Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 40 , Issue 4
Showing 1-24 articles out of 24 articles from the selected issue
Review
  • Kotoku Kawaguchi, Saori Yoshida, Ryo Hatano, Shinji Asano
    2017 Volume 40 Issue 4 Pages 381-390
    Published: April 01, 2017
    Released: April 01, 2017
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    Ezrin/radixin/moesin (ERM) proteins function as general cross-linkers between plasma membrane proteins and the actin cytoskeleton and are involved in the functional expression of membrane proteins on the cell surface. They also integrate Rho guanosine 5′-triphosphatase (GTPase) signaling to regulate cytoskeletal organization by sequestering Rho-related proteins. They act as protein kinase A (PKA)-anchoring proteins and sequester PKA close to its target proteins for their effective phosphorylation and functional regulation. Therefore, ERM proteins seem to play important roles in the membrane transport of electrolytes by ion channels and transporters. In this review, we focus on the pathophysiological roles of ERM proteins in in vivo studies and introduce the phenotypes of their knockout and knockdown mice.

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Regular Articles
  • Tohru Sakai, Miyuki Ohhata, Misaki Fujii, Sayaka Oda, Yasuna Kusaka, M ...
    2017 Volume 40 Issue 4 Pages 391-395
    Published: April 01, 2017
    Released: April 01, 2017
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    Propolis is a bee product with various biological properties. C57BL/6 mice were fed a high-fat diet and treated with propolis for 14 weeks. Body weight in mice treated with 2% propolis was less than that in control mice from 3 weeks after the start of treatment until 14 weeks except for the 7th week. Mice treated with propolis showed significantly lower epididymal fat weight and subcutaneous fat weight. Infiltration of epididymal fat by macrophages and T cells was reduced in the propolis group. Supplementation of propolis increased feces weight and fat content in feces, suggesting that mechanisms of weight reduction by propolis partly include a laxative effect and inhibition of fat absorption.

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  • Akiko Matsuyama, Shingo Takatori, Yoko Sone, Eiko Ochi, Mitsuhiro Goda ...
    2017 Volume 40 Issue 4 Pages 396-401
    Published: April 01, 2017
    Released: April 01, 2017
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    Angiogenesis, which is the generation of new vascular networks from existing blood vessels, occurs under normal and pathophysiological conditions. Perivascular nerves, which innervate mature vasculatures, maintain vascular tone and regulate tissue blood flow. However, little is known whether perivascular nerves innervate newborn blood vessels. Therefore, the aim of this study was to investigate the distribution and characterization of perivascular nerves in neovasculatures, which were generated by the mouse corneal micropocket method. Under anesthesia, a pellet containing basic fibroblast growth factor (bFGF) (100 ng/pellet) was implanted into a mouse cornea in one side of the eyeball. Nerve growth factor (NGF) was locally (2 or 20 ng) applied with the pellet, or subcutaneously (40 ng/h for 7 d) administered with an osmotic mini-pump. After the implantation, vascular endothelial cells, smooth muscle cells, and perivascular nerves in the cornea were immunohistochemically studied. Neovessels generated from existing limbal vessels were observed in pellet-implanted cornea. Immunostaining of neovasculatures showed the presence of CD31-like immunoreactive (LI) endothelial cells and α-smooth muscle actin-LI vascular smooth muscles. Perivascular nerves immunostained by protein gene product (PGP) 9.5, an axonal marker, were found in the existing limbal vessels, but they were not observed in neovasculatures. Local and subcutaneous treatment of NGF inhibits bFGF-derived angiogenesis and resulted in loop-shaped vessels that had many anastomoses, and produced innervation of PGP 9.5-LI perivascular nerves around bFGF-derived neovessels. These findings suggest that neovasculatures have no innervation of perivascular nerves, and that NGF facilitates innervations of perivascular nerves to regulate the blood flow in neovessels.

