Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 46, Issue 7
Displaying 1-24 of 24 articles from this issue
Communication to the Editor
  • Daiki Masukawa, Ryo Takahagi, Yuka Nakao, Yoshio Goshima
    2023 Volume 46 Issue 7 Pages 869-873
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Adrenergic receptors (ADRs) are widely distributed in the peripheral and central nervous systems. We previously reported that L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor of dopamine, sensitizes adrenergic α1 receptor (ADRA1) through a G protein-coupled receptor GPR143. Chimeric analysis, in which the transmembrane (TM) domains of GPR143 were replaced with those of GPR37, revealed that the second TM region was essential for the potentiation of phenylephrine-induced extracellular signal-regulated kinase (ERK) phosphorylation by GPR143. In HEK293T cells expressing ADRA1B, phenylephrine-induced ERK phosphorylation was augmented by the co-expression of GPR143, compared to the mock vector. Immunoprecipitation analysis revealed that a synthetic transactivator of the transcription peptide fused with TM2 of GPR143 (TAT-TM2) disrupts the interaction between GPR143 and ADRA1B. This TAT-TM2 peptide suppressed the augmentation of phenylephrine-induced ERK phosphorylation by GPR143 in HEK293T cells co-expressing ADRA1B and GPR143. These results indicate that the interaction between GPR143 and ADRA1B is required for the potentiation of ADRA1B-mediated signaling by GPR143. The TM2 region of GPR143 is a crucial dimeric interface for the functional coupling between ADRA1B and GPR143.

Regular Articles
  • Michiko Yoshii, Ai Kitazaki, Koichiro Ozawa
    2023 Volume 46 Issue 7 Pages 874-882
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Hypercholesterolemia is a major complication of arteriosclerosis. Mast cells in arteriosclerosis plaques induce inflammatory reactions and promote arterial sclerosis. In this study, we evaluated the pharmacological effects of simvastatin (SV)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors on the degranulation of rat basophilic leukemia (RBL)-2H3 cells, which are commonly used as mast cell models. SV significantly decreased the degranulation induced by three types of stimulation: antigen antibody reaction (Ag-Ab), thapsigargin (Tg) serosal endoplasmic reticulum calcium ATPase (SERCA) inhibitor, and A23187 calcium ionophore. SV had a stronger inhibitory effect on degranulation induced by Ag-Ab stimulation than the other two stimulations. However, SV did not inhibit increase of intracellular Ca2+ concentrations. Mevalonate or geranylgeraniol co-treatment with SV completely prevented the inhibitory effect of SV on the degranulation induced by these stimulations. Immunoblotting results showed that SV inhibited protein kinase C (PKC) delta translocation induced by Ag-Ab but not by Tg or A23187. SV induced a reduction in active Rac1, and actin filament rearrangement. In conclusion, SV inhibits RBL-2H3 cell degranulation by inhibiting downstream signaling pathways, including the sequential degranulation pathway. These inhibitory effects were completely reversed by the addition of geranylgeraniol and might be induced by changes in the translocation of the small guanosine 5′-triphosphatase (GTPase) families Rab and Rho, which are related to vesicular transport PKC delta translocation and actin filament formation, respectively. These changes are caused by the inhibition of HMG-CoA reductase by SV following the synthesis of geranylgeranyl pyrophosphates, which play important roles in the activation of small GTPases, Rab.

  • Xiaoyu Yu, Chun Du, Yifei Cui, Yang Jiang, Di Feng
    2023 Volume 46 Issue 7 Pages 883-892
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Supplementary material

