a normal form. Furthermore, abnormal sperm samples were classified into ten types based on their defective sites. The percentage of abnormal sperm with an amorphous head, bent head, no head, hairpin loop, short tail, and two tails in ICR mice was significantly lower than that in C57BL/6J mice. In contrast, the percentage of coil-tailed sperm in ICR mice was significantly higher than that in C57BL/6J mice. These results suggest that C57BL/6J mice have a limited ability to remove the cytoplasm during spermiation and ICR mice have fewer sperm abnormalities than C57BL/6J mice. The characteristics of male reproductive traits among mouse strains should be taken into consideration in sperm analysis, as the negligence of this could generate an increased potential for a misleading in toxicology evaluation.

Huperzia serrata and the Constitute of Huperzine A Attenuate MK-801-Induced Cognitive Dysfunction in Mice via PKC-Erk Pathway Vol.4, No.5, p.155-161
Takuya Ohba , Honoka Fujimori , Shinsuke Nakamura , Yoshihiro Hayashi , Hiroyuki Kono , Masamitsu Shimazawa , Hideaki Hara
Received: December 11, 2020
Accepted: July 28, 2021
Released: September 30, 2021
Abstract Full Text PDF[2M]

N-methyl-D-aspartate (NMDA) receptor has important role in synapse function and neurotransmission by the high permeability of Ca2+. In Alzheimer's disease (AD), NMDA mediated neurotransmission is impaired by decreasing NMDA receptor subunits and increasing glutamate, which is an NMDA receptor ligand. Therefore, the NMDA antagonist (memantine) is used as a therapeutic drug of AD. Huperzia serrata is a traditional Chinese herbal medicine, and the constitute huperzine A has been reported to inhibit NMDA receptor. In this study, we clarified the effect of Huperzia serrata and the constitute huperzine A on MK-801-induced cognitive dysfunction. Memantine, Huperzia serrata and huperzine A were administered orally once a day and Y-maze test was performed at day 7 to investigate cognitive function. After Y-maze test, mice brains were collected and evaluated the expression levels of glutamate receptors and Ca2+ signalling associated protein. Memantine (5 mg/kg, p.o.), Huperzia serrata (1000 mg/kg, p.o.) and huperzine A (0.7 mg/kg, p.o.) were improved collect alternation behaviour in Y-maze test. Treatment of memantine, Huperzia serrata and huperzine A also improved the total arm entries increased by MK-801. The expression level of NMDA receptor subunit NR2A was increased by Huperzia serrata and huperzine A treatment. In addition, the decreased expression level of PKCα and phosphorylation of Erk1/2 by MK-801 were improved. These results suggest that Huperzia serrata and the constitute huperzine A improve cognitive dysfunction through NMDA receptor function and glutaminergic signaling pathway.

Comparison of the Components of Three Types of Miso (Fermented Soybean Paste) by 1H NMR Metabolomic Analysis Vol.4, No.5, p.148-154
Masumi Motonaga , Hiromitsu Watanabe , Yoshitaka Tayama , Ryo Shimizu , Seigo Sanoh , Yaichiro Kotake , Shigeyuki Kitamura , Shigeru Ohta , Kazumi Sugihara
Received: August 04, 2021
Accepted: September 05, 2021
Released: September 27, 2021
Abstract Full Text PDF[1M]

Miso is a traditional Japanese fermented food made by fermenting steamed soybean with koji (fermented cereals with Aspergillus oryzae). Many types of miso are produced in Japan, including miso with rice and/or barley depending on the region where it is produced. In this study, we used 1H NMR metabolomic analysis to compare the characteristics of the components (metabolites) of miso with different ingredients. Three types of miso were compared: soybean miso, rice miso, and barley miso. After measuring the 1H NMR of the aqueous solution of each miso, multivariate analysis of the spectral integration data was performed to compare the characteristic metabolites. Principal component analysis (PCA) showed a separation between soybean miso and rice, barley miso. Orthogonal Projection to Latent Structure Discriminant Analysis (OPLS-DA) extracted ethanol, saccharides such as glucose, and amino acids as metabolites contributing to the separation. Ethanol and glucose were higher in rice and barley miso, especially glucose in barley miso and ethanol in rice miso. Soybean miso was characterized by its high content of amino acids, including branched-chain amino acids. It was suggested that the characteristics of these ingredients were influenced not only by differences in ingredients, but also by the fermentation period and other factors. Although the number of metabolites that can be analyzed by 1H NMR metabolomic analysis is smaller than that by GC/MS or LC/MS, it does not require any pretreatment and is easy to measure, so it can be applied to the comparison of food components and quality control, as in the analysis of miso components.

Regular Article
Antiviral Effects of the Anti-Occludin Monoclonal Antibody on Persistent Hepatitis C Virus Infection in a Human Liver Chimeric Mouse Model Vol.4, No.5, p.142-147
Yoshimi Shimizu , Takeru Suzuki , Yoshitaka Shirasago , Masuo Kondoh , Tetsuro Suzuki , Takaji Wakita , Masayoshi Fukasawa
Received: August 19, 2021
Accepted: September 02, 2021
Released: September 16, 2021
Abstract Full Text PDF[1M]

Worldwide, ~71 million people are infected with hepatitis C virus (HCV). Claudin-1 (CLDN1) and occludin (OCLN), both tetraspanins of epithelial tight junctions, are entry receptors for HCV. Previously, we generated anti-CLDN1 and anti-OCLN monoclonal antibodies (mAbs), both of which strongly inhibit HCV entry into hepatocytes. However, the relevance of CLDN1 and OCLN in persistent HCV infection remains unclear. In the present study, we evaluated the involvement of CLDN1 and OCLN in persistent HCV infection using the mAbs against CLDN1 (clone 3A2) and OCLN (clone 1-3). Interestingly, both mAbs significantly reduced intracellular HCV RNA levels in a cell culture system. Additionally, the anti-OCLN mAb reduced serum HCV levels in chronic HCV-infected human liver chimeric (PXB) mice (often used as an in vivo HCV infection model), whereas the anti-CLDN1 mAb did not have this effect. These results suggest that the OCLN molecule contributes to maintaining persistent HCV infection in vivo. In further investigation, we determined whether combinations of NS5B inhibitor, nesbuvir, and the anti-OCLN mAb had anti-HCV effects on persistent HCV-infected PXB mice. Administration of nesbuvir and control IgG caused a breakthrough of serum HCV levels in all mice, whereas nesbuvir and anti-OCLN mAb combinations caused the breakthrough at a later phase in only one of three mice. Thus, anti-OCLN mAb seems to suppress the occurrence of resistant viruses against nesbuvir. Based on these results, we suggest that the anti-OCLN mAb, which could be combined with direct-acting antiviral agents, might be a potential candidate antiviral agent in HCV therapeutics.