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  • Yasuhiro Yamamoto, Hiromi Koma, Sayaka Nishii, Tatsurou Yagami
    2017 Volume 40 Issue 4 Pages 402-412
    Published: April 01, 2017
    Released: April 01, 2017
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    Heat shock protein 70 (Hsp70) is not only a molecular chaperone in cytosol, but also presents in synaptic plasma membranes. To detect plasmalemmal Hsp70 (pl-Hsp70), neurons were immunostained with anti-Hsp70 antibody without permeabilization and fixation. Dotted immunofluorescent signals at neuronal cell bodies and neurites indicated the localization of Hsp70 on the neuronal cell surface. To target only pl-Hsp70, but not cytosolic Hsp70, the anti-Hsp70 antibody was applied without permeabilization in the primary culture of rat cortical neurons. The antibody induced neuronal cell death in a concentration-dependent manner. The anti-Hsp70 antibody activated ubiquitin-proteasome pathway, but inactivated caspase-3. A lag time was required for the neurotoxicity of anti-Hsp70 antibody. Hydrogen peroxide was increased in the anti-Hsp70 antibody-treated neurons during the lag time. Catalase suppressed the anti-Hsp70 antibody-reduced cell viability via the plausible inhibition of hydrogen peroxide generation. One of down-streams of hydrogen peroxide exposure is activation of the mitogen-activated protein kinase (MAPK) signaling cascade. The neurotoxicity of anti-Hsp70 antibody was partially ascribed to c-Jun N-terminal kinase among MAPKs. In conclusion, the anti-Hsp70 antibody targeted pl-Hsp70 on the neuronal cell surface and induced neuronal cell death without complement. Furthermore, hydrogen peroxide appeared to mediate the neuronal cell death, which was accompanied with the enhancement of the ubiquitin–proteasome pathway and the suppression of caspase in a different fashion from the known cell death.

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  • Akiyoshi Kikkawa, Yoshihisa Kitamura, Tetsuya Aiba, Koichi Hiraki, Tos ...
    2017 Volume 40 Issue 4 Pages 413-418
    Published: April 01, 2017
    Released: April 01, 2017
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    Lamotrigine has acute antidepressant effects in patients with bipolar disorder. However, there is little information regarding appropriate serum levels of lamotrigine and the time until remission after the start of lamotrigine therapy in patients with bipolar II depression. This was a naturalistic and unblinded prospective pilot study. Twelve patients’ depressive symptoms were evaluated using the Montgomery–Åsberg Depression Rating Scale (MADRS) at the start of treatment and at the time of remission, and blood samples were obtained at the time of remission. Mahalanobis distance was used to analyze the relationship between the MADRS improvement rate and the serum lamotrigine level. Furthermore, we calculated the Spearman’s rank correlation coefficient for the relationship between the MADRS improvement rate and the serum lamotrigine level, and produced box plots of the serum lamotrigine level at remission and the time until remission. The Mahalanobis distance for the patient that was co-administered lamotrigine and valproic acid differed significantly from those of the other patients (p<0.001). There was no linear relationship between the serum lamotrigine level and the MADRS improvement rate among the patients that did not receive valproic acid. The median time from the start of lamotrigine therapy until remission was 6 weeks. The serum lamotrigine level does not have an important impact on the acute therapeutic effects of lamotrigine on bipolar II depression. In addition, we consider that different treatment options should be considered for non-responders who do not exhibit any improvement after the administration of lamotrigine for approximately 6 weeks.

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  • Mun Hwan Oh, Hye Jin Lee, Sang Hyun Jo, Bernie Byunghoon Park, Sae-Bom ...
    2017 Volume 40 Issue 4 Pages 419-424
    Published: April 01, 2017
    Released: April 01, 2017
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    The parallel artificial membrane permeability assay (PAMPA) is widely used in early-stage drug discovery to discriminate compounds by intestinal permeability. The purpose of the current study was to establish a cassette (n-in-1) PAMPA to enable permeability screening of lipophilic compounds. A double-sink PAMPA consisting of a pH gradient (i.e., pH 6.5 and 7.4 for the donor and receiver compartments, respectively) and a lipophilic sink (i.e., a surfactant in the receiver solution) was utilized with cassette incubation of 10 reference compounds. Sample analysis was conducted using selected reaction monitoring (SRM) with a triple quadrupole LC-MS/MS system. Correlation between PAMPA permeability and human intestinal absorption (HIA) of the reference compounds yielded two false negatives, namely propranolol (PPN) and verapamil (VER); these two compounds showed a substantially lower recovery (ca. 10%) than other reference compounds (>69%). This cassette PAMPA was repeated subsequently with polysorbate 80 added to the donor compartments, which resulted in a significant increase in both the recovery and the permeability of the false negatives. Accordingly, the permeability class of all reference compounds could be unambiguously differentiated using this cassette PAMPA. Also, a strong linear correlation (r=0.9845) was observed between the cassette and discrete permeability of all reference compounds.