    Ovarian cancer (OC) is one of the most common tumors in female reproductive organs with a five-year survival rate of less than 45%. Metastasis is a crucial contributor to OC development. ETS transcription factor (ELK3), as a transcriptional factor, have been involved in multiple tumor development. However, its role in OC remains elusive. In this study, we observed high expression of ELK3 and AEG1 in human OC tissues. OVCAR-3 and SKOV3 cells were treated with hypoxia to mimic tumor microenvironment in vivo. We found that the expression of ELK3 was significantly increased in cells under hypoxia compared with normoxia. ELK3 knockdown inhibited cell migration and invasion abilities under hypoxia. Moreover, ELK3 knockdown decreased β-catenin expression and inhibited the activation of Wnt/β-catenin pathway in SKOV3 cells under hypoxia. Astrocyte-elevated gene-1 (AEG1) has been reported to promote OC progression. Our results showed that the mRNA level of AEG1 was decreased when ELK3 knockdown under hypoxia. Dural luciferase assay confirmed that ELK3 bound to gene AEG1 promoter (−2005–+15) and enhanced its transcriptional activity under hypoxia. Overexpression of AEG1 increased the migration and invasion abilities of SKOV3 cell with ELK3 knockdown. In the absence of ELK3, the activation of β-catenin was recovered by AEG1 overexpression. To sum up, we conclude that ELK3 promotes AEG1 expression by binding to its promoter. ELK3 could promote migration and invasion of OC cells by targeting AEG1, which provides a potential basis for therapeutic approaches to OC.

  • Takeshi Chiba, Tomoji Maeda
    2023 Volume 46 Issue 7 Pages 893-897
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Supplementary material

    Human milk exosomes (HMEs) enhance intestinal barrier function and contribute to an improvement in inflammation and mucosal injury, such as necrotizing enteritis (NEC), in infants. Here, we aimed to elucidate the intracellular factors involved in HME-induced expression of zonula occludens-1 (ZO-1), a tight junction protein, in Caco-2 human intestinal epithelial cells. HME treatment for 72 h significantly increased transepithelial electrical resistance in these cells. The mean ZO-1 protein levels in cells treated with HME for 72 h were significantly higher than those in the control cells. The mRNA and protein levels of regulated in development and DNA damage response 1 (REDD1) in HME-treated cells were significantly lower than those in the control cells. Although HME treatment did not increase the mechanistic target of rapamycin (mTOR) level in Caco-2 cells, it significantly increased the phosphorylated mTOR (p-mTOR) level and p-mTOR/mTOR ratio. The ZO-1 protein levels in cells treated with an inducer of REDD1, cobalt chloride (CoCl2) alone were significantly lower than those in the control cells. However, ZO-1 protein levels in cells co-treated with HME and CoCl2 were significantly higher than those in cells treated with CoCl2 alone. Additionally, REDD1 protein levels in cells treated with CoCl2 alone were significantly higher than those in the control cells. However, REDD1 protein levels in cells co-treated with HME and CoCl2 were significantly lower than those in cells treated with CoCl2 alone. This HME-mediated effect may contribute to the development of barrier function in the infant intestine and protect infants from diseases.

  • Kazuki Nagashima, Norihiko Fujii, Saori Oka, Atsushi Yamashita, Fumio ...
    2023 Volume 46 Issue 7 Pages 898-906
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Supplementary material

    Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein β-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of β-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean β-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

  • Takaki Kamiya, Daiki Hira, Ryo Nakajima, Kazuha Shinoda, Atsuko Motomo ...
    2023 Volume 46 Issue 7 Pages 907-913
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Tramadol is metabolized by CYP2D6 to an active metabolite, which in turn acts as an analgesic. This study aimed to investigate the impact of CYP2D6 genotype on the analgesic effect of tramadol in clinical practice. A retrospective cohort study was performed in patients treated with tramadol for postoperative pain after arthroscopic surgery for rotator cuff injury during April 2017–March 2019. The impact of CYP2D6 genotypes on the analgesic effects was assessed by the numeric rating scale (NRS) pain scoring and analyzed by the Mann–Whitney U test. Stepwise multiple linear regression analysis was performed to identify predictive factors for the area under the time-NRS curve (NRS-AUC), which was calculated using the linear trapezoidal method. Among the 85 enrolled Japanese patients, the number of phenotypes with CYP2D6 normal metabolizer (NM) and intermediate metabolizer (IM) was n = 69 (81.1%) and n = 16 (18.9%), respectively. The NRS and NRS-AUC in the IM group were significantly higher than those in the NM group until Day 7 (p < 0.05). The multiple linear regression analysis indicated that the CYP2D6 polymorphism was a prediction factor of the high NRS-AUC levels in Days 0–7 (β = 9.52, 95% CI 1.30–17.7). In IM patients, the analgesic effect of tramadol was significantly reduced one week after orthopedic surgery in clinical practice. Therefore, dose escalation of tramadol or the use of alternative analgesic medications can be recommended for IM patients.