Regular Article
Gastrointestinal Absorption of Ternatins, Polyacylated Anthocyanins Derived from Butterfly Pea (Clitoria ternatea L.) Petals in Rats Vol.4, No.4, p.136-141
Takashi Ichiyanagi , Masayuki Nashimoto , Norihiko Terahara
Received: July 28, 2021
Accepted: August 19, 2021
Released: September 02, 2021
Abstract Full Text PDF[1M]

Ternatins, polyacylated anthocyanins that contain two or more aromatic acyl groups, are found in the petals of butterfly pea (Clitoria ternatea L.). We examined the gastrointestinal absorption of ternatins in rats after oral administration of the extract of the butterfly pea petals. Ingested ternatins were absorbed rapidly in the gastrointestinal tract in their original acylated forms. Nine ternatins were detected, together with preternatin A3, in rat blood plasma at 15 min after oral administration. After a single oral dose of 0.0527 mmol/kg ternatin, the maximum plasma concentration and area under the plasma concentration curve for total ternatin was 0.141 ± 0.035 μM and 16.398 ± 1.542 μM·min, respectively, during the 8-h period post-administration. The absorption of ternatins in blood plasma tended to negatively correlate with increasing order of molecular weight; however, ternatins carrying symmetrical substitution patterns and glucosyl terminals on the both side chains at the 3' and 5' positions of the aglycone were exceptionally bioavailable.

Regular Article
The Effect of Medroxyprogesterone Acetate on the Secretion of Lipoprotein Lipase in Mouse Mammary Tumor Cells Vol.4, No.4, p.130-135
Tomoyasu Fujii , Jun Kamishikiryo , Tetsuo Morita
Received: July 07, 2021
Accepted: August 20, 2021
Released: August 31, 2021
Abstract Full Text PDF[1M]

Progesterone (P4) is a corpus luteum hormone associated with the development of the mammary gland and uterus. The actions of P4 on lipid metabolism in breast cancer are unclear. In this study, we investigated medroxyprogesterone acetate (MPA) on the secretion of lipoprotein lipase (LPL) from mouse mammary tumor FM3A cells. The tumor cells were incubated with MPA and other agents. The treated cells were used in the mitogen-activated protein kinase (MAPK) and LPL activity assay. The supernatant was used in the LPL activity assay and western blotting. The increased secretion of LPL in the tumor cells treated with MPA was observed. The MPA-stimulated secretion of LPL was suppressed by a protein kinase A (PKA) inhibitor. The activity of MAPK increased in the tumor cells treated with MPA, and various MAPK inhibitors suppressed the stimulatory secretion of LPL. The effect of MPA on LPL secretion was markedly suppressed by an inhibitor of the mechanistic target of rapamycin complex (mTORC) 1 and 2, KU0063794, but not the mTORC1 inhibitor, rapamycin. Furthermore, a small interfering RNA-mediated decrease in Rictor's expression, a pivotal component of mTORC2, suppressed the stimulatory secretion of LPL. These results suggest that the stimulatory secretion of LPL in the tumor cells treated with MPA is closely associated with activation of mTORC2, possibly via the MAPK signaling associated with PKA activation.

Regular Article
Association between Whole Blood Manganese and Dry Skin in Hemodialysis Patients Vol.4, No.4, p.124-129
Masataka Deguchi , Hirofumi Machida , Hiroyuki Yasui , Jun Hiraoka , Keigo Nishida , Keiichi Hiramoto , Hidehiko Jose , Misao Takeuchi , Kazuya Ooi
Received: July 18, 2021
Accepted: July 20, 2021
Released: August 18, 2021
Abstract Full Text PDF[874K]

Background and aim: Abnormalities in blood metal concentrations and pruritus occur frequently in hemodialysis patients. Pruritus significantly impairs the quality of life for these patients, and may be related to abnormal blood metal concentrations. Therefore, we measured the blood metal concentrations in hemodialysis patients and non-dialysis control patients to examine the relationship between blood metal level, pruritus, and skin condition. Methods: Hemodialysis patients were divided into “scratching” and “non-scratching” groups based on the severity of their symptoms. Blood was collected and the skin condition was measured at the start of hemodialysis in the patient group. Concentrations of metals such as magnesium, calcium, manganese, iron, copper, and zinc in serum and whole blood were determined by an inductively coupled plasma-mass spectrometer. Skin condition was assessed by measuring transepidermal water loss and stratum corneum moisture content. Results: The whole blood manganese level in hemodialysis patients was 8 times higher than that in non-dialysis patients (168 ± 42 ng/mL vs 22 ± 15 ng/mL), and a negative correlation was found between manganese level and stratum corneum moisture content. The stratum corneum moisture content in the scratching group was significantly lower than that in the non-scratching group. Conclusion: Patients with higher levels of whole blood manganese exhibited dry skin.

