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Displaying 261-260 of 282 articles
Volume 41(2018)Issue 11Pages1716-1721
Analysis of Glycoforms and Amino Acids in Infliximab and a Biosimilar Product Using New Method with LC/TOF-MS
Masahiro Tsuda, Yuki Otani, Atsushi Yonezawa, Sho Masui, Yasuaki Ikemi, Masaya Denda, Yuki Sato, Shunsaku Nakagawa, Tomohiro Omura, Satoshi Imai, Takayuki Nakagawa, Makoto Hayakari, Kazuo Matsubara
Biosimilar products of therapeutic antibodies have been launched all over the world. They can relieve some of the economic burden of medicines. Although clinical trials have demonstrated the equivalency of biosimilar products with their reference product, biosimilar products are not commonly used in clinical practice. One reason is that the structural difference between the reference product and a biosimilar one remains unclear. We analyzed glycoforms and amino acids of an infliximab biosimilar product approved in Japan compared to that of the reference product (Remicade®). By combination of papain digestion and LC/ time-of-flight (TOF)-MS, we established a valuable method to analyze these therapeutic antibodies. Nine glycoforms were detected in infliximab, and a difference in amino acids was observed. In the glycoforms of MMF, MGnF/GnMF, GnGn, GnGnF, AGnF/GnAF, and AAF, the relative intensities were significantly different between the reference and biosimilar product. Furthermore, we elucidated that the content rate of the C-terminal lysine was different among glycoforms. In conclusion, our analytical method can analyze not only amino acids but also carbohydrate chains of therapeutic antibodies, and will provide a useful strategy to evaluate bio-medicines including biosimilar antibodies.
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How can you know the structural differencebetween biosimilar and reference product? The structure of biosimilar productsis not the same as their original product because of post-translationalmodifications. In the article by Tsuda et al., a valuable method with a papaindigestion and LC/TOF-MS analysis was established. Their new method can analyzenot only carbohydrate chains but also amino acids of bio-medicines including biosimilarantibodies. This technology will provide a useful strategy to evaluatebio-medicines including biosimilar antibodies.
Volume 41(2018)Issue 10Pages1567-1573
Characteristics of Bone Strength and Metabolism in Type 2 Diabetic Model Nagoya Shibata Yasuda Mice
Hiroaki Tanaka, Toshihiro Miura, Takenori Yamash*ta, Misao Yoneda, Satoshi Takagi
We evaluated the suitability of Nagoya Shibata Yasuda (NSY) mice as an animal model for examining the influence of a glucose metabolism disorder on bone integrity, using Institute of Cancer Research (ICR) mice as controls. We selected six NSY and ICR mice each that were matched for weight, and measured serum glucose levels, serum insulin levels, and conducted an oral glucose tolerance test. Histological sections of the femurs of both mouse lines were prepared, and the bone strength, mass, and microstructure of the femur were compared, along with bone metabolism. Serum glucose levels were significantly higher in the NSY mice than in the control mice, but body weight and serum insulin levels did not differ between the groups. Bone mass, microstructure, and strength of the femur, and bone metabolism were lower in the NSY mice than in the control mice. In the cortical bone of the femur in the NSY mice, several parts were not stained with eosin, demonstrating a strong negative correlation between serum glucose levels and bone mineral density; however, there was a negative correlation between serum glucose levels and bone metabolic markers. The bone turnover rate in the NSY mice was decreased by hyperglycemia, resulting in a thinner and shorter femur, reduced cortical and trabecular areas, and lower bone mass compared to those of the control mice. Collectively, these results suggest deteriorated bone strength of the femur in NSY mice, serving as a useful model for studying the link between glucose metabolism and bone integrity.
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Type2 diabetes mellitus (T2DM) animal models are often used in basic research ondiabetic osteoporosis. The article by Tanaka et al. elucidatedhyperglycemia contributed to a decrease in bonemetabolism turnover in the T2DM animal model NSY mice, resulting in a thinnerand shorter femur, lower cortical and trabecular area, and lower bone mass.Their results suggest that these effects contribute to the deteriorated bone strength of the femur in NSY mice, and the NSY mouse could serve as an appropriate T2DM animal model forexamining the specific effect of hyperglycemia on bone integrity, using the ICRmouse as a control.
Volume 41(2018)Issue 9Pages1456-1462
Effects of Supplementary Seleno-L-methionine on Atopic Dermatitis-Like Skin Lesions in Mice
Tomohiro Arakawa, Takahiro Sugiyama, Haruka Matsuura, Tomofumi Okuno, Hirofumi Ogino, Fumitoshi Sakazaki, Hitoshi Ueno
Effects of selenium supplementation on atopic dermatitis (AD) were investigated by administering seleno-L-methionine (SeMet) using a mouse model of AD caused by repeated application of 2,4,6-trinitrochlorobenzene (TNCB). BALB/c mice were sensitized with TNCB to the abdomen on day −7; then, TNCB was applied repeatedly to each ear three times a week from days 0 to 23. SeMet was orally administered to the mice from days 0 to 23. The efficacy of SeMet on AD was assessed by measuring ear thickness, histologic evaluation, serum total immunoglobulin (Ig) E levels, and expression of interleukin (IL)-4 in the ear and superficial parotid lymph node. Ear thickness was remarkably increased by repeated application of TNCB, and SeMet significantly suppressed ear thickness in BALB/c mice. SeMet inhibited epidermal hyperplasia and dense infiltration of inflammatory cells. The number of TNCB-induced mast cells was significantly decreased by SeMet. Serum total IgE levels that increased by the repeated application of TNCB were significantly suppressed by SeMet. Repeated application of TNCB induced expression of IL-4, a T-helper (Th) 2 cytokine, in the ear and superficial parotid lymph node of BALB/c mice and its expression was significantly inhibited by SeMet. These results demonstrated that SeMet supplementation suppresses AD-like skin lesions in BALB/c mice and inhibits the expression of total IgE and IL-4.