    Editor's pick

    Pharmacogenomic (PGx) testing can predict therapeutic responses or adverse effects based on genetic variants and is expected to be the preemptive precision medicine for patient management. Tramadol is metabolized by CYP2D6 to an active metabolite, which in turn acts as an analgesic. From the retrospective cohort study in Japanese patients with postoperative pain after orthopedic arthroscopic surgery, the authors demonstrated that the analgesic effect in the early postoperative period was depended on CYP2D6 gene polymorphism. These results suggested the clinical utility of preemptive treatment based on the PGx test, which can consider dose adjustment or drug change in advance.

  • Tatsuhiro Akaishi, Shohei Yamamoto, Kazuho Abe
    2023 Volume 46 Issue 7 Pages 914-920
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Supplementary material

    Microglia-related neuroinflammation contributes to the pathogenesis of a variety of neurodegenerative disorders such as Alzheimer’s disease. The synthetic flavonoid, 3′,4′-dihydroxyflavonol (3,3′,4′-trihydroxyflavone), has been shown to protect brain or myocardial ischemia reperfusion-induced cell death and prevent the aggregation of amyloid-β protein, a process that causes progressive neurodegeneration in Alzheimer’s disease. Here, we explored the anti-neuroinflammatory ability of 3′,4′-dihydroxyflavonol in lipopolysaccharide (LPS)-activated MG6 microglial cells. 3′,4′-Dihydroxyflavonol attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LPS-induced phosphorylation of mammalian target of rapamycin (mTOR), nuclear factor-κB (NF-κB), and protein kinase B (AKT) (which are all associated with the neuroinflammatory response in microglia) were attenuated by 3′,4′-dihydroxyflavonol treatment. Treatment with the mTOR inhibitor, rapamycin, NF-κB inhibitor, caffeic acid phenethyl ester, or AKT inhibitor, LY294002, also attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LY294002 treatment attenuated LPS-induced phosphorylation of mTOR and NF-κB in MG6 cells. Hence, our study suggests that 3′,4′-dihydroxyflavonol can attenuate the neuroinflammatory response of microglial cells by suppressing the AKT–mTOR and NF-κB pathways.

  • Yasuhiro Mori, Akifumi Yamamoto, Ayumi Nakagawa, Tomohiro Hikima, Akih ...
    2023 Volume 46 Issue 7 Pages 921-928
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
    Advance online publication: May 11, 2023
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    Intravitreal injection therapy of anti-vascular endothelial growth factor (VEGF) antibody or steroids is the mainstream for patients with age-related macular degeneration (AMD). However, since intravitreal injection is invasive administration, side effects such as endophthalmitis are major problems. In this study, we selected eye drops as a non-invasive treatment method, and aimed to develop eye drops that can deliver TAK-593 (VEGF receptor tyrosine kinase inhibitor) to the posterior segment of the eye. Since TAK-593 is a poorly water-soluble drug, the TAK-593 emulsion was formulated. The solubility of TAK-593 in various oils was measured, and the oil used for the emulsion was selected. Furthermore, viscosity enhancers were added to the emulsion in order to improve the drug delivery into the eye. As viscosity enhancer, xanthan gum was selected based on the properties and the viscosity of the emulsion. The delivery of TAK-593 to the posterior eye was increased by the formulation concentration and the addition of viscosity enhancers. In the laser-induced choroidal neovascularization model, TAK-593 emulsion eye drops showed the same angiogenesis-suppression efficacy as anti-VEGF antibody intravitreal injection. From these results, it was revealed that TAK-593 with an effective drug concentration can be delivered to the posterior eye by non-invasive eye drop administration.

  • Xiaojuan Mao, Xiaomin Xie, Jun Ma, Yulin Wei, Zhiyong Huang, Tiantian ...
    2023 Volume 46 Issue 7 Pages 929-938
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Supplementary material