Extracellular Elaidate, a Trans Fatty Acid, Tends to be Incorporated into Triglycerides and Incorporated Elaidate is Released by Lipolysis Vol.4, No.4, p.120-123
Kenichi Ishibashi , Gen-ichi Atsumi
Received: July 21, 2021
Accepted: July 22, 2021
Released: August 18, 2021
Abstract Full Text PDF[1M]

Intake of elaidate, an industrially produced trans fatty acid, is associated with the development of cardiovascular disease. Recently, we revealed that persistent exposure to elaidate impairs the insulin responsiveness of adipocytes. Moreover, extracellular elaidate is incorporated into phospholipids and triglycerides and exists mainly as triglycerides in adipocytes. Because fatty acids in adipocytes are not only used as an energy source but also released as cytokines to regulate cellular function and whole-body metabolism, we hypothesized that elaidate is released from adipocytes. Here, we examined the intracellular behavior of elaidate to explain that incorporated elaidate exists mainly as triglycerides, and whether it is released from adipocytes. Extracellular elaidate was incorporated into triglycerides rather than phospholipids, and elaidate incorporated into triglycerides did not decrease during the study period. Under lipolytic stimulation, incorporated elaidate—together with other fatty acids—was released from adipocytes. These results imply that adipocytes act as a reservoir of elaidate.

Inhibitory Activity and Proposed Binding Model of γ-Glutamyl Cysteine, the Precursor of Glutathione, on Angiotensin Converting Enzyme Vol.4, No.4, p.116-119
Chisato Okajima , Naoki Imakawa , Kazuya Nagano , Masayoshi Arai , Mitsuhiro Arisawa , Misa Muraoka , Hirofumi Tsujino , Yoshihiko Hirata , Kazumasa Hirata
Received: June 11, 2021
Accepted: July 21, 2021
Released: August 13, 2021
Abstract Full Text PDF[1M]

Owing to their various physiological activities, thiol compounds, such as l-cysteine with UV-protection properties and captopril that inhibits the catalytic activity of angiotensin converting enzyme (ACE), are currently used as supplements and pharmaceuticals. Glutathione (GSH) plays an important role in intracellular protective effects and is currently used for the treatment of cataract and detoxification from metal poisoning. In contrast to GSH, the GSH precursor γ-glutamyl cysteine (γ-EC) has been reported to exhibit neuroprotective effects, thus making it an attractive key biological protective molecule. However, its characteristics are largely unknown. Here, we evaluated the ACE inhibitory function of γ-EC and its mechanism by comparing it with that of GSH in vitro. ACE inhibitory analysis showed that the IC50 of GSH and γ-EC against ACE were 8.3 μM and 3.9 × 102 μM, respectively. These data suggested that γ-EC exerted ACE inhibitory activity, but it was weaker than that of GSH. Docking simulation showed that the ACE inhibitory activity of both compounds was due to the interaction of their carboxyl groups of Glu with Zn2+ in the active center of ACE. Moreover, GSH could fit more compactly in the pocket of ACE, forming more hydrogen bonds with the enzyme than γ-EC. By analyzing its kinetics and in vivo efficacy, we hope that γ-EC could be used as a promising compound for lowering blood pressure in applications with moderate activity, such as functional foods.

Methotrexate Induces Hyperplasia of Enterochromaffin Cells in Mouse Jejunum Vol.4, No.4, p.112-115
Takuji Machida , Airi Tanaka , Megumi Kobayashi , Kohei Imai , Masahiko Hirafuji , Kenji Iizuka
Received: May 12, 2021
Accepted: July 26, 2021
Released: August 13, 2021
Abstract Full Text PDF[1M]

5-Hydroxytryptamine (5-HT) is synthesized by L-tryptophan hydroxylase (TPH) and is stored mainly in enterochromaffin cells of the mucosal epithelium. We previously reported that administration of methotrexate, an anticancer agent, to rats caused hyperplasia of enterochromaffin cells, and nitric oxide (NO) might be involved in the underlying mechanism. The aim of this study was to clarify the effect of methotrexate on hyperplasia of enterochromaffin cells in mice. C57BL/6J mice were intraperitoneally injected with methotrexate or saline as a control. Methotrexate caused an increase in the number of TPH-expressing cells (i.e., enterochromaffin cells) in the jejunum. Methotrexate also increased inducible, but not constitutive, NOS mRNA expression. Our results indicate that methotrexate potentiates 5-HT synthesis in mice, as we previously found in rats.

Regular Article
Elevated Expression of CELSR1 is Associated with Peritoneal Metastasis in Human Scirrhous Gastric Cancers Vol.4, No.4, p.103-111
Toshifumi Hara , Yuri Nomura , Masaaki Hattori , Keichiro Mihara , Kazuyoshi Yanagihara , Yoshifumi Takei
Received: July 17, 2021
Accepted: July 19, 2021
Released: August 13, 2021
Abstract Full Text PDF[4M]

Cancer metastasis is the leading cause of death in patients with any type of solid cancer. To develop effective cancer treatments, it is essential to understand the molecular mechanisms underlying cancer metastasis. Previously we established peritoneal metastasis cell models derived from human scirrhous gastric cancer patients. In this article, we focus on the CELSR1 gene, which is involved in Wnt signaling but whose association with peritoneal metastasis is still unclear. We unveiled gene alterations and the prognostic relevance of the CELSR1 gene in cancer patients by analyzing public resources for cancer genomic and patient cohorts. RT-qPCR and immunoblot analyses revealed that CELSR1 expression was significantly elevated in our cell lines, which had high peritoneal metastatic property, compared with their parental cell lines, which had lower peritoneal metastatic property. Some of the gene alterations in the coding region of CELSR1 were observed in our metastatic cell lines, but they were not associated with the metastatic property or with patient prognosis. Knockdown of CELSR1 via the shRNA technique significantly decreased migration and invasion in the cell lines having high peritoneal metastatic property, whereas the knockdown did not significantly affect proliferation. These results show that CELSR1 plays an important role in peritoneal metastasis and that CELSR1 is a novel peritoneal metastasisassociated gene. The results also suggest that CELSR1 is a proper molecular target for therapy against peritoneal metastasis of scirrhous gastric cancers.