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Seleno-L-methionine(SeMet) is a major form of selenium compounds in foods. The article by Arakawa et al. demonstrated that ear thicknesswas increased by repeated application of 2,4,6-trinitrochlorobenzene (TNCB) to theear, and SeMet significantly suppressed ear thickness in mice. SeMet inhibitedepidermal hyperplasia and dense infiltration of inflammatory cells. Serum totalimmunoglobulin (Ig) E levels were suppressed by SeMet. Interleukin (IL) 4expression in the ear and superficial parotid lymph node was inhibited bySeMet. These results demonstrated that SeMet suppresses atopic dermatitis-likeskin lesions and inhibits the expression of total IgE and IL-4.
Volume 41(2018)Issue 7Pages1078-1083
Doxorubicin Expands in Vivo Secretion of Circulating Exosome in Mice
Sherif E. Emam, Hidenori Ando, Amr Selim Abu Lila, Shinya Kobayashi, Taro Shimizu, Keiichiro Okuhira, Yu Ishima, Tatsuhiro Ishida
Modulation of tumor immunity is a known factor in the antitumor activity of many chemotherapeutic agents. Exosomes are extracellular nanometric vesicles that are released by almost all types of cells, which includes cancer cells. These vesicles play a crucial role in tumor immunity. Many in vitro studies have reproduced the aggressive secretion of exosomes following treatment with conventional anticancer drugs. Nevertheless, how chemotherapeutic agents including nanomedicines such as Doxil® affect the in vivo secretion of exosomes is yet to be elucidated. In this study, the effect of intravenous injection of either free doxorubicin (DXR) or liposomal DXR formulation (Doxil®) on exosome secretion was evaluated in BALB/c mice. Exosomes were isolated from serum by using an ExoQuick™ kit. Free DXR treatment markedly increased serum exosome levels in a post-injection time-dependent manner, while Doxil® treatment did not. Exosomal size distribution and marker protein expressions (CD9, CD63, and TSG101) were studied. The physical/biological characteristics of treatment-induced exosomes were comparable to those of control mice. Interestingly, splenectomy significantly suppressed the copious exosomal secretions induced by free DXR. Collectively, our results indicate that conventional anticancer agents induce the secretion of circulating exosomes, presumably via stimulating immune cells of the spleen. As far as we know, this study represents the first report indicating that conventional chemotherapeutics may induce exosome secretion which might, in turn, contribute partly to the antitumor effect of chemotherapeutic agents.
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Exosomesare extracellular vesicles released by various types of cells, including immunecells and cancer cells. The article by Emam et al evaluated in vivo secretion of exosomes followingintravenous injection of free doxorubicin (DXR) or liposomal DXR (Doxil®)was evaluated using normal mice. Free DXR treatment markedly increased theserum exosome level, while Doxil® treatment did not change. Interestingly,splenectomy significantly suppressed the exosomal secretions induced by freeDXR treatment. Their results suggest that conventional anticancer agents inducethe secretion of exosomes via stimulating immune cells of the spleen, and mightcontribute to the antitumor effect of chemotherapeutic agents.
Volume 41(2018)Issue 6Pages925-936
A Novel Mechanism of γ-Irradiation-Induced IL-6 Production Mediated by P2Y11 Receptor in Epidermal Keratinocytes
Airi Ohsaki, Yuki Miyano, Rei Tanaka, Sei-ichi Tanuma, Shuji Kojima, Mitsutoshi Tsukimoto
Skin inflammation is caused by excessive production of cytokines and chemokines in response to an external stimulus, such as radiation, but the mechanisms involved are not completely understood. Here, we report a novel mechanism of γ-irradiation-induced interleukin-6 (IL-6) production mediated by P2Y11 receptors in epidermal cells. After irradiation of HaCaT cells derived from human epidermal keratinocytes with 5 Gy of γ-rays (137Cs: 0.78 Gy/min), IL-6 production was unchanged at 24 h after γ-irradiation, but was increased at 48 h. IL-6 mRNA was increased at 30 h, and IL-6 production was increased at 33 h after irradiation. The production of IL-6 was sustained at least for 4 d after irradiation. P2Y11 receptor antagonist NF157 inhibited IL-6 production in irradiated cells. Treatment with ATP, a ligand of P2Y11 receptor caused IL-6 production within 24 h. ATP-induced IL-6 production was also suppressed by NF157. Extracellular ATP level was increased after irradiation. The p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) signaling was involved in the production of IL-6 at the downstream of P2Y11 receptor activation. In addition, the cell cycle was arrested at the G2/M phase, and DNA repair foci were not disappeared at 48 h after γ-irradiation. The protein level of histone methylation enzyme G9a, which inhibits IL-6 production, was decreased after γ-irradiation. In conclusion, we suggest that γ-irradiation induces sustained IL-6 production in HaCaT cells from 33 h after irradiation, which is mediated through P2Y11 receptor-p38 MAPK-NF-κB signaling pathway and G9a degradation. This is a novel mechanism of cytokine production in γ-irradiated cells.