    Chlorogenic acid (CGA), derived from dicotyledons and ferns, has been demonstrated with anti-inflammatory, anti-bacterial, and free radical-scavenging effects and can be used to treat pulmonary fibrosis (PF). However, the specific mechanism by which CGA treats PF needs to be further investigated. In this study, in vivo experiment was firstly performed to evaluate the effects of CGA on epithelial–mesenchymal transition (EMT) and autophagy in bleomycin (BLM)-induced PF mice. Then, the effects of CGA on EMT and autophagy was assessed using transforming growth factor beta (TGF-β) 1-induced EMT model in vitro. Furthermore, autophagy inhibitor (3-methyladenine) was used to verify that the inhibitory mechanism of CGA on EMT was associated with activating autophagy. Our results found that 60 mg/kg of CGA treatment significantly ameliorated lung inflammation and fibrosis in mice with BLM-induced PF. Besides, CGA inhibited EMT and promoted autophagy in mice with PF. In vitro studies also demonstrated that 50 µM of CGA treatment inhibited EMT and induced autophagy related factors in TGF-β1-induced EMT cell model. Moreover, the inhibitory effect of CGA on autophagy and EMT in vitro was abolished after using autophagy inhibitor. In conclusion, CGA could inhibit EMT to treat BLM-induced PF in mice through, activating autophagy.

  • Kenjiro Matsumoto, Mayu Kamide, Kunitoshi Uchida, Mitsuki Takahata, Ru ...
    2023 Volume 46 Issue 7 Pages 939-945
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Transient receptor potential (TRP) channels play a significant role in taste perception. TRP ankyrin 1 (TRPA1) is present in the afferent sensory neurons and is activated by food-derived ingredients, such as Japanese horseradish, cinnamon, and garlic. The present study aimed to investigate the expression of TRPA1 in taste buds, and determine its functional roles in taste perception using TRPA1-deficient mice. In circumvallate papillae, TRPA1 immunoreactivity colocalised with P2X2 receptor-positive taste nerves but not with type II or III taste cell markers. Behavioural studies showed that TRPA1 deficiency significantly reduced sensitivity to sweet and umami tastes, but not to salty, bitter, and sour tastes, compared to that in wild-type animals. Furthermore, administration of the TRPA1 antagonist HC030031 significantly decreased taste preference to sucrose solution compared to that in the vehicle-treated group in the two-bottle preference tests. TRPA1 deficiency did not affect the structure of circumvallate papillae or the expression of type II or III taste cell and taste nerve markers. Adenosine 5′-O-(3-thio)triphosphate evoked inward currents did not differ between P2X2- and P2X2/TRPA1-expressing human embryonic kidney 293T cells. TRPA1-deficient mice had significantly decreased c-fos expression in the nucleus of the solitary tract in the brain stem following sucrose stimulation than wild-type mice. Taken together, the current study suggested that TRPA1 in the taste nerve contributes to the sense of sweet taste in mice.

    Editor's pick

    TRP ankyrin 1 (TRPA1) is present in the afferent sensory neurons and is activated by food-derived ingredients, such as Japanese horseradish, cinnamon, and garlic. The present study aimed to investigate the expression of TRPA1 in taste buds, and determine its functional roles in taste perception using TRPA1-deficient mice. TRPA1-immunoreactivities are detectable in the taste nerve. TRPA1 deficiency significantly reduced sweet sensitivity compared to that in WT mice as per the two-bottle preference tests. Authors found that TRPA1 in the taste nerve contributes to the sense of sweet taste in mice.

  • Takayuki Koga, Kie Inoue, Fuka Hirayama, Makoto Hiromura, Kiyonaga Fuj ...
    2023 Volume 46 Issue 7 Pages 946-954
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
    Advance online publication: May 13, 2023
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    Allergic contact dermatitis (ACD) is a common skin disorder caused by contact with allergens. The optimal treatment for ACD is to avoid contact with allergens. However, in some cases, avoiding exposure is not possible when the allergens are unknown. Therefore, establishing treatment methods other than allergen avoidance is important. We previously reported that the continuous administration of methionine, an essential amino acid, in a mouse model of atopic dermatitis alleviated its symptoms. In the present study, we investigated the effect of methionine on a mouse model of ACD caused by 1-fluoro-2,4-dinitrobenzene (DNFB). Differences in the effect of methionine were observed in DNFB-induced ACD model mice based on the mouse strain used. This difference was attributed to the suppression of hepatic dimethylglycine (DMG) production, which is associated with the suppression of hepatic betaine-homocysteine methyltransferase (Bhmt) expression by ACD. Although we did not reveal the mechanism underlying DMG suppression, our study suggests the presence of interactions between the liver and skin in dermatitis, such as the regulation of hepatic metabolic enzyme expression in dermatitis and the alleviation of dermatitis symptoms by the hepatic metabolism status of DMG.