Immunomodulatory Activity of an Aqueous Extract of Mycelium of an Endophytic-Like Fungus, Ustilago esculenta Vol.4, No.4, p.98-102
Makoto Tsuiji , Junko Nagasaka , Yoshimasa Yajima , Keita Nishiumi , Yasutaka Kojima , Miho Amemiya , Wakana Ogura , Takaaki Moriyama , Tomoko Sakai , Teruaki Oku , Tsutomu Tsuji
Received: March 08, 2021
Accepted: June 30, 2021
Released: July 30, 2021
Abstract Full Text PDF[1M]

An aqueous extract of mycelium of Ustilago esculenta, an endophytic-like fungus, was intraperitoneally administered to mice with a model antigen (NP-BSA) and aluminum adjuvant (Alum). Blood was periodically collected from the tail veins of the mice, and the antibody titer against NP-BSA and/or BSA in the serum was measured by ELISA. The aqueous U. esculenta extract had no significant effect on the IgM and IgG antibody titers in the primary response but greatly lowered those in the secondary response. When the antibody titers of the IgG subtypes were separately measured, the ratios of IgG2b/IgG1 and IgG2c/IgG1 titers in the mice treated with the U. esculenta extract were markedly lower than those in the control mice treated with NP-BSA/Alum. The Bio-PlexTM cytokine assay revealed that the concentrations of interferon-γ (IFN-γ) and interleukin-12 (IL-12 (p70)) in peripheral blood from U. esculenta extract-treated mice were decreased as compared with those from the control mice three weeks after the initial immunization. These results suggest that the aqueous extract of mycelium of U. esculenta suppressed antibody production in the secondary response and that changes of the Th1/Th2 balance were involved in this process.

Regular Article
Suramin Inhibits Mitochondrial ADP/ATP Carrier, Not Only from the Cytosolic Side But Also from the Matrix Side, of the Mitochondrial Inner Membrane Vol.4, No.3, p.92-97
Yoshinobu Fujiwara , Takeshi Ito , Atsumi Toiyama , Takenori Yamamoto , Naoshi Yamazaki , Mitsuru Shindo , Yasuo Shinohara
Received: May 12, 2021
Accepted: June 01, 2021
Released: June 17, 2021
Abstract Full Text PDF[1M]

Suramin was earlier reported to show inhibitory effects on the mitochondrial ADP/ATP carrier. However, two important questions, i) whether it shows a specific inhibition of the ADP/ATP carrier when applied to isolated mitochondria, and ii) whether it inhibits the mitochondrial ADP/ATP carrier only from the cytosolic side or from the matrix side, as has been observed with its canonical inhibitors of carboxyatractyloside or bongkrekic acid, remain to be answered. In the present study, we sought exact answers to these questions. As for the first question, suramin showed certain inhibitory effects on the mitochondrial respiratory chain; and at a concentration of 25 μM it showed strong inhibition of the mitochondrial ADP/ATP carrier. This property was due to its weaker inhibitory effects on the mitochondrial ADP/ATP carrier than those of carboxyatractyloside or bongkrekic acid. As for the second question, suramin inhibited the ADP/ATP carrier from both sides of the mitochondrial inner membrane. Thus, suramin was concluded to be utilizable as a new type of inhibitor for the ADP/ATP carrier; but we must pay attention to its side-effects, especially when it is applied to whole mitochondria.

Regular Article
Increased Expression of Heparan Sulfate 6-O-Sulfotransferase-2 Promotes Collagen Production in Cardiac Myofibroblasts Vol.4, No.3, p.85-91
Kotaro Kasai , Yuma Horii , Takanori Hironaka , Kyosuke Mae , Tomoyuki Ueno , Akiomi Nagasaka , Michio Nakaya
Received: April 22, 2021
Accepted: May 14, 2021
Released: June 07, 2021
Abstract Full Text PDF[1M]

Fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) proteins. These excessive ECM proteins are produced by myofibroblasts, which are differentiated mainly from resident fibroblasts in response to tissue injury. In addition to the ECM proteins, the amounts of heparan sulfate, one of the sugar chains, and the proteoglycans attached with heparan sulfate chains are reported to be increased in the fibrotic tissues. However, the contribution of heparan sulfate and heparan sulfate proteoglycans to the development of fibrosis remains unclear. In this study, we found that heparan sulfate 6-O-sulfotransferase-2 (Hs6st2), a type of heparan sulfate transferase, is remarkably induced during fibrosis in the heart, liver, and kidney of mice. We also demonstrated that Hs6st2 was specifically expressed in myofibroblasts of mice with cardiac and liver fibrosis. Hs6st2 knockdown in cardiac myofibroblasts reduced the mRNA expression of fibrosis-related factors, such as Collagen1a1. In summary, this study revealed that Hs6st2 is specifically expressed in myofibroblasts in fibrotic tissues, promotes fibrosis, and can be a good target for the treatment for fibrosis.

Regular Article
Verification of MA-T Safety and Efficacy Against Pathogens Including SARS-CoV-2 Vol.4, No.3, p.78-84
Takekatsu Shibata , Ryuta Urakawa , Chikako Ono , Yukihiro Akeda , Takayoshi Sakai , Shigeto Hamaguchi , Kiyoto Takamori , Tsuyoshi Inoue , Kazunori Tomono , Kiyoshi Konishi , Yoshiharu Matsuura
Received: March 17, 2021
Accepted: May 11, 2021
Released: May 26, 2021
Abstract Full Text PDF[731K]

Matching transformation system (MA-T) is an on-demand aqueous chlorine dioxide solution. It is a disinfectant developed to maximize the safety of chlorine dioxide radical in water and its effectiveness against various microorganisms. In this study, we examined the safety and effectiveness of MA-T for its use in various infectious disease countermeasures, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and consider if MA-T can be implemented in society. To validate the safety of MA-T, we conducted safety tests and efficacy tests in accordance with GLP-based reliability criteria. To evaluate the efficacy, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) confirmation tests against various bacteria, and virus inactivation test against various viruses including SARS-CoV-2 by TCID50 method were performed. The results of safety tests showed that MA-T was at least as safe as Japanese tap water. As a result of efficacy tests for microorganisms, MA-T was effective against many bacteria. Efficacy tests for virus showed that MA-T inactivates SARS-CoV-1, Middle East respiratory syndrome coronavirus (MERS-CoV), rotavirus A (RVA), hepatitis C virus (HCV), dengue virus (DENV), and hepatitis B virus (HBV). MA-T also inactivated 99.98% of SARS-CoV-2, which is equivalent to ethanol for disinfection. MA-T has proven to be a safe and effective disinfectant. MA-T is a next-generation disinfectant that has the potential to be safer and more effective than conventional chlorine disinfectants and other disinfectants. It also proved to be an effective disinfectant against SARS-CoV-2, which is currently causing pandemic all over the world.