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  • Marlene Raimunda Andreola Perazzoli, Camila Katerin Perondi, Cesar Mil ...
    2017 Volume 40 Issue 4 Pages 425-434
    Published: April 01, 2017
    Released: April 01, 2017
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    Gallic acid (3,4,5-trihydroxybenzoic acid, GA), a natural phenolic acid has been reported as a strong antioxidant. Therefore the present study was designed to evaluate the effects of GA and dodecyl gallate (DGA) against acute and chronic carbon tetrachloride (CCl4)-induced hepatotoxicity. For acute model, rats were orally treated with GA and DGA for 7 d prior to CCl4 by intraperitoneally (i.p.) injection. For the chronic model, rats were orally treated with GA or DGA and CCl4 i.p. twice a week for four weeks. In both acute and chronic models, the CCl4-treated groups showed significantly increase in serum hepatic enzyme activities and histopathologic alterations, as well as a disruption in antioxidative status. In contrast, the treatment with GA and DGA restored serum hepatic enzymes activities, improved histopathologic alterations, increased glutathione (GSH) and decreased lipid peroxidation levels. The activities of liver antioxidant enzymes were increased by GA and DGA only in acute model. The expression of p53 gene increased about 3.5 times after GA and DGA treatments, which could result in cell death of damaged hepatocytes preventing of a lifelong liver failure. Thus, these results suggest that GA and DGA has the potential to prevent liver damages as the case of fibrosis condition.

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  • Li Wang, Ren Zhang, Jian Chen, Qihui Wu, Zaoyuan Kuang
    2017 Volume 40 Issue 4 Pages 435-443
    Published: April 01, 2017
    Released: April 01, 2017
    [Advance publication] Released: January 20, 2017
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    Tumor necrosis factor-alpha (TNF-α) plays an important role in the developing process of inflammatory bowel disease. Tight junction protein zonula occludens-1 (ZO-1), one of epithelial junctional proteins, maintains the permeability of intestinal barrier. The objective of this study was to investigate the mechanism of the protective effect of baicalin on TNF-α-induced injury and ZO-1 expression in intestinal epithelial cells (IECs). We found that baicalin pretreatment significantly improved cell viability and cell migration following TNF-α stimulation. miR-191a inhibitor increased the protective effect of baicalin on cell motility injured by TNF-α. In addition, miR-191a down-regulated the mRNA and protein level of its target gene ZO-1. TNF-α stimulation increased miR-191a expression, leading to the decline of ZO-1 mRNA and protein. Moreover, pretreatment with baicalin reversed TNF-α induced decrease of ZO-1 and increase of miR-191a, miR-191a inhibitor significantly enhanced ZO-1 protein expression restored by baicalin. These results indicate that baicalin exerts a protective effect on IEC-6 (rat small intestinal epithelial cells) cells against TNF-α-induced injury, which is at least partly via inhibiting the expression of miR-191a, thus increasing ZO-1 mRNA and protein levels.

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  • Noriaki Nagai, Fumihiko Ogata, Saori Deguchi, Akina Ueno, Naohito Kawa ...
    2017 Volume 40 Issue 4 Pages 444-450
    Published: April 01, 2017
    Released: April 01, 2017
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    We attempted to design a combination ointment containing solid tranilast nanoparticles and dissolved sericin as a wound-healing drug (TS-combination ointment), and evaluated its usefulness as therapy for wound-healing deficits in streptozotocin-induced diabetic rat (STZ rat) using kinetic analyses as an index. Solid tranilast nanoparticles were prepared by bead mill methods with low-substituted methylcellulose; the mean particle size of the tranilast nanoparticles was 70 nm. The ointment was designed to contain the tranilast nanoparticles plus sericin powder and/or Carbopol® 934. Skin wound healing in STZ rats begins significantly later than in normal rats. Although the skin wound healing rate in STZ rats treated with an ointment containing tranilast nanoparticles was lower than in STZ rats treated with vehicle, the ointment was effective in reducing redness. An ointment containing sericin enhanced the skin-healing rate, but the preventive effect on redness was weak. On the other hand, the combination of tranilast and sericin increased both the skin healing rate and reduction in redness. In conclusion, we have adapted kinetic analyses to skin wound healing in rats, and found these analyses to be useful as an index of wound healing ability by a wound-healing drug. In addition, we show that treatment with the TS-combination ointment enhances the skin wound healing rate and reduces redness. These findings provide information significant to the search for new wound-healing therapies and for the design of wound-healing drugs.