    Editor's pick

    Allergic contact dermatitis is a common dermatitis and is induced by contact with allergens. The authors found that methionine suppressed this dermatitis in some strains of mice. They also found that the strength of the effect depended on the suppression of liver betaine homocysteine methyltransferase (Bhmt) expression by the dermatitis. Although the mechanism has not been elucidated, the authors propose that while dermatitis suppresses liver Bhmt expression in some strains of mice, the fact that the metabolic state of the liver affects the suppression of dermatitis suggests that there is at least a skin-liver interaction, especially a dermatitis-liver interaction.

  • Atsuo Fujito, Keiichi Hiramoto, Masashi Imai, Shota Tanaka, Kazuya Ooi
    2023 Volume 46 Issue 7 Pages 955-963
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
    Advance online publication: May 17, 2023
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    Anticancer drugs exhibit many side effects, including skin pigmentation, which often lowers patient QOL. However, the mechanism of pigmentation caused by anticancer drugs remains unknown. The purpose of this study was to elucidate the mechanism of anticancer drug-induced skin pigmentation using 5-fluorouracil (5-FU), a widely used anticancer drug. Specific pathogen-free, 9-week-old Hos:HRM-2 male mice were intraperitoneally administered 5-FU daily for 8 weeks. Skin pigmentation was observed at the end of the study. Mice treated with 5-FU were also administered inhibitors of cAMP, α-melanocyte-stimulating hormone (α-MSH), and adrenocorticotropic hormone (ACTH) for analysis. Administration of oxidative stress, nuclear factor-kappa B (NF-κB), cAMP, and ACTH inhibitors reduced pigmentation in 5-FU-treated mice. These results indicate that the oxidative stress/NF-κB/ACTH/cAMP/tyrosinase pathway plays an important role in pigmentation in 5-FU-treated mice.

    Editor's pick

    This study aimed to investigate the mechanism underlying the skin pigmentation caused by anticancer drugs using 5-fluorouracil (5-FU), a widely used anticancer drug known to cause this complication. Authors believe our study makes a significant contribution to the literature because anticancer drug-induced skin pigmentation remarkably affects the quality of life of cancer patients, has no established treatment, and has an unknown mechanism. This result provides new insight into the role of the ACTH/cAMP/tyrosinase pathway in 5-FU-mediated skin pigmentation and may help manage this complication.

  • Yusuke Tabuchi, Masayuki Tsujimoto, Kosuke Yamamoto, Ryo Shimizu, Tada ...
    2023 Volume 46 Issue 7 Pages 964-968
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Trastuzumab is a humanized monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2) that is indicated for the treatment of HER2-positive breast cancer. The administration of biologics, such as trastuzumab, frequently causes infusion reactions (IRs) with fever and chills. This study aimed to clarify the risk factors for IRs in trastuzumab therapy. Between March 2013 and July 2022, 227 patients with breast cancer who started trastuzumab therapy were included in this study. The severity of IRs was graded according to the Common Terminology Criteria for Adverse Events, Version 5.0. The incidence of IRs in trastuzumab therapy was 27.3% (62/227). Dexamethasone administration was significantly different between the IR and non-IR groups in patients receiving trastuzumab therapy (univariate analysis, p < 0.001; multivariate analysis, p = 0.0002). Without dexamethasone, the severity of IRs in the pertuzumab combination group (Grade 1, 8/65; Grade 2, 23/65) was significantly higher than that in the non-pertuzumab group (Grade 1, 9/37; Grade 2, 3/37; p < 0.05). Our findings suggest that the risk of IRs is significantly higher in patients not premedicated with dexamethasone in trastuzumab therapy and that the concomitant use of pertuzumab without dexamethasone increases the severity of IRs caused by trastuzumab.