Effect of Cadmium on the Expression of ABCB1 Transporter in Human Proximal Tubular Cells Vol.4, No.2, p.74-77
Chikage Mori , Jin-Yong Lee , Maki Tokumoto , Masahiko Satoh
Received: March 15, 2021
Accepted: April 01, 2021
Released: April 20, 2021
Abstract Full Text PDF[1M]

Cadmium (Cd) is an ecotoxic heavy metal that predominantly causes renal failure. Proximal tubular cell damage is typical of chronic Cd toxicity. Proximal tubular cells play important roles in maintaining a stable balance of body chemicals through the functions of various transporters. ABC transporter subfamily B member 1 [ABCB1; also called MDR1 (multiple drug resistance 1)], one of the ATP binding cassette (ABC) multidrug efflux transporters, is expressed in the proximal tubular epithelium and is involved in the extracellular clearance of various chemicals. In this study, we demonstrate that Cd significantly increases the levels of ABCB1 mRNA and P-glycoprotein protein (the ABCB1 gene product) in the HK-2 human proximal tubular cells. Our results suggest that Cd affects the transportation function in the proximal tubules. However, ABCB1 knockdown did not affect Cd toxicity in the HK-2 cells. Therefore, the Cd-induced increase in ABCB1 may affect the transportation function in the kidney but not the Cd toxicity.

Time-Dependent Changes in the Gene Expression Levels in the Mouse Kidney by Long-Term Exposure to Cadmium Vol.4, No.2, p.69-73
Jin-Yong Lee , Chikage Mori , Maki Tokumoto , Masahiko Satoh
Received: February 16, 2021
Accepted: March 10, 2021
Released: April 12, 2021
Abstract Full Text PDF[1M]

Cadmium (Cd) is an environmental hazardous heavy metal that causes renal dysfunction triggered by its toxicity to proximal tubular cells. Our previous study demonstrated that Cd changed the activities of various transcription factors (TFs) in the mouse kidney. In this study, we investigated whether long-term exposure to Cd affected the expression levels of downstream genes of these TFs. C57BL/6J female mice were fed chow containing 300 ppm Cd for 12 months. After 4, 8, and 12 months of Cd exposure, total RNA was extracted from the mouse kidney. The results confirmed that Cd exposure dramatically increased the expression of metallothionein-2 (Mt2) in the mouse kidney. Cd exposure increased the mRNA levels of Slc13a1, Vegfa, and Vegfb among the downstream genes regulated by Cd-activated TFs. Thy1 expression was decreased by Cd exposure, even though the upstream TF was activated by Cd. Furthermore, Cd exposure decreased the mRNA levels of Agtrap, Tert, Fgfr4, Foxq1, Abcb1b, Cd274, Pck1, and Egr1 among the downstream genes regulated by Cd-suppressed TFs. The expression of Pklr increased at 4-month Cd exposure, but decreased at 12-month exposure. Although our previous study indicated Cd exposure suppressed the retinoic acid receptor TF in the mouse kidney, in the present study, it was found that the downstream gene Tnfrsf10b was up-regulated by Cd exposure. For many of the genes whose expressions were affected by long-term Cd exposure, the relationship with Cd renal toxicity has not been reported so far. Our results may provide useful clues into the molecular mechanism of Cd renal toxicity.

Regular Article
Extracellular Adenosine Induces IL-6 Production through Activation of A2B Receptor and Epidermal Growth Factor Receptor in Human Keratinocyte HaCaT Cells Vol.4, No.2, p.64-68
Ken Watanabe , Sei-ichi Tanuma , Mitsutoshi Tsukimoto
Received: January 12, 2021
Accepted: March 15, 2021
Released: March 25, 2021
Abstract Full Text PDF[1M]

Epidermal cells produce cytokines as a part of the body’s response to various external stimuli. Though extracellular ATP-induced activation of P2 receptors is involved in cytokine production in epidermal cells, it is not known whether activation of P1 receptors by extracellular adenosine leads to IL-6 production in epidermal cells. Here, we show that activation of adenosine A2B receptor induces IL-6 production via phosphorylation of epidermal growth factor receptor (EGFR) in human keratinocyte HaCaT cells. We found that treatment of HaCaT cells with 100 μM adenosine or with A2B receptor-specific agonist BAY60-6583 induced IL-6 production, and the production of IL-6 was suppressed by pretreatment with A2B receptor-specific antagonist PSB603. Adenosine- induced IL-6 production was also suppressed by A2B receptor knockdown. In addition, adenosine- and BAY60-6583-induced IL-6 production was suppressed by treatment with EGFR antagonist AG1478. Furthermore, adenosine and BAY60-6583 induced EGFR phosphorylation, and this phosphorylation was suppressed by A2B receptor knockdown. Thus, our data indicate that the A2B receptor-EGFR pathway has a role in IL-6 production. This in turn suggests that extracellular adenosine is involved in skin inflammation.