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The article by Ohsaki et al. proposeda novel mechanism of cytokine production in γ-irradiated cells. γ-Rayirradiation induced sustained IL-6 production in HaCaT epidermal cells from 33h after irradiation. Extracellular ATP-induced activation of P2Y11 receptor wasinvolved in the production of IL-6. At the downstream of P2Y11 receptoractivation, the p38 MAPK and NF-kB signaling was involved in IL-6 production. Theprotein level of G9a, which inhibits IL-6 production, was decreased after g-irradiation. These findings should be helpful to understand thepathogenesis of radiation-induced inflammation, as well as thepotential side effects of therapeutic irradiation.
Volume 41(2018)Issue 5Pages761-769
Protective Effect of Ipragliflozin on Pancreatic Islet Cells in Obese Type 2 Diabetic db/db Mice
Toshiyuki Takasu, Shoji Takakura
Ipragliflozin is a selective sodium glucose cotransporter 2 (SGLT2) inhibitor that increases urinary glucose excretion and subsequently improves hyperglycemia in patients with type 2 diabetes mellitus (T2DM). To assess the beneficial effect of ipragliflozin on the mass and function of pancreatic β-cells under diabetic conditions, obese T2DM db/db mice were treated with ipragliflozin for 5 weeks. Glucose and lipid metabolism parameters, pathological changes in pancreatic islet cells and insulin content were evaluated. Pathological examination of pancreatic islet cells comprised measuring the ratios of insulin- and glucagon-positive cells and levels of oxidative stress markers. Hemoglobin A1c, plasma glucose, non-esterified fatty acid and triglyceride levels in ipragliflozin-treated groups were reduced compared to the diabetic control (DM-control) group. Histopathological examination of pancreatic islet cells revealed strong insulin staining and reduced glucagon staining in the ipragliflozin 10 mg/kg-treated group compared with the DM-control group. The ratio of α- to β-cell mass was lower in the ipragliflozin 10 mg/kg-treated group than the DM-control group and was similar to that of the non-diabetic control group. The density of immunostaining for 4-hydroxy-2-nonenal, an oxidative stress marker, in pancreatic islets was significantly lower in the ipragliflozin 10 mg/kg-treated group than the DM-control group. Pancreatic insulin content tended to be higher in the ipragliflozin-treated groups than the DM-control group. Our findings demonstrate the benefit of ipragliflozin treatment in improving glucolipotoxicity and reducing oxidative stress in pancreatic islet cells. Treatment with ipragliflozin may protect against the progressive loss of islet β-cells in patients with T2DM.
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Ipragliflozinis a selective sodium glucose cotransporter 2 inhibitor that increases urinaryglucose excretion, and subsequently improves glucolipotoxicity. The article byTakasu et al. demonstrated that the treatment with ipragliflozin decreasedpancreatic cells positive for 4-hydroxy-2-nonenal (4HNE), an oxidative stressmarker, in obese type 2 diabetes mellitus (DM) db/db mice. Histopathologicalexamination of pancreatic islet cells revealed strong insulin staining, whereasreduced glucagon staining, accordingly pancreatic insulin content tended to behigher in the ipragliflozin 10 mg/kg-treated group compared with the DM-controlgroup. It was demonstrated that ipragliflozin has a protective effect on thepancreas by reducing oxidative stress.
Volume 41(2018)Issue 4Pages612-618
Virion-Packaged Pyruvate Kinase Muscle Type 2 Affects Reverse Transcription Efficiency of Human Immunodeficiency Virus Type 1 by Blocking Virion Recruitment of tRNALys3
Kumkum Rahman Mouree, Naoki Kishimoto, Nozomi Iga, Chie Kirihara, Kengo Yamamoto, Nobutoki Takamune, Shogo Misumi
Human immunodeficiency virus type 1 (HIV-1) recruits diverse cellular factors into viral particles during its morphogenesis, which apparently play roles in modulating its infectivity. In our study, proteomic techniques demonstrated that a key glycolytic protein, pyruvate kinase muscle type 2 (PKM2), is incorporated into viral particles. Here, we show that virion-packaged PKM2 significantly reduces viral infectivity by affecting the incorporation level of a cellular tRNALys3 into virions. Enhanced expression of PKM2 in HIV-1-producing cells led to a higher incorporation level of PKM2 into progeny virions without affecting the viral maturation process. Compared with the control virus, the high-level-PKM2-packaging virus showed decreased levels of both reverse transcription products and cellular tRNALys3 packaging, suggesting that the shortage of intravirion tRNALys3 suppresses reverse transcription efficiency in target cells. Interestingly, the enhanced expression of PKM2 also suppressed the virion recruitment of other nonpriming cellular tRNAs such as tRNALys1,2 and tRNAAsn, which are known to be selectively packaged into virions, without affecting the steady level of the cytoplasmic pool of those tRNAs in producer cells, suggesting that PKM2 specifically impedes the selective incorporation of tRNAs into virions. Taken together, our findings indicate that PKM2 is a vital host factor that negatively affects HIV-1 infectivity by targeting the tRNALys3-mediated initiation of reverse transcription in target cells.
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Humanimmunodeficiency virus type 1 (HIV-1) recruits diverse cellular factors intoviral particles during its morphogenesis, which apparently play roles inmodulating its infectivity. The article by Mouree et al. evaluated that a keyglycolytic protein, pyruvate kinase muscle type 2 (PKM2) is incorporated intoviral particles. Furthermore, the virion-packaged PKM2 significantly reducesthe viral infectivity by affecting the selective packaging of intraviriontRNALys3, which primes the initiation of reverse transcription, along withother nonpriming tRNAs, such as tRNALys1,2 and tRNAAsn, without affecting thecytoplasmic level of these tRNAs. These findings proposed that PKM2 is a vitalhost factor that negatively affects HIV-1 infectivity by targeting thetRNALys3-mediated initiation of reverse transcription in target cells.