  • Shiro Heitaku, Tomohiko Sasase, Tomohiro Sotani, Mimi Maki, Sohei Kats ...
    2023 Volume 46 Issue 7 Pages 969-978
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is the only enzyme that converts inactive glucocorticoids to active forms and plays an important role in the regulation of glucocorticoid action in target tissues. JTT-654 is a selective 11β-HSD1 inhibitor and we investigated its pharmacological properties in cortisone-treated rats and non-obese type 2 diabetic Goto-Kakizaki (GK) rats because Asians, including Japanese, are more likely to have non-obese type 2 diabetics. Systemic cortisone treatment increased fasting plasma glucose and insulin levels and impaired insulin action on glucose disposal rate and hepatic glucose production assessed by hyperinsulinemic-euglycemic clamp, but all these effects were attenuated by JTT-654 administration. Cortisone treatment also reduced basal and insulin-stimulated glucose oxidation in adipose tissue, increased plasma glucose levels after administration of the pyruvate, the substrate of gluconeogenesis, and increased liver glycogen content. Administration of JTT-654 also inhibited all of these effects. Cortisone treatment decreased basal and insulin-stimulated 2-deoxy-D-[1-3H]-glucose uptake in 3T3-L1 adipocytes and increased the release of free fatty acids and glycerol, a gluconeogenic substrate, from 3T3-L1 adipocytes, and JTT-654 significantly attenuated these effects. In GK rats, JTT-654 treatment significantly reduced fasting plasma glucose and insulin levels, enhanced insulin-stimulated glucose oxidation in adipose tissue, and suppressed hepatic gluconeogenesis as assessed by pyruvate administration. These results demonstrated that glucocorticoid was involved in the pathology of diabetes in GK rats, as in cortisone-treated rats, and that JTT-654 ameliorated the diabetic conditions. Our results suggest that JTT-654 ameliorates insulin resistance and non-obese type 2 diabetes by inhibiting adipose tissue and liver 11β-HSD1.

    Editor's pick

    11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is the only enzyme that converts inactive glucocorticoids to active forms and plays an important role in regulating glucocorticoid action in target tissues. Excess glucocorticoids cause insulin resistance in the liver and adipose tissue. Several 11β-HSD1 inhibitors have been reported, but all have been evaluated in obese diabetes models. In the present study, the authors investigated the pharmacological properties of JTT-654, a selective 11β-HSD1 inhibitor, in cortisone-treated rats and non-obese Goto-Kakizaki rats with type 2 diabetes, because many Asians, including Japanese, have non-obese type 2 diabetes. Information gained from detailed analysis of the mechanism of action of JTT-654 may provide a new therapeutic approach for the treatment of non-obese type 2 diabetic patients.

  • Jing-nan Fu, Shu-chang Liu, Yi Chen, Jie Zhao, Ning Lu, Tao Ma
    2023 Volume 46 Issue 7 Pages 979-986
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
    Advance online publication: May 13, 2023
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    Supplementary material

    The liver is the primary site of inflammation caused by bacterial endotoxins in sepsis, and septic acute liver injury (SALI) is usually associated with poor outcomes in sepsis. Forsythiaside A (FTA), an active constituent of Forsythia suspensa, has been reported to have anti-inflammatory properties, antioxidant properties, and protective properties against neuroinflammation, sepsis, and edema. Therefore, the purpose of the present study was to examine FTA’s potential effects on lipopolysaccharide (LPS)-induced SALI in mice. Our results indicated that pretreatment with FTA significantly attenuated aspartate aminotransferase (AST) and aminoleucine transferase (ALT) levels in plasma, ameliorated histopathological damage, inhibited hepatocyte apoptosis, diminished the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the liver from mice exposed to LPS. Furthermore, our data showed that the administration of LPS resulted in robust endoplasmic reticulum (ER) stress response, as evidenced by glucose-regulated protein 78 (GRP78) upregulation, phosphorylated-protein kinase R-like ER kinase (p-PERK) activation, elF2α phosphorylation, and activating transcription factor 4 (ATF4) and CHOP overexpression in the liver. This, in turn, led to nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation, including the cleavage of caspase-1, secretion of IL-1β, and pyroptotic cell death in the liver specimens. Importantly, the ER stress response induced by the LPS challenge was blocked by FTA administration. Correspondingly, NLRP3 inflammasome activation was significantly ameliorated by the pretreatment with FTA. Thus, we demonstrated that FTA pretreatment could protect mice from LPS-induced SALI, and its protective effects were possibly mediated by inhibiting ER stress response and subsequent NLRP3 inflammasome activation.