Implication of NF-κB Activation on Ozone-Induced HO-1 Activation Vol.4, No.2, p.59-63
Sumihito Togi , Misa Togi , Satoshi Nagashima , Yuichi Kitai , Ryuta Muromoto , Jun-ichi Kashiwakura , Toshiaki Miura , Tadashi Matsuda
Received: February 05, 2021
Accepted: March 09, 2021
Released: March 25, 2021
Abstract Full Text PDF[2M]

The controlled and moderate oxidative stress such as ozone induces both inflammatory and anti-inflammatory response. This balance is important for homeostasis of living organisms. Furthermore, it has been shown that this conflict response is mainly regulated by two transcriptional factors, nuclear transcriptional factor κB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2). NF-κB is involved in inflammatory responses by regulating expression of cyclooxygenase-2 (COX-2) and various inflammatory cytokines while Nrf2 is involved in anti-inflammatory responses by controlling expression of numerous antioxidant enzymes such as heme oxygenase- 1 (HO-1). We here demonstrate the molecular mechanisms of the crosstalk between NF-κB and Nrf2 activation during the moderate oxidative stress induced by ozone. We first confirmed the activation of NF-κB and Nrf2 signaling during the moderate oxidative stress in HeLa cells. Induction of NF-κB-mediated COX-2 mRNA expression was observed at the early phase after stimulation (30-60 min after ozone treatment). However, induction of HO-1 mRNA expression was observed at the late phase of stimulation (6 h after stimulation). To reveal the crosstalk between NF-κB and Nrf2, we tested whether reduction of NF-κB expression affects ozone-induced Nrf2 activation by knocking down of NF-κB in HeLa cells. Importantly, the HO-1 induction by ozone was remarkably decreased by a reduction in NF-κB expression. These results suggest that the moderate oxidative stress by ozone initially induces NF-κB activation, and this NF-κB activation is required for HO-1 induction at the late phase of the moderate stress.

Relationship Between Affinity of Kn2-7 to CpG DNA and the Ability of Kn2-7 to Enhance Cellular Uptake of CpG DNA by RAW264.7 Cells Vol.4, No.2, p.55-58
Saeka Nishihara , Mayu Wakita , Kiyoshi Kawasaki
Received: January 27, 2021
Accepted: February 13, 2021
Released: March 18, 2021
Abstract Full Text PDF[1M]

Some α-helical antimicrobial peptides enhance the activation of immune cells induced by the recognition of DNA containing unmethylated cytosine-guanine motifs (CpG DNA). We recently found that an α-helical antimicrobial peptide FIKRIARLLRKIF, known as Kn2-7, increased CpG DNA-dependent responses in mouse macrophage- like RAW264.7 cells, and we also found that enhanced cellular uptake of CpG DNA by Kn2-7 was necessary but insufficient to augment CpG DNA-dependent responses. In this study, we clarified the relationship between the affinity of Kn2-7 to CpG DNA and the ability of Kn2-7 to enhance cellular uptake of DNA. Electrophoretic mobility analysis on a polyacrylamide gel revealed that Kn2-7 binds to CpG DNA more effectively than Kn2-7RA in which arginine residues of Kn2-7 were substituted with alanine residues, and also found that Kn2-7 binds to CpG DNA less effectively than Kn2-7KR in which lysine residues of Kn2-7 were substituted with arginine residues. The DNA-binding abilities of Kn2-7, Kn2-7RA, and Kn2-7KR correlated well with their abilities to enhance the cellular uptake of CpG DNA. In contrast, Kn2-7LA in which leucine residues of Kn2-7 were substituted with alanine residues exhibited a similar DNA-binding ability to Kn2-7, but it did not enhance cellular uptake of CpG DNA. Our results indicate that affinity to DNA is necessary for the ability of Kn2-7 to enhance cellular uptake of CpG DNA, but hydrophobicity of Kn2-7 is also necessary for the enhancement of cellular uptake.

Regular Article
Methionine is a Key Regulator in the Onset of Atopic Dermatitis in NC/Nga Mice Vol.4, No.1, p.47-54
Takayuki Koga , Fuka Hirayama , Tomomitsu Satoh , Yuji Ishii , Nobuhiro Kashige , Makoto Hiromura , Fumio Soeda , Akihisa Toda
Received: October 30, 2020
Accepted: January 23, 2021
Released: February 10, 2021
Abstract Full Text PDF[1M]

Atopic dermatitis (AD) is a skin disorder that presents with itching and scratching and frequently progresses to a chronic state. AD often develops in patients with an individual or family history of allergic diseases. In addition, AD may develop in patients exposed to environmental stimuli such as air pollutants or dust. However, there can be differences in the magnitude of symptoms between patients even with the same genetic background or exposure to similar environmental conditions. NC/Nga mice have been used as a model for AD. They show AD-like symptoms in an age-dependent manner, even in the absence of AD-inducing agents. In addition, similar to humans, the magnitude of AD symptoms in this model varies between individual mice. However, the mechanisms underlying these differences are unclear. In addition, little is known about the relationship between AD skin symptoms and other organs and tissues. Here, we performed a metabolome analysis on sera from NC/Nga mice to identify factors potentially related to the severity of AD symptoms. The analysis showed a correlation between reduced serum methionine levels and increased severity of AD. In addition, treatment with excess methionine before the onset of AD symptoms suppressed the development of AD. In contrast, administration of methionine after the onset of AD symptoms did not. Importantly, cysteine and taurine, irreversible metabolites of methionine, did not suppress AD development. These results show that methionine, but not its metabolites, is a key factor in the onset, rather than the development of AD.