Volume 41(2018)Issue 3Pages419-426
Identification and Characterization of a Novel NADPH Oxidase 1 (Nox1) Inhibitor That Suppresses Proliferation of Colon and Stomach Cancer Cells
Tsuyoshi Yamamoto, Hirofumi Nakano, Kazuro Shiomi, Kiyofumi Wanibuchi, Hisashi Masui, Takashi Takahashi, Yasuteru Urano, Tohru Kamata
Reactive oxygen species (ROS) generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)1 mediate cellular signalings involved in normal physiological processes, and aberrant control of Nox1 has been implicated in the pathogenesis of various diseases. Therefore, Nox1 could have great potential as a therapeutic target. Here, we identified a novel Nox1 inhibitor, NOS31 secreted from Stretomyces sp. and analyzed its chemical structure. Furthermore, NOS31 was found to selectively inhibit Nox1-mediated ROS generation, with only a marginal effect on other Nox isoforms (Nox2–5) and no ROS scavenging activity. This compound blocked both Nox organizer 1 (NOXO1)/Nox activator 1 (NOXA1)-dependent and phorbol 12-myristate 13-acetate-stimulated Nox1-mediated ROS production in colon cancer cells. NOS31 inhibited the proliferation of several colon carcinoma and gastric cancer cell lines that upregulate the Nox1 system, whereas it had no appreciable effect on normal cells with low levels of Nox1. The finding suggests that NOS31 is a unique, potent Nox1 inhibitor of microbial origin and raises its possibility as a therapeutic agent for inhibiting gastrointestinal cancer cell growth.
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NADPHoxidase (Nox) isozymes are implicated in the diseases associated with oxidativestress, and search of their selective inhibitors has been a focus of attention.By screening microbial metabolites, Nakano and colleagues identified a novelNox1 inhibitor (NOS31) produced from actinomyces. NOS31 inhibited Nox1-mediatedhydrogen peroxide production with high Nox1 selectivity and suppressedproliferation of colon and gastric cancer cells that up-regulate Nox1. Thus,NOS31 may have the therapeutic potential for treating cancer involving theover-activation of Nox1.
Volume 41(2018)Issue 2Pages239-246
Exploration of the Key Factors for Optimizing the in Vivo Oral Delivery of Insulin by Using a Noncovalent Strategy with Cell-Penetrating Peptides
Noriyasu Kamei, Chikako Shigei, Ryota Hasegawa, Mariko Takeda-Morish*ta
This present study aimed to determine the optimal oral insulin delivery conditions that would maximize the utility of cell-penetrating peptides (CPPs) by using a noncovalent strategy. We first compared the effectiveness of two potential CPPs, penetratin and its analog PenetraMax, as absorption enhancers for insulin. The combined effect was evaluated under in vivo oral administration conditions. Both D-forms of CPPs were highly effective for increasing the oral absorption of insulin, and D-PenetraMax showed a more rapid onset of absorption enhancement effects compared with those of D-penetratin. However, synergistic absorption enhancement effects after combination treatment were not observed. Next, we tried a theoretical approach to establish optimized oral insulin delivery conditions. A surface plasmon resonance (SPR)-based analysis demonstrated that binding between insulin and penetratin (2 mM) might be saturated at 100–500 µM penetratin, while the bound concentration of penetratin could increase in accordance with an increased concentration of mixed insulin. To test this hypothesis, we investigated the effectiveness of different insulin doses in the gastric pH-neutralized mice. The results showed that the dissociation of noncovalent complexes of insulin and CPPs at the low gastric pH was prevented in these mice. Our findings clearly suggested that a noncovalent strategy with CPPs represents an effective approach for the L-form of CPP to increase the concentration of CPP-bound insulin to attain greater absorption of insulin, although this approach may not be appropriate for the D-form of CPP. Our findings will contribute to the development of oral dosage forms of insulin for noncovalent strategies involving CPP.
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Based on thebinding characteristics, Kamei et al. attempted to increase the boundconcentration of penetratin, cell-penetrating peptide (CPP), by increasing theconcentration of mixed insulin, however the effect of L-penetratin on the oralabsorption of insulin was not boosted by increasing the dose of insulin. Theinvestigation in the gastric pH-neutralized mice showed that the dissociationof noncovalent complexes of insulin and CPPs at the low gastric pH wasprevented in these mice, and clearly suggested that a noncovalent theoreticalstrategy with CPPs represents an effective approach for the L-form of CPP toattain greater absorption of insulin.