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  • Mai Sotoyama, Shinya Uchida, Shimako Tanaka, Akio Hakamata, Keiichi Od ...
    2017 Volume 40 Issue 4 Pages 451-457
    Published: April 01, 2017
    Released: April 01, 2017
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    Orally disintegrating tablets (ODTs) are formulated to disintegrate upon contact with saliva, allowing administration without water. Olopatadine hydrochloride, a second-generation antihistamine, is widely used for treating allergic rhinitis. However, it has a bitter taste; therefore, the development of taste-masked olopatadine ODTs is essential. Some studies have suggested that citric acid could suppress the bitterness of drugs. However, these experiments were performed using solutions, and the taste-masking effect of citric acid on ODTs has not been evaluated using human gustatory sensation tests. Thus, this study evaluated citric acid’s taste-masking effect on olopatadine ODTs. Six types of olopatadine ODTs containing 0–10% citric acid were prepared and subjected to gustatory sensation tests that were scored using the visual analog scale. The bitterness and overall palatability of olopatadine ODTs during disintegration in the mouth and after spitting out were evaluated in 11 healthy volunteers (age: 22.8±2.2 years). The hardness of the ODTs was >50 N. Disintegration time and dissolution did not differ among the different ODTs. The results of the gustatory sensation tests suggest that citric acid could suppress the bitterness of olopatadine ODTs in a dose-dependent manner. Olopatadine ODTs with a high content of citric acid (5–10%) showed poorer overall palatability than that of those without citric acid despite the bitterness suppression. ODTs containing 2.5% citric acid, yogurt flavoring, and aspartame were the most suitable formulations since they showed low bitterness and good overall palatability. Thus, citric acid is an effective bitterness-masking option for ODTs.

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  • Aimi Watanabe, Kazuhiro Yamamoto, Takeshi Ioroi, Sachi Hirata, Kenichi ...
    2017 Volume 40 Issue 4 Pages 458-464
    Published: April 01, 2017
    Released: April 01, 2017
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    Signal transducer and activator of transcription (STAT) 3 is a key factor in homeostasis of the oral mucosa by regulating the production of inflammatory cytokines. Sunitinib is a substrate of P-glycoprotein (multidrug resistance (MDR)-1/ABCB1) and breast-cancer resistance protein (BCRP/ABCG2). In this retrospective study, we evaluated the association between sunitinib-induced stomatitis and STAT3, ABCB1, and ABCG2 polymorphisms in patients with metastatic renal cell carcinoma (mRCC). Fifty-two Japanese patients with RCC treated with sunitinib were retrospectively genotyped to elucidate a potential association between STAT3, ABCB1, and ABCG2 polymorphisms and stomatitis development. Stomatitis occurred in 22 out of 52 patients. The TT+TC genotypes at STAT3 rs744166 had an odds ratio of 5.00 against CC genotype for the stomatitis development (95% confident interval, 0.97–25.8). In the Kaplan–Meier method for the cumulative incidence of stomatitis, a statistically significant difference was observed between the TT+TC and CC genotypes in STAT3 rs744166 (p=0.037). Both multiple logistic regression analysis and Cox proportional-hazards regression analysis show STAT3 rs744166 TT+TC genotypes and serum creatinine in each patient were significant independent factors for stomatitis development. In conclusion, STAT3 polymorphism may be a novel risk factor for sunitinib-induced stomatitis in patients with mRCC.

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  • Cheng-Niu Wang, Ying-Jie Wang, Hao Wang, Lu Song, Yu Chen, Jin-Liang W ...
    2017 Volume 40 Issue 4 Pages 465-472
    Published: April 01, 2017
    Released: April 01, 2017
    [Advance publication] Released: January 25, 2017
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    Alzheimer’s disease (AD) is a most serious age-related neurodegenerative disorder accompanied with significant memory impairments in this world. Recently, microRNAs (miRNAs) have been reported to be invlolved in the pathophysiology of AD. Previous studies have shown that miRNA-206 (miR-206) is implicated in the pathogenesis of AD via suppressing the expression of brain-derived neurotrophic factor (BDNF) in the brain. Here, we examined the miR-206-3p and miR-206-5p expression in the hippocampus and cortex of Abeta precursor protein (APP)/presenilin-1 (PS1) transgenic mice treated with donepezil, a drug approved for treating AD in clinic. We found that the expression of miR-206-3p was significantly up-regulated in the hippocampus and cortex of APP/PS1 mice, while donepezil administration significantly reversed this dysfunction. In addition, enhancing the miR-206-3p level by the usage of AgomiR-206-3p significantly attenuated the anti-dementia effects of donepezil in APP/PS1 mice. Together, these results suggested that miR-206-3p is involved in the anti-dementia effects of donepezil, and could be a novel pharmacological target for treating AD.