Browning Effect of Brominated Flame Retardant, TBBP-A, on Undifferentiated Adipocytes Vol.4, No.1, p.41-46
Masahiro Yamasaki , Shinya Hasegawa , Masahiko Imai , Tetsuya Fukui , Noriko Takahashi
Received: December 19, 2020
Accepted: January 27, 2021
Released: February 08, 2021
Abstract Full Text PDF[2M]

Recent studies have suggested that exposure to brominated flame retardants (BFR) may play a pivotal role in the development of high-fat diet-induced obesity and metabolic disorders in the liver. Ketone bodies produced by β-oxidation are utilized by acetoacetyl-CoA synthetase (AACS), a cytosolic ketone body-utilizing enzyme. Previously, we reported that the gene expression of AACS is upregulated in high-fat diet-induced obesity. Here, we examined the effects of BFR, tetrabromobisphenol A (TBBP-A), on gene expression in adipocyte cell line (ST-13). Treatment of differentiated cells with TBBP-A for 48 h did not have any remarkable effects on lipid accumulation and mRNA expression of AACS, PPAR-γ, SCOT, and FAS, whereas in undifferentiated cells, mRNA expression of for the lipid and ketone body utilizing-factors (AACS, perilipin-1, and FAS) and brown adipose tissue (BAT) related factors (UCPs, PRDM16, CIDEA, and LSD-1) was upregulated. These observations suggest that TBBP-A may dysregulate lipid metabolism in undifferentiated adipocytes during ketone body utilization via AACS.

Inhibitory Effect of Anionic Uremic Toxins and Creatinine on the Renal Transport of Methotrexate in Rats Vol.4, No.1, p.36-40
Yuichi Ichimura , Natsumi Kudoh , Shiho Ito , Masako Oda , Hiroshi Saitoh
Received: June 26, 2020
Accepted: January 25, 2021
Released: February 08, 2021
Abstract Full Text PDF[846K]

Various substances called uremic toxins (UTs) accumulate in the blood of patients with chronic kidney disease (CKD) and induce unfavorable effects on the body. It has been reported that some kinds of UTs are excreted extensively in the urine via renal transporters. This characteristic of UTs often becomes a factor for influencing pharmacokinetics of drugs in CKD patients. Even now, however, information on the interactions between UTs and drugs in the process of renal excretion remains limited. Methotrexate (MTX) is widely used for the treatment of rheumatoid and leukemia. It is known that MTX is predominantly excreted in the urine and that this process is mediated by organic anion transporters (OATs). In this study, we investigated the inhibitory effects of two anionic UTs, indoxyl sulfate (IS) and indoleacetic acid (IA), as well as creatinine (Cr) on the renal transport of methotrexate (MTX) using rat renal cortical slices. IS and IA, both substrates for OATs, significantly inhibited the uptake of 50 μM MTX in a concentration-dependent manner at 0.1 mM and 1 mM. In the presence of 1 mM Cr, a cationic guanidino compound, the uptake of MTX was significantly decreased, indicating that Cr is capable of interfering with OATs. In conclusion, it was suggested that the urinary excretion of MTX is extensively suppressed through interactions via OATs when IS, IA, and Cr exist a high concentrations in the blood of CKD patients.

Regular Article
Rab7-Dependent Endocytic Pathways Play an Important Role in Nutrient Absorption during Pre-Weaning Growth Vol.4, No.1, p.27-35
Aya Takimoto
Received: December 04, 2020
Accepted: December 27, 2020
Released: February 08, 2021
Abstract Full Text PDF[17M]

Enterocytes in neonatal rodent endocytose milk materials as macromolecule and digest them into small nutrient molecules. Bulk endocytic membrane flow sustaining nutrient uptake inevitably brings the plasma membrane into large lysosomes, as known as supranuclear vesicles or apical vacuoles which exhibit a unique morphology in infant enterocytes. Endocytic delivery to the large vacuoles required the function of Rab7 GTPase. The Rab7-deficient neonatal enterocytes became filled with abnormal gigantic vacuoles as they migrated from the intervillus pocket to the distal region of the villi and they became defective in taking up macromolecules. Infant animals lacking Rab7 in enterocytes exhibited growth retardation. These results showed that the Rab7-dependent endocytic pathways play an important role in nutrient absorption during pre-weaning growth.

Regular Article
Relationship Between Serum Potassium, Magnesium, and Calcium in Patients Receiving Cetuximab Therapy Vol.4, No.1, p.22-26
Kaito Yamashiro , Atsushi Hirata , Ryosuke Ota , Fumihiko Ogata , Takehiro Nakamura , Naohito Kawasaki
Received: January 09, 2021
Accepted: January 20, 2021
Released: February 04, 2021
Abstract Full Text PDF[1M]

Cetuximab (Cmab) is known to cause electrolyte abnormalities, including hypomagnesemia, hypokalemia, and hypocalcemia. However, little is known about differences in these electrolyte levels between hypomagnesemia and non-hypomagnesemia group in patients receiving Cmab. Therefore, the aim of this study was to clarify the relationship between these serum electrolyte levels in patients undergoing Cmab therapy. A retrospective study was conducted to investigate patients for advanced or recurrent colorectal cancer and head and neck cancer, treated with a regimen composed of Cmab from 2012 to 2020 at the Kindai University Nara Hospital. A total of 113 patients were identified from the medical records, and 24 patients who met the inclusion criteria were enrolled in this study. In the non-hypomagnesemia group, significant positively correlations were observed between calcium and potassium (p = 0.018), between potassium and magnesium (p = 0.008), and between magnesium and calcium (p = 0.038). Simultaneously, in the hypomagnesemia group, no statistically significant correlations were recorded between these electrolytes. The incidences of hypokalemia, hypomagnesemia, and hypocalcemia were 25.0% (6/24), 29.2% (7/24), and 25.0% (6/24), respectively. Additionally, the onset of hypokalemia was significantly associated with the onset of hypocalcemia (p = 0.009). These data suggest that it is important to monitor these electrolyte levels, especially in patients who received Cmab with combination therapy.