Volume 40(2017)Issue 12Pages2158-2165
Evaluation of Drug-Induced Photosensitivity Using the Japanese Adverse Drug Event Report (JADER) Database
Satoshi Nakao, Haruna Hatahira, Sayaka Sasaoka, Shiori Hasegawa, Yumi Motooka, Natsumi Ueda, Junko Abe, Akiho f*ckuda, Misa Naganuma, Hiroyuki Kanoh, Mariko Seishima, Motoyuki Ishiguro, Yasutomi Kinosada, Mitsuhiro Nakamura
Drug-induced photosensitivity (DIP) refers to the development of cutaneous disorders caused by the combined effects of different medications and light. The aim of this study was to obtain new information on drug risk comparisons and on DIP onset profiles, including seasonal variations, for clinically used prescription drugs. We analyzed reports of DIP recorded in the Japanese Adverse Drug Event Report (JADER) database using a reporting odds ratio (ROR). We also used Weibull proportional-hazards models for each drug to examine the patterns of DIP. The JADER database contains 430587 reports recorded from April 2004 to November 2016. The ROR values (95% confidence interval [CI]) of losartan/hydrochlorothiazide (HCTZ), valsartan/HCTZ, and ketoprofen were 214.5 (162.1–283.9), 104.7 (66.3–165.5), and 117.9 (76.6–181.5), respectively. For time-to-onset analysis, the median durations (interquartile range) for DIP caused by losartan/HCTZ, valsartan/HCTZ, and ketoprofen were 56 (41–78), 49 (38–88), and 8 (2–14) days, respectively. The lower limit of the 95% CI for the Weibull shape parameter β value for losartan/HCTZ was greater than 1. More than half of the reports of DIP onset following the administration of ketoprofen were recorded within 10 d of treatment initiation. The seasonal variation of photosensitivity reactions was shown to follow an annual sinusoidal pattern with a peak in April and May. Based on the results, losartan/HCTZ, valsartan/HCTZ, and ketoprofen should be used carefully in clinical practice to avoid DIP.
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Drug-induced photosensitivity (DIP) is a cutaneous adverse event caused by the combined effects of a medication and exposure to light. The article by Nakao et al. evaluated the association between drugs and DIP by using the reporting odds ratio and time-to-onset analysis data from the Japanese Adverse Drug Event Report (JADER) database. More than half of the reports of DIP onset following ketoprofen administration were recorded within 10 days of initiation of treatment. The seasonal variation of DIP was shown to follow an annual sinusoidal pattern with peaks observed in April and May. The results of this study suggest close monitoring of patients taking suspected drugs, especially during the peak season of photosensitivity reactions.
Volume 40(2017)Issue 11Pages1968-1975
The Truncated Isoform of the Receptor Tyrosine Kinase ALK Generated by Alternative Transcription Initiation (ALKATI) Induces Chromatin Structural Changes in the Nucleus in a Kinase Activity-Dependent Manner
Yuki Takakura, Noritaka Yamaguchi, Takuya Honda, Mariko Morii, Ryuzaburo Yuki, Yuji Nakayama, Naoto Yamaguchi
Anaplastic lymphoma kinase (ALK) is a receptor-type tyrosine kinase that promotes cell growth upon stimulation with ligands such as midkine and pleiotrophin. Recently, a truncated isoform of ALK was identified in a variety of tumors. This isoform is expressed from a novel ALK transcript initiated from a de novo alternative transcription initiation (ATI) site in ALK intron 19 (referred to as ALKATI). ALKATI, which consists of only the intracellular kinase domain, localizes to the nucleus as well as the cytoplasm. However, its nuclear role is unknown. In this study, we determined that ALKATI promoted chromatin structural changes in the nucleus in a kinase activity-dependent manner. We found that expression of ALKATI increased the level of the heterochromatin marker Lys9 tri-methylated histone H3. In addition, we demonstrated that ALKATI phosphorylated the nuclear protein A-kinase anchoring protein 8 (AKAP8) and altered its subcellular localization from the insoluble fraction to the soluble fraction. These results suggest that ALKATI induces chromatin structural changes and heterochromatinization through phosphorylation of AKAP8 in the nucleus.
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Because tyrosine kinases mainly localize to the cytoplasm or the plasma membrane, most studies have focused on their roles in the cytoplasm. However, emerging evidence has revealed that tyrosine kinases also localize to the nucleus and regulate nuclear events, such as DNA damage responses, gene expression, and chromatin structural changes. In this paper, Takakura et al. showed that the truncated isoform of the receptor tyrosine kinase ALK (ALK ATI), which only has the intracellular kinase domain, regulates chromatin structural changes, heterochromatinization, and gene expression in the nucleus. This paper is the first report shedding light on the nuclear roles of ALK.
Volume 40(2017)Issue 9Pages1468-1474
Up-Regulation of the Voltage-Gated KV2.1 K+ Channel in the Renal Arterial Myocytes of Dahl Salt-Sensitive Hypertensive Rats
Kazunobu Ogiwara, Susumu Ohya, Yoshiaki Suzuki, Hisao Yamamura, Yuji Imaizumi
Salt-sensitive hypertension induces renal injury via decreased blood flow in the renal artery (RA), and ion channel dysfunction in RA myocytes (RAMs) may be involved in the higher renal vascular resistance. We examined the effects of several voltage-gated K+ (KV) channel blockers on the resting tension in endothelium-denuded RA strips and delayed-rectifier K+ currents in RAMs of Dahl salt-sensitive hypertensive rats (Dahl-S) fed with low- (Dahl-LS) and high-salt diets (Dahl-HS). The tetraethylammonium (TEA)-induced contraction in RA strips were significantly larger in Dahl-HS than Dahl-LS. Correspondingly, TEA-sensitive KV currents were significantly larger in the RAMs of Dahl-HS than Dahl-LS. Among the TEA-sensitive KV channel subtypes, the expression levels of KV2.1 transcript and protein were significantly higher in the RA of Dahl-HS than Dahl-LS, while those of KV1.5, KV7.1, and KV7.4 transcripts was comparable in two groups. KV2.1 currents detected as the guangxitoxin-1E-sensitive component were larger in the RAMs of Dahl-HS than Dahl-LS. These suggest that the up-regulation of the KV2.1 channel in RAMs may be involved in the compensatory mechanisms against decreased renal blood flow in salt-sensitive hypertension.