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  • Tsugunobu Andoh, Daisuke Uta, Mitsuru Kato, Kazufumi Toume, Katsuko Ko ...
    2017 Volume 40 Issue 4 Pages 473-478
    Published: April 01, 2017
    Released: April 01, 2017
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    Paclitaxel is a chemotherapeutic agent that causes peripheral neuropathy as its major dose-limiting side effect. However, the peripheral neuropathy is difficult to manage. A study we recently conducted showed that repetitive administration of aucubin as a prophylactic inhibits paclitaxel-induced mechanical allodynia. However, the mechanisms underlying the anti-allodynic activity of aucubin, which is a major component of Plantaginis Semen, was unclear. In addition to mechanical allodynia, aucubin inhibited spontaneous and mechanical stimuli-induced firing in spinal dorsal horn neurons; however, catalpol, a metabolite of aucubin, did not show these effects. Furthermore, paclitaxel induced the expression of CCAAT/enhancer-binding protein homologous protein, a marker of endoplasmic reticulum (ER) stress, in the sciatic nerve and a Schwann cell line (LY-PPB6 cells); however, this effect was inhibited by aucubin. These results suggest that aucubin inhibits paclitaxel-induced mechanical allodynia through the inhibition of ER stress in peripheral Schwann cells.

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  • Zhenzhong Wang, Chao Wang, Ziyu Wu, Jun Xue, Baixin Shen, Wei Zuo, Zen ...
    2017 Volume 40 Issue 4 Pages 479-485
    Published: April 01, 2017
    Released: April 01, 2017
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    Prostatic cancer (PCa) is a leading cause of cancer related death in males and is often regarded as a kind of androgen-sensitive cancer. Artesunate (ART), a semi-synthetic derivative of the Chinese herb Artemisia annua, is such an anti-cancer agent. However, the effects and mechanism of ART on PCa cells remains unclear. The study aims to elaborate the mechanism of the involvement of androgen receptor (AR) in anti-prostatic cancer (PCa) of artesunate (ART). PCa cells 22rvl were used in vivo and in vitro, and the viability and apoptosis were conducted using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, respectively. Ectopic expressions of AR and DNA methyltransferase (DNMT) were detected in cells in overexpression or interference of AR or DNMT3b. ART dose-dependently suppressed tumor growth, inhibited cell viability, enhanced apoptosis, decreased AR expression, and increased the expression and the catalytic activity of DNMT3b in 22rv1 cells either in transplanted mice or in vitro. Furthermore, AR downregulated DNMT3b expression, and overexpression of AR or interference of DNMT3b could reverse ART-induced cytotoxicity and apoptosis in 22rvl cells, whereas overexpression of DNMT3b could not change the effect profiles of ART on the cells. The results indicated that ART suppressed tumor growth of prostatic cancer cells through AR-DNMT3b pathway, underlying ART will allow for the utilization of this Chinese therapeutic agent for the potential treatment of prostate cancer.

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  • Chuan Li, Masahiko Imai, Masahiro Yamasaki, Shinya Hasegawa, Noriko Ta ...
    2017 Volume 40 Issue 4 Pages 486-494
    Published: April 01, 2017
    Released: April 01, 2017
    [Advance publication] Released: January 18, 2017
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    Vitamin A is an essential nutrient that is obtained from the daily diet. The major forms of vitamin A in the body consist of retinol, retinal, retinoic acid (RA), and retinyl esters. Retinal is fundamental for vision and RA is used in clinical therapy of human acute promyelocytic leukemia. The actions of retinol and retinyl palmitate (RP) are not known well. Recently, we found that retinol is a potent anti-proliferative agent against human refractory cancers, including gallbladder cancer, being more effective than RA, while RP was inactive. In the current study, we determined serum retinol concentrations in xenograft mice bearing tumors derived from four refractory cancer cell lines. We also examined the effects of vitamin A on proliferation of human gallbladder cancer cells in vivo. Serum retinol concentrations were significantly lower in xenograft mice with tumors derived from various refractory cancer cell lines as compared with control mice. The growth of tumors was inhibited with increasing serum retinol concentrations obtained post-administration of RP. In addition, pre-administration of RP increased serum retinol concentrations and suppressed tumor growth. These results indicate that administration of RP can maintain retinol concentrations in the body and that this might suppress cancer cell growth and attachment. The regulation of vitamin A concentration in the body, which is critical biomarker of health, could be beneficial for cancer prevention and therapy.