  • Toshiyuki Shizuya, Seiko Miyazaki
    2023 Volume 46 Issue 7 Pages 987-996
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    The indications for antineoplastic agents are limited in Japan compared with those in the United States. This may be because it takes longer to add indications and the number of additions of indications is lower in Japan than in the United States. To clarify the differences in the timing and number of additions of indications for antineoplastic agents, the agents approved from 2001 to 2020 and sold as of the end of 2020 in Japan and the United States were identified and their additions of indications were compared. Of the 81 antineoplastic agents analyzed, the proportion of agents with additional indications was 71.6 and 63.0%, and the number of additions of indications (median/average per agent) was 2/3.52 and 1/2.43, for the United States and Japan, respectively. The median date of approval for addition of indications was August 10, 2017 and July 3, 2018 for the United States and Japan (p = 0.015), indicating that the indications were added earlier in the United States. The proportion of priority review and orphan drug designation for addition of indications was lower in Japan (55.6 and 34.7%) than in the United States (80.9 and 57.8%) (p < 0.001). When indications were developed with global clinical trials or designated as orphan drugs in the United States, delays in application and approval in Japan against the United States were small (p < 0.020). New indications for antineoplastic agents should be added promptly for Japanese patients because malignancy is the leading cause of death in Japan.

Notes
  • Ge Liu, Keisuke Obara, Kento Yoshioka, Yoshio Tanaka
    2023 Volume 46 Issue 7 Pages 997-1003
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10−6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10−5 M), diltiazem (10−5 M), and nifedipine (10−7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10−5 M) was strongly suppressed by SKF-96365 (3 × 10−5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10−5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.

  • Masao Nakamura, Akira Shiga, Ami Iimori, Takumi Matsuzaki
    2023 Volume 46 Issue 7 Pages 1004-1009
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
    Advance online publication: April 22, 2023
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    Human lactoferrin (hLF) is a glycosylated globular iron-binding protein with high functional versatility that elicits anticancer, neuroprotective, and anti-inflammatory effects. Some of the diverse functions of hLF are induced after its internalization into various cells via cell surface endocytosis receptors, such as proteoglycans, which contain glycosaminoglycan (GAG) chains. We have previously demonstrated that an hLF derivative comprising the N-terminal half of hLF (referred to as the N-lobe) is internalized by intestinal enterocyte Caco-2 cells. However, the relationship between the intracellular uptake of the N-lobe and its pharmacological activity remains poorly understood. Here, we report that the N-lobe is efficiently internalized by lung cancer cells via endocytic pathways, suppressing their proliferation. Moreover, the N-lobe showed higher intracellular uptake than hLF. We found that the N-lobe was internalized into the human lung cancer cell lines PC-14 and PC-3 via clathrin- and/or caveolae-mediated endocytosis. Intracellular uptake of the N-lobe was inhibited when an equimolar concentration of chondroitin sulfate (CS)-E, a GAG subtype involved in malignant transformation and tumor metastasis, was added. The inhibitory effect of the N-lobe on PC-14 cell proliferation decreased with the addition of CS-E in a dose-dependent manner, suggesting that the CS-recognizing sequence on the N-lobe is necessary for its internalization or that the CS proteoglycan on cancer cells acts as an endocytosis receptor. These results suggest that the efficient endocytic uptake of the N-lobe is important for its antiproliferation effects on lung cancer cell lines. Thus, the N-lobe presents a promising drug candidate for cancer treatment.

  • Satoshi Sato, Hiroyoshi Ariga, Hiroshi Maita
    2023 Volume 46 Issue 7 Pages 1010-1014
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Supplementary material

    Recently, the finding of recurrent mutations in the spliceosome components in cancer has indicated that the spliceosome is a potential target for cancer therapy. However, the number of small molecules known to affect the cellular spliceosome is currently limited probably because of the lack of a robust cell-based approach to identify small molecules that target the spliceosome. We have previously reported the development of a genetic reporter to detect the cellular levels of small nuclear ribonucleoproteins (snRNPs), which are subunits of the spliceosome, using a split luciferase. However, the original protocol was designed for small scale experiments and was not suitable for compound screening. Here, we found that the use of cell lysis buffer used in blue native polyacrylamide gel electrophoresis (BN-PAGE) dramatically improved the sensitivity and the robustness of the assay. Improved assay conditions were used to discover a small molecule that altered the reporter activity. Our method may be used with other cellular macromolecular complexes and may assist in the discovery of small bioactive molecules.