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Salt-sensitivehypertension induces renal injury via decreased blood flow in the renal artery.Voltage-gated, delayed-rectifier K+ (KV) channels playkey roles in the regulation of vascular tone, and their dysfunction in arterialmyocytes may be involved in the higher vascular resistance. In their report, Ogiwara et al. described that there is a largecontribution of KV2.1 to the resting tension maintenance of renalartery in Dahl salt-sensitive hypertensive rats and suggested the up-regulationof the KV2.1 channel in renal artery might be involved in thecompensatory mechanisms against decreased renal blood flow in salt-sensitivehypertension.
Volume 40(2017)Issue 8Pages1183-1191
Characterization of Membrane Integrity and Morphological Stability of Human Salivary Exosomes
Nahoko Kumeda, Yuko Ogawa, Yoshihiro Akimoto, Hayato Kawakami, Masafumi Tsujimoto, Ryohei Yanosh*ta
Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.
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In their report, Kumeda et al. examined Membrane integrity and morphological stability of salivary exosomes using dipeptidyl peptidase IV and CD9 as membrane makers, and Alix and Tsg101 as lumen markers. Neither localization nor levels of these marker proteins were changed in isolated exosomes after long-term storage at 4°C or multiple freeze-thaw cycles. Moreover, intact exosomes could be isolated from whole saliva refrigerated for a month. Components inside the exosomes were stable even after solubilization of membrane components with detergents such as Triton X-100. These results indicate that human saliva-derived exosomes are very stable, maintaining their membrane integrity over a long storage period.
Volume 40(2017)Issue 7Pages1029-1034
Antioxidative Protection of Squalene Adjuvant and Rabies Vaccine with Adjuvant
Anna Ondrejková, Judit Süli, Jarmila Harvanová, Róbert Ondrejka, Marián Prokeš
The authors verified the possibility of antioxidative protection of squalene adjuvant emulsions by the antioxidants α-tocopherol and β-carotene. They determined the influence of β-carotene on the stability and antigenic effectiveness of adjuvant emulsion in combination with rabies vaccine. The composition of the adjuvant emulsions or vaccines was: 2.5% squalene; 6% detergents; 0.5% antioxidant; 91% water phase. The oxidative injury after UV-irradiation was followed by the detection of the peroxide value of the emulsions. The stability of the emulsions was evaluated by the determination of the emulsion’s particle size. The level of rabies antibodies (RAB) in mice sera until day 90 after vaccination, was determined by the rapid fluorescent focus inhibition test. In the in vitro system of squalene adjuvant, α-tocopherol acted as a prooxidant, while β-carotene effectively reduced the oxidative injury. The hom*ogenization of the squalene adjuvant during a prolonged period from 8 to 10 min did not change the particle size. The oxidation processes were efficiently reduced by β-carotene during the preparation process and also during the 70-d storage. The vaccine with β-carotene induced a gradual increase in the RAB levels with the highest value on day 28. While the inactivated rabies vaccine with adjuvant without β-carotene developed a rapid formation of RAB, the application of the vaccine with β-carotene induced a slower but more uniform production of RAB. The level of RAB was significantly higher after the application of the vaccine with β-carotene and reached the protective value of 0.5 IU/mL, in contrast to the vaccine without β-carotene.
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The composition of the adjuvant emulsions was 2.5%squalene, 6% detergents, 0.5% antioxidant – α-tocopherol or β-carotene and 91%water phase. Antioxidant effectivity was testing by determination of peroxidevalue. α-tocopherol acted as a prooxidant, β-carotene was an effectiveantioxidant. Effectiveness of rabies vaccine with squalene adjuvant was testingon mice. Adjuvanted vaccine with β-carotene was compared to vaccine withoutantioxidant and induced a slower but prolonged immunity response with protectivelevels of rabies antibodies (0.5 IU/mL).
Volume 40(2017)Issue 6Pages815-823
Effective-Loading of Platinum–Chloroquine into PEGylated Neutral and Cationic Liposomes as a Drug Delivery System for Resistant Malaria Parasites
Shaimaa Ibrahim, Tatsuaki Tagami, Tetsuya Ozeki
The trans platinum–chloroquine diphosphate dichloride (PtCQ) is a new type of antimalarial drug used to fight parasites resistant to traditional drugs. PtCQ is synthesized by mixing platinum and chloroquine diphosphate (CQ). This study examines two efficient methods for forming a nanodrug, PtCQ-loaded liposomes, for use as a potential antimalarial drug-delivery system: the thin drug–lipid film method to incorporate the drug into a liposomal membrane, and a remote-loading method to load the drug into the interior of a cationic liposome. The membranes accordingly comprised PEGylated neutral or cationic liposomes. PtCQ was efficiently loaded into PEGylated neutral and cationic liposomes using the thin drug–lipid film method (encapsulation efficiency, EE: 76.1±6.7% for neutral liposomes, 1 : 14 drug-to-lipid weight ratio; 70.4±9.8% for cationic liposomes, 1 : 14 drug-to-lipid weight ratio). More PtCQ was loaded into PEGylated neutral liposomes using the remote-loading method than by the thin drug–lipid film method and the EE was maximum (96.1±4.5% for neutral liposomes, 1 : 7 (w/w)). PtCQ was encapsulated in PEGylated cationic liposomes comprising various amounts of cationic lipids (0–20 mol%; EE: 96.9–92.3%) using the remote-loading method. PEGylated neutral liposomes and cationic liposomes exhibited minimum leakage of PtCQ after two months’ storage at 4°C, and further exhibited little release under in vitro culture conditions at 37°C for 72 h. These results provide a useful framework for the design of future liposome-based in vivo drug delivery systems targeting the malaria parasite.