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  • Chuan Li, Masahiko Imai, Shinya Hasegawa, Masahiro Yamasaki, Noriko Ta ...
    2017 Volume 40 Issue 4 Pages 495-503
    Published: April 01, 2017
    Released: April 01, 2017
    [Advance publication] Released: January 18, 2017
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    Among the constituents of the essential nutrient vitamin A, retinol is a potent suppressor of refractory cancer cell growth linked to tumor progression, showing greater efficacy than retinoic acid (RA). However, the mechanisms of retinol action on human refractory cancer are not known well. In the current study, we examined the actions of retinol on proliferation of human gallbladder cancer NOZ C-1 cells. Retinol and RA inhibited the proliferation of human NOZ C-1 cells in dose-dependent manner, while RA was less potent than retinol. Cell incorporation of RA was approximately two-fold higher than retinol and was not correlated with anti-proliferative activity. Retinol did not affect caspase-3 activity or mRNA expression of Bax and Bcl-2, which are associated with apoptosis. In addition, protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK)/ERK and p-Akt/Akt were not significantly changed by retinol treatment. In contrast, retinol treatment significantly increased the mRNA expression of endoplasmic reticulum (ER) stress factors (heme oxygenase 1 (HMOX1), CCAAT/enhancer-binding protein homologous protein (CHOP), 78 kDa glucose-regulated protein (GRP78), and DnaJ (Hsp40) homolog, subfamily B, member 9 (DNAJB9)). Furthermore, the number of cells in the G0/G1 phase was increased, while the number of cells in the S phase were decreased by retinol treatment. Retinol increased expression of the autophagy-associated protein, LC3-II. These results indicate that retinol is a potent suppressor of gallbladder cancer cell growth by mechanisms that involve ER stress, which results in autophagy and cell cycle delay. This suggests that retinol might be useful for anticancer prevention and therapy in the clinic.

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    Editor’s picks

    In their report, Li et al. described that vitamin A (retinol) inhibits cancer cell growth via endoplasmic reticulum (ER) stress. Retinal is a more potent suppressor of refractory human gallbladder cancer cell growth linked to tumor progression than retinoic acid (RA). Although cellular incorporation of RA is higher than retinol, it was not correlated with anti-proliferative activity. Retinol did not induce apoptosis or suppress MEK/ERK and PI3K/Akt pathways. However, it significantly increased the expression of ER stress related genes and autophagy-associated protein (LC3-II) and the number of cells in the G0/G1 phase. This result indicates that retinol suppresses gallbladder cancer cell growth by mechanisms involving ER stress, autophagy, and cell cycle delay. Retinol might be useful for the prevention and treatment of cancer.



  • Toru Tanaka, Naoki Maekawa, Taito Kashio, Kohei Izawa, Ryosuke Ishiba, ...
    2017 Volume 40 Issue 4 Pages 504-509
    Published: April 01, 2017
    Released: April 01, 2017
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    Roundabout4 (Robo4) is an endothelial cell-specific receptor that regulates vascular stability. Recently, Robo4 has been shown to regulate vascular permeability in inflammation. However, the mechanisms regulating the Robo4 gene in the context of inflammation are poorly understood. In this study, we found that intravenous injection of tumor necrosis factor (TNF) α increased Robo4 expression in mouse organs. In vitro analyses showed that TNFα increased Robo4 expression in human primary endothelial cells, but not in cells pretreated with a nuclear factor (NF)-κB inhibitor. Reporter assays using wild-type and mutant Robo4 promoters indicated that TNFα activated the Robo4 promoter and that both the −2753 and −2220 NF-κB motifs were essential for this activation. Electrophoretic mobility shift assays demonstrated that the NF-κB p65–p50 heterodimer bound to these motifs. These findings were further supported by chromatin immunoprecipitation assays in endothelial cells. Taken together, these results indicated that TNFα induced Robo4 expression by facilitating NF-κB p65–p50 heterodimer binding to the −2753 and −2220 motifs in the Robo4 promoter in endothelial cells in the context of inflammation.