  • Sachi Shibata, Kento Takayama, Ken-ichi Tsurusaki, Shigeyuki Kon
    2023 Volume 46 Issue 7 Pages 1015-1020
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    The Shimanami Leaf®, produced at Innoshima Island in Onomichi City, Hiroshima Prefecture, Japan, is a leafy vegetable that does not require pesticide use and has a high nutritional value. Although the leaf has abundant dietary fiber and other nutrients, reports on its biological regulatory functions are lacking. Therefore, this study aimed to elucidate the effects of Shimanami leaf intake on bowel movement and gut microbiota in mice. We examined the effects of Shimanami leaves on fecal weight, fecal water content, and intestinal microbiota composition. On day 10 of administration, the Shimanami leaf-treated group exhibited significantly higher fecal weight and water content than the control group. Next-generation sequencing analysis revealed that the ingestion of Shimanami leaf increased the abundances and diversity of intestinal bacteria, including members from Lactococcus, Streptococcus, and Muribaculaceae. Our findings suggest that Shimanami leaf supplementation improves bowel movement and promotes defecation.

  • Tomohiro Hikima, Haruhiko Ezaki
    2023 Volume 46 Issue 7 Pages 1021-1023
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    We aimed to investigate eye damage caused by ocular iontophoresis (IP) based on an in vitro eye irritation test using a reconstructed human corneal cell. In this study, the LabCyte CORNEA-MODEL was selected as the reconstructed corneal cell. The test procedure was performed according to Test Guideline No.492 of the Organisation for Economic Co-operation and Development, which was partially revised for the IP. From the relationship between the cell viability of the cornea model and the electric field intensity [current density (mA/cm2) × application time (min)] of the IP, we predicted that the intensity values of 465 mA/cm2 × min and 930 mA/cm2 × min caused reversible eye irritation and irreversible eye damage, respectively. However, further studies are required to improve the accuracy and reproducibility of the prediction. This report provides essential knowledge on the clinical safety of ocular IP.

  • Hiroshi Akiyama, Airi Ishibashi, Tsuyoshi Kai, Ayaka Kikuchi, Takaaki ...
    2023 Volume 46 Issue 7 Pages 1024-1026
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    It is necessary to evaluate the efficiency of reduction for cyanide and cyanoglycosides during the manufacturing process from raw material beans to sweetened bean paste in a food hygiene control system from the viewpoint of food safety. Analytical methods for cyanide and cyanoglycoside determination in sweetened bean paste by HPLC with fluorescence detection were developed. In analysis of collection time of free cyanide in the free cyanide assay, the recovery was improved by extending the collection time, the recovery rate was >80% by 2 h. The accuracy, repeatability and intra-laboratory precision of the free cyanide assay were 82.3, 2.0, and 2.4%, respectively. The method for cyanoglycoside analysis was evaluated by 5 repeated spiked recovery experiments at a concentration of 10 ppm. The accuracy, repeatability and intra-laboratory precision of the cyanoglycoside method were 82.2, 1.9, and 3.4%, respectively. These analytical methods will enable the analysis of cyanide and cyanoglycosides in sweetened bean paste without using steam distillation method in the pretreatment.

  • Osamu Nakagawasai, Kotaro Yamada, Kohei Takahashi, Yuko Oshima, Sena H ...
    2023 Volume 46 Issue 7 Pages 1027-1030
    Published: July 01, 2023
    Released on J-STAGE: July 01, 2023
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    Globin digest (GD) inhibits dietary hypertriglyceridemia; however, its effects on physical fatigue remain unknown. Therefore, this study aimed to investigate the potential anti-fatigue effects of GD. Repeated administration of GD and valine (Val)-Val-tyrosine (Tyr)-proline (Pro), a component of GD, for five days prevented the forced walking-induced decrease in locomotion. Furthermore, GD treatment reversed the forced walking-induced increase in blood lactate levels in mice and increased phosphorylated AMP-activated protein kinase (p-AMPK) in the soleus muscle, suggesting that the anti-fatigue effect of GD involves AMPK activation in the soleus muscle through reduced blood lactate.

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