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Intheir report, Ibrahim et al. described that generating platinum chloroquinediphosphate dichloride (PtCQ)-loaded polyethylene glycol (PEG)-modified(PEGylated) cationic liposomes exhibiting high drug encapsulation and high drugretention. PtCQ was encapsulated in PEGylated cationic liposomes comprisingvarious amounts of cationic lipids using the remote-loading method. PEGylatedneutral liposomes and cationic liposomes exhibited minimum leakage of PtCQafter two months’ storage at 4˚C, and further exhibited little release under invitro culture conditions at 37˚C for 72 hours. These results provide a usefulframework for the design of future liposome-based in vivo drug delivery systemstargeting the drug-resistant malaria parasite.
Volume 40(2017)Issue 5Pages594-597
Cost-Outcome Description of PEG-IFN-α2b+RBV for Hepatitis C: Results Based on the Interferon Database
Maiko Akutagawa, Yohei Kawasaki, Atsuko Kawasaki, Kazuki Ide, Hiroshi Yamada, Naohiko Masaki
Economic evaluation of drugs is used in decision-making on medical care and public policy. Recently, real-world data (RWD) have been used in the analysis. In this study, we discuss the risk and benefits of using RWD for economic evaluation. We conducted a cost-outcome description with RWD from a nationwide registry providing information on hepatitis treatment in Japan and estimated the utility of the analysis. We evaluated the cost-outcome description of peginterferon plus ribavirin (PEG-IFN-α2b+RBV) treatment in hepatitis C virus (HCV)-infected patients. Simulations were based on a Markov model. The cohorts were set using data from the registry and we assumed a societal perspective for the calculation of costs. The dose and drug cost were chosen based on the Japanese Guidelines for the Management of Hepatitis C Virus Infection or package inserts. Model details and parameters were as described in previous studies. The simulations were performed for a period of 10 years with no discount rate. We estimated 2.5 million JPY per Quality Adjusted Life Year (QALY) in 48-week PEG-IFN-α2b+RBV treatment for a period of 10 years. The results of this study are in agreement with previous HCV treatment economic evaluation studies in Japan. We analyzed the statistics of the HCV-infected patients at each disease stage using the data in our registry and calculated the costs. The results of this study more closely reflect a real-world clinical situation compared to the widely used randomized clinical trial method, which estimates clinical trial results and scenarios.
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Economicevaluation has been recently carried out using real-world data instead ofclinical trial data. Akutagawa et al. conducted a cost-outcome description basedon a nationwide registry providing information on hepatitis treatment in Japanand estimated the utility of the analysis. Specifically, they evaluated thecost-outcome description of a 48-week peginterferon plus ribavirin treatment inpatients infected by the hepatitis C virus. Simulations were based on a Markovmodel. After setting the cohorts using data from the registry, and assuming asocietal perspective for the calculation of costs, they estimated 2.5 millionJPY per quality-adjustedlife years(QALY) for treatments over a 10-year period. They analyzedpatients’ statistics at each disease stage using their registry data andcalculated the costs. Their results reflect more closely a real-world clinicalsituation as compared to the widely used clinical trial method.
Volume 40(2017)Issue 4Pages495-503
Growth Inhibition of Refractory Human Gallbladder Cancer Cells by Retinol, and Its Mechanism of Action
Chuan Li, Masahiko Imai, Shinya Hasegawa, Masahiro Yamasaki, Noriko Takahashi
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 hom*ologous protein (CHOP), 78 kDa glucose-regulated protein (GRP78), and DnaJ (Hsp40) hom*olog, 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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In their report, Li et al. described that vitaminA (retinol) inhibits cancer cell growth via endoplasmic reticulum (ER) stress. Retinal is a more potent suppressor ofrefractory humangallbladder cancer cell growth linked to tumor progression than retinoic acid(RA). Although cellular incorporation of RA is higher than retinol, it was not correlated withanti-proliferative activity. Retinol did not induce apoptosis or suppress MEK/ERK andPI3K/Akt pathways. However, it significantlyincreased the expression of ER stressrelated genes and autophagy-associated protein (LC3-II) and the number of cells inthe G0/G1 phase. This resultindicates that retinol suppresses gallbladder cancer cell growth by mechanismsinvolving ER stress, autophagy, and cell cycle delay.Retinol might be useful for the prevention and treatment of cancer.