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  • Masayuki Munekane, Masashi Ueda, Shinji Motomura, Shinichiro Kamino, H ...
    2017 Volume 40 Issue 4 Pages 510-515
    Published: April 01, 2017
    Released: April 01, 2017
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    Many zinc (Zn) complexes have been developed as promising oral antidiabetic agents. In vitro assays using adipocytes have demonstrated that the coordination structures of Zn complexes affect the uptake of Zn into cells and have insulinomimetic activities, for which moderate stability of Zn complexes is vital. The complexation of Zn plays a major role improving its bioavailability. However, investigation of the speciation changes of Zn complexes after oral administration is lacking. A dual radiolabeling approach was applied in order to investigate the speciation of bis(5-chloro-7-iodo-8-quinolinolato)zinc complex [Zn(Cq)2], which exhibits the antidiabetic activity in diabetic mice. In the present study, 65Zn- and 131I-labeled [Zn(Cq)2] were synthesized, and their biodistribution were analyzed after an oral administration using both invasive conventional assays and noninvasive gamma-ray emission imaging (GREI), a novel nuclear medicine imaging modality that enables analysis of multiple radionuclides simultaneously. The GREI experiments visualized the behavior of 65Zn and [131I]Cq from the stomach to large intestine and through the small intestine; most of the administered Zn was transported together with clioquinol (5-chloro-7-iodo-8-quinolinol) (Cq). Higher accumulation of 65Zn for [Zn(Cq)2] than ZnCl2 suggests that the Zn associated with Cq was highly absorbed by the intestinal tract. In particular, the molar ratio of administered iodine to Zn decreased during the distribution processes, indicating the dissociation of most [Zn(Cq)2] complexes. In conclusion, the present study successfully evaluated the speciation changes of orally administered [Zn(Cq)2] using the dual radiolabeling method.

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  • Kenji Kizawa, Takeshi Fujimori, Tomomitsu Kawai
    2017 Volume 40 Issue 4 Pages 516-523
    Published: April 01, 2017
    Released: April 01, 2017
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    The human hair shaft is covered with multiple scale-like cuticular layers. During the terminal differentiation stage of immature cuticular cells within the hair follicle, cysteine-rich calcium binding S100A3 protein is predominantly translated, and its arginine residues are converted to citrullines by peptidylarginine deiminases (PADI). In this study, we found several naturally occurring compounds (e.g., hinokitiol, escletin, and quercetin) elevate S100A3 citrullination in a human colorectal adenocarcinoma cell line (SW480). Selected compounds similarly promoted cuticular differentiation within isolated human hair follicles. Their promotive activities correlated with the previously reported inhibitory activities of arachidonate 12-lipoxygenase (ALOX12) in vitro. Microarray analysis revealed that ALOX12 inhibitor remarkably up-regulated heparin-binding epidermal growth factor-like growth factor (HBEGF). ALOX12 inhibitor and recombinant HBEGF similarly regulated expression of PADI genes in SW480 cells. In isolated hair follicles, arachidonic acid strongly promoted S100A3 citrullination along with elevation of HBEGF. These results suggest that ALOX12 inhibition efficiently triggers hair cuticle maturation by modulating arachidonate metabolism in concert with HBEGF.

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  • Maya Kudo, Hisae Yoshitomi, Maki Momoo, Shiori Suguro, Yoshie Yamagish ...
    2017 Volume 40 Issue 4 Pages 524-530
    Published: April 01, 2017
    Released: April 01, 2017
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    L-Citrulline (L-Cit), a free amino acid from watermelon, has effects on hypertension and anti-oxidization; however, there are few reports of effects related to obesity. This study investigated the effects and mechanism of L-Cit on anti-obesity in obese/diabetic KK-Ay mice and high-fat diet fed Sprague–Dawley (SD) rats. L-Cit induced significant reduction of food intake, body weight and fat tissue mass in obese/diabetic KK-Ay mice. Moreover, blood glucose level did not change but free fatty acid level and serum insulin level were significantly decreased by treatment with L-Cit, suggesting that L-Cit improved glucose and fatty metabolism in obesity model mice. As well as obese/diabetic KK-Ay mice, there was a significant decrease in food intake and a tendency of body weight to decrease in high-fat diet fed SD rats treated with L-Cit. Also, levels of proopiomelanocortin (POMC), a food intake suppression peptide, increased in the hypothalamus. Our study suggests that L-Cit improves metabolic syndrome through decreased body weight by appetite suppression.