Fiber-Knob Region of Adenovirus Type 5 Vector Promotes Migration of A549 Cells Vol.4, No.1, p.17-21
Junpei Kano , Naoya Koizumi , Airi Terada , Ayana Matsuoka , Takamasa Hirai , Tetsuya Nomura , Hiroyuki Mizuguchi , Naoki Utoguchi
Received: November 05, 2020
Accepted: January 11, 2021
Released: February 02, 2021
Abstract Full Text PDF[1M]

Adenoviral vectors based on adenovirus type 5 (Ad5) are commonly used for gene therapy. The Ad5 fiber-knob region primarily interacts with the coxsackievirus and adenovirus receptor (CAR). Reportedly, when stimulated, this receptor participates in the regulation of cell-to-cell adhesion and cell migration. In oncogene therapy, cell migration can have adverse effects by promoting metastasis and infiltration. Alternatively, cell migration may enhance the therapeutic effect of gene therapy by promoting the healing of injured tissues. However, the effect of binding of the Ad fiber-knob region to CAR of target cells has not been investigated in detail. Therefore, the aim of the present study was to investigate the effects of the Ad5 vectors on cell migration with the use of wound healing and migration assays. The results showed that infection with the Ad5 vectors promoted the migration of A549 cells, as determined quantifiably. Furthermore, when the Ad5 fiber-knob protein was applied to A549 cells, the same results were obtained. Together, the results revealed that binding of the Ad fiber-knob protein to CAR causes cell migration as a functional change in target cells. Studying the effect of the Ad fiber-knob protein will lead to the development of a gene transfer vector with greater safety and therapeutic effects.

Regular Article
Comparative Study of Different Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinase Inhibitors Affecting Lung Cancer Cell Lines Stably Overexpressing EGFR Mutations Vol.4, No.1, p.12-16
Tomohiro Ariyama , Yuichiro Kanno , Sonomi Takizawa , Kiyomitsu Nemoto , Toshihiro Ishii
Received: October 15, 2020
Accepted: January 05, 2021
Released: January 28, 2021
Abstract Full Text PDF[1M]

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective drugs against non-small cell lung cancer (NSCLC) cells harboring common EGFR mutations such as in-frame exon 19 deletions (Del19) and the exon 21 L858R point mutations (L858R). However, currently used EGFR-TKIs are less effective against cells showing uncommon EGFR mutations. Because there is less information available on the sensitivities of the uncommon EGFR mutations, it is important to evaluate the effect of EGFR-TKIs on uncommon mutations. In this study, we sought to establish H1299 NSCLC cell lines stably expressing mutated EGFRs having Del18, G719S, or L861Q that are uncommon mutations as well as Del19 or L858R common mutations, and evaluated whether these cell lines could be applied as a prediction system for the therapeutic effects of EGFR-TKIs. In fact, the levels of phosphorylated EGFR in these cell lines were assessed after treatment with various EGFR-TKIs (4 approved and 4 unapproved drugs). Gefitinib, erlotinib, afatinib, and osimertinib, approved drugs, were effective against Del19, L858R, and L861Q mutations. However, these EGFR-TKIs were less effective against G719S and Del18 mutations. The unapproved drugs neratinib and poziotinib were effective against Del19, L858R, Del18, and L861Q mutations. Interestingly, canertinib and sapitinib had effects against Del19, Del18, and L861Q mutations and no effect against L858R mutation. These results indicate that the established cell lines are suitable for assessing the effects of the EGFR-TKIs on EGFR mutations, including uncommon mutations, and that some of the EGFR-TKIs used are also effective against uncommon mutations.

Regular Article
Sorghum (Sorghum bicolor (L.) Moench) Extract Enhances Thiazolidinedione-Induced 3T3-L1 Preadipocyte Differentiation but Inhibits Adipogenic Genes Vol.4, No.1, p.6-11
Makoto Sakurai , Ikumi Aizu , Marina Tonosaki , Yuko Oba , Mei Nagata , Katsuyoshi Kamiie , Atsuko Masumi
Received: November 20, 2020
Accepted: January 17, 2021
Released: January 28, 2021
Abstract Full Text PDF[1M]

Sorghum bicolor (L.) Moench is known as a healthful food. We examined whether a water-soluble sorghum extract (SE) from S. bicolor has an anti-diabetic effect through a mechanism that improves insulin sensitivity or anti-adipogenesis. Although the treatment of SE did not affect the adipogenesis of 3T3-L1 adipocytes induced by isobutyl methylxanthine/dexamethasone/insulin (MDI), it significantly enhanced MDI/thiazolidinedione (TZD)-induced adipogenesis in 3T3-L1 adipocyte differentiation. Real-time polymerase chain reaction analysis showed that treatment with SE reduced the expression of adiponectin, adipocyte protein 2 (aP2), and resistin in 3T3-L1 adipocyte cells. SE suppressed the expression of transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT enhancer-binding protein α in both MDI- and MDI/TZDs-treated 3T3-L1 adipocytes. SE treatment reduced tumor necrosis factor α protein in cell lysates from MDI-induced 3T3-L1 adipocytes but not those induced by MDI/TZD. Our results suggest that SE can serve as an effective food source that improves insulin sensitivity and an anti-obesity agent.

Regular Article
Ropinirole Prevents Light-Induced Retinal Photoreceptor Damage in Mice Vol.4, No.1, p.1-5
Miruto Tanaka , Yuki Inoue , Yuta Yoshino , Yoshiki Kuse , Norifumi Tanida , Koichi Takahashi , Yohei Miyamoto , Hideaki Hara
Received: November 25, 2020
Accepted: January 08, 2021
Released: January 21, 2021
Abstract Full Text PDF[3M]

Photoreceptor cell death leads to blindness in dry age-related macular degeneration (AMD) patients. However, no current therapy exists to treat drug for dry AMD patients. Here, we investigated the neuroprotective effects of ropinirole, a dopamine agonist used to treat Parkinson’s disease, in vitro and in vivo murine dry AMD models. Ropinirole prevented photoreceptor cell death induced by excessive light exposure in vitro. Next, we exposed mice to intensive white fluorescent light, performed morphological analysis of the outer nuclear layer (ONL), and examined electroretinogram (ERG) recordings. Although light exposure reduced ONL thickness and ERG amplitudes, the oral administration of ropinirole improved ONL thickness and a- and b-wave amplitudes, similar to pramipexole treatment. Furthermore, the continuous administration of ropinirole using an osmotic pump attenuated the decrease in retinal thickness induced by chronic light exposure. These findings indicated that ropinirole represents a novel candidate drug for dry AMD treatment.