Volume 40(2017)Issue 3Pages297-302
An Activatable Fluorescent γ-Polyglutamic Acid Complex for Sentinel Lymph Node Imaging
Masayori Hagimori, Eri Hatabe, Kohei Sano, Hirotaka Miyazaki, Hitoshi Sasaki, Hideo Saji, Takahiro Mukai
Sentinel lymph nodes (SLN) are the first lymph nodes (LN) where cancer cells metastasize from the primary tumor. We designed fluorophore-quencher-based activatable nanoparticles for SLN imaging. We selected TAMRA as a fluorophore and BHQ2 or QSY7 as a quencher. Ternary anionic complexes were constructed with generation 4th polyamidoamine dendrimer (G4) modified with TAMRA and p-SCN-Bn-DTPA (DTPA), polyethyleneimine (PEI) modified with BHQ2 or QSY7, and γ-polyglutamic acid (γ-PGA) by the electrostatic self-assembly system. TAMRA-G4-DTPA/PEI-BHQ2/γ-PGA and TAMRA-G4-DTPA/PEI-QSY7/γ-PGA complexes had a particle size of about 40 nm and a ζ-potential of −50 mV, and showed fluorescence resonance energy transfer (FRET) quenching. Fluorescence microscopy studies demonstrated that TAMRA-G4-DTPA/PEI-QSY7/γ-PGA complex produced intracellular fluorescent signals in the lysosome. During in vivo fluorescent imaging, TAMRA-G4-DTPA/PEI-QSY7/γ-PGA complex enabled the detection of mouse popliteal LN. The fluorophore-quencher conjugated γ-PGA complex based on FRET quenching would be useful for fluorescence-based optical imaging of SLN.
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Sentinellymph nodes (SLN) are the first LN where cancer cells metastasize from theprimary tumor. As an activatablefluorescence probe to detect the SLNs, Hagimori et al. developed ternaryanionic nanoparticles constructed with fluorophore (TAMRA)-labeledpolyamidoamine dendrimer conjugated with diethylenetriaminepentaacetic acid(TAMRA-G4-DTPA), quencher-labeled polyethyleneimine (PEI-QSY7 or PEI-BHQ2), andg-polyglutamic acid, namely TAMRA-G4-DTPA/PEI-QSY7/g-PGA and TAMRA-G4-DTPA/PEI-BHQ2/g-PGA by the electrostatic self-assembly system. Thefluorescence of these complexes was quenched by a strong stacking interactionof TAMRA and quenchers, but was dequenched by dissociation of complexes whentaken up by inflammatory cells (high populations in LN). They performed fluorescenceimaging at 24 h after intradermal injection of TAMRA-G4-DTPA/PEI-QSY7/g-PGA into mouse footpads. Then, TAMRA fluorescencesignal was clearly visualized in popliteal lymph node with high contrast.
Volume 40(2017)Issue 1Pages82-87
Cobalt Chloride Induces Expression and Function of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Renal Proximal Tubular Epithelial Cell Line HK-2
Katsuki Nishihashi, Kei Kawashima, Takami Nomura, Yumiko Urakami-Takebayashi, Makoto Miyazaki, Mikihisa Takano, Junya Nagai
The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.
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In their report, Nishihashi etal. describe in detail that cobalt chloride-induced activation of hypoxia-induciblefactor-1 (HIF-1) increases not only mRNA and protein expression but also functionof breast cancer resistance protein (BCRP/ABCG2) in a human renal proximaltubular epithelial cell line. HIF-1 consists of an inducible a-subunit(HIF-1a)and a constitutive b-subunit(HIF-1b).The stabilization of HIF-1a underhypoxia and various biochemical stimuli leads to nuclear translocation,dimerization with HIF-1band binding to hypoxia response element (HRE) sequences in the promoter of varioustarget genes. The present results indicate that HIF-1 plays an important rolein modulating BCRP-mediated transport activity in renal proximal tubular epithelialcells.
Volume 39(2016)Issue 11Pages1868-1875
Methicillin-Resistant Staphylococcus epidermidis Is Part of the Skin Flora on the Hands of Both Healthy Individuals and Hospital Workers
Kiyoshi Watanabe, Hidemasa Nakaminami, Chihiro Azuma, Ippei Tanaka, Keisuke Nakase, Norifumi Matsunaga, Kiyoshi Okuyama, Kanako Yamada, Kenta Utsumi, Takeshi Fujii, Norihisa Noguchi
Staphylococcus epidermidis, a major skin flora on hands, acts as a reservoir of various antimicrobial resistance determinants including staphylococcal cassette chromosome mec (SCCmec) and contributes to multidrug resistance for S. aureus. The aim of this study was understanding the characteristics of commensal S. epidermidis on the hands of hospital workers and healthy individuals. A total of 23 hospital workers (physicians, nurses, and hospital pharmacists), 13 community pharmacists, and 24 healthy individuals (students) were studied. Commensal bacteria on hands were recovered using a glove-juice method. For methicillin-resistant S. epidermidis (MRSE), we performed SCCmec typing, pulsed-field gel electrophoresis (PFGE), and determined the antimicrobial susceptibility. The detection rates of MRSE in community pharmacists (92.3%) and students (87.5%) were higher than those in hospital workers (66.7 to 81.8%). SCCmec type IV strains were predominant in both hospital workers and students. PFGE analysis strongly suggested that the MRSE of hospital workers and students were normal inhabitants of each subject. The antimicrobial resistance rates and levels in MRSE of hospital workers were higher than those of students. Our findings showed that MRSE was frequently colonized on the hands of healthy individuals as well as hospital workers.
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In their report, Watanabe et al. described the prevalence of methicillin-resistantStaphylococcus epidermidis (MRSE), oneof the major nosocomial pathogens, on the hands of healthy individuals andhospital workers. The detection ratesof MRSE in community pharmacists (92.3%) and students (87.5%) were higher thanthose in hospital workers (66.7% to 81.8%). Pulsed-field gel electrophoresis (PFGE)analysis strongly suggested that the MRSE strains on the hands of hospital workers and students werenormal inhabitants of each subject. Their results lead to a new finding that MRSEis commonly colonized on the hands of not only hospital workers but also of healthy individuals.