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Notes
  • Yukako Ito, Kengo Matsumoto, Noriyuki Osakama, Ryosuke Yoshioka, Shinj ...
    2017 Volume 40 Issue 4 Pages 531-534
    Published: April 01, 2017
    Released: April 01, 2017
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    The possibility of using dissolving microneedles (DMs) as a skin allergy test device was studied in rats. Poly-L-arginine was used as a model allergen. Dextran was used to prepare three kinds of DM array chips containing different doses of poly-L-arginine: 17.1±0.5 µg (low-dose DM), 42.2±0.8 µg (medium-dose DM), and 87.4±1.1 µg (high-dose DM); each 1.0 cm2 chip contained 300 DMs. The mean lengths of the low-, medium-, and high-dose DM were 489±3, 485±3, and 492±1 µm and mean diameters of the base were 301±2, 299±1, and 299±2 µm, respectively. Furthermore, for the low-, medium-, and high-dose DM, the administered doses of poly-L-arginine were estimated to be 9.3±1.9, 31.1±1.3, and 61.9±4.7 µg and the scratching behavior per 30 min was 9.8±3.4, 60.4±8.3, and 95.7±10.6 times, respectively. These results demonstrate the dose dependence of the immunoreactivity of the poly-L-arginine DMs, suggesting that DMs can be used an alternative skin allergy device.

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  • Jeong Ah Hwang, Nok Hyun Park, Yong Joo Na, Hae Kwang Lee, John Hwan L ...
    2017 Volume 40 Issue 4 Pages 535-539
    Published: April 01, 2017
    Released: April 01, 2017
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    Pigmentation reflects skin darkening caused by melanin production, but excessive melanin synthesis may cause problems, such as melasma, solar lentigo, dark spots, and freckles. Considerable effort has been devoted to alleviating these undesired symptoms through the development of safe and effective depigmenting agents. Coumestrol, a plant-derived natural isoflavone with an estrogen-like structure and actions, is known to have anti-aging ability, but its potential depigmenting efficacy has not been evaluated. In the present study, we investigated the effects of coumestrol on melanin synthesis in normal melan-a murine melanocytes. Coumestrol significantly reduced melanin synthesis in a concentration-dependent manner up to a concentration of 25 µM without causing cytotoxicity. It also brightened tissue in an artificial skin model (MelanoDerm) that incorporates both human keratinocytes and melanocytes. Interestingly, although coumestrol did not inhibit tyrosinase activity or transcript level in melan-a cells, it clearly decreased the expression level of tyrosinase protein at a concentration of 25 µM. This coumestrol-induced reduction in tyrosinase protein levels was prevented by pretreatment with the proteasome inhibitor MG-132 or the lysosomal proteolysis inhibitor chloroquine. Collectively, our findings indicate that coumestrol exerts an inhibitory effect on melanin synthesis in melan-a cells, at least in part, through degradation of tyrosinase. These findings suggest that coumestrol is a good candidate for use in depigmentary reagents from a cosmetic and clinical perspective.

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  • Shinya Yanamoto, Hasan Babazada, Shinya Sakai, Yuriko Higuchi, Fumiyos ...
    2017 Volume 40 Issue 4 Pages 540-545
    Published: April 01, 2017
    Released: April 01, 2017
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    Glycosaminoglycans (GAGs) play important roles in various biological processes such as cell adhesion and signal transduction, as well as promote anti-inflammatory activity. We previously revealed that glycol-split heparin (HP)-aliphatic amine conjugates form self-assembled nanoparticles and suppress the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in lipopolysaccharide (LPS)-stimulated macrophages much more strongly than native HP (J. Control. Release, 194, 2014, Babazada et al.). Considering that HP is not the only GAG to have anti-inflammatory activity, the present study was initiated to examine whether conjugation of GAGs with aliphatic amines is generally effective in their activity augmentation against LPS-stimulated macrophages. We newly synthesized the stearylamine conjugates of chondroitin sulfate (CS), hyaluronic acid (HA), and low-molecular-weight heparin (LH), and investigated the effect of the position and degree of sulfation and molecular weight of GAGs on their anti-inflammatory activity. All of the conjugates formed self-assembled nanoparticles in aqueous solution. The IC50 value for suppression of TNF-α production from the macrophages was the smallest with the derivative of LH, followed by HP, CS, and HA. The degree of sulfation appeared to be important in determining their anti-inflammatory activity, which would correspond to previous results using the derivatives of site-selectively desulfated HP. Comparison of HP and LH derivatives revealed that fractionated smaller heparin has greater anti-inflammatory activity.

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