論文 - 榊原 陽一
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Yoshimura Toshihiro, Kurogi Katsuhisa, Liu Ming-Cheh, Suiko Masahito, Sakakibara Yoichi
Journal of Electrophoresis 60 ( 1 ) 5 - 14 2016年12月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：日本電気泳動学会
<i>S</i>-nitrosylation, a post-translational modification of the thiol group of cysteine residues by nitric oxide (NO), has emerged as a new mode of signal transduction and regulation of protein function. It has recently been shown that <i>S</i>-nitrosylation may result in various protein dysfunctions. However, an improved <i>S</i>-nitrosylation analysis method is needed to achieve high sensitivity and quantitative accuracy. We hypothesized that an analysis method using fluorescence dye could detect <i>S</i>-nitrosylated proteins at a higher sensitivity than that of the conventional method. In this study, we developed a procedure for analyzing <i>S</i>-nitrosylated proteins using CyDye (the CyDye switch method). This CyDye switch method for detecting <i>S</i>-nitrosylated proteins was developed based on the biotin-switch method for analyzing <i>S</i>-nitrosylated proteins. We analyzed NO donor-induced <i>S</i>-nitrosylated proteins in a model protein and at the cellular level. We demonstrated that this CyDye switch method could detect specific <i>S</i>-nitrosylated proteins using SDS-PAGE and mass spectrometry. Our results indicate that the optimized CyDye switch method is suitable for the detection of the post-translational <i>S</i>-nitrosylation of proteins.
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Sulfation of benzyl alcohol by the human cytosolic sulfotransferases (SULTs): a systematic analysis 査読あり
Zhang L., Kurogi K., Kurogi K., Liu M., Liu M., Schnapp A., Williams F., Sakakibara Y., Suiko M., Liu M.
Journal of Applied Toxicology 36 ( 9 ) 1090 - 1094 2016年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Applied Toxicology
Copyright © 2015 John Wiley & Sons, Ltd.The aim of the present study was to identify human cytosolic sulfotransferases (SULTs) that are capable of sulfating benzyl alcohol and to examine whether benzyl alcohol sulfation may occur in cultured human cells as well as in human organ homogenates. A systematic analysis revealed that of the 13 known human SULTs, SULT1A1 SULT1A2, SULTA3, and SULT1B1 are capable of mediating the sulfation of benzyl alcohol. The kinetic parameters of SULT1A1 that showed the strongest benzyl alcohol-sulfating activity were determined. HepG2 human hepatoma cells were used to demonstrate the generation and release of sulfated benzyl alcohol under the metabolic settings. Moreover, the cytosol or S9 fractions of human liver, lung, kidney and small intestine were examined to verify the presence of benzyl alcohol sulfating activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.
DOI： 10.1002/jat.3268
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Yamamoto A., Yamamoto A., Debrah-Pinamang M., DiModica N., Kurogi K., Kurogi K., Naqvi A., Hui Y., Hui Y., Sakakibara Y., Suiko M., Liu M.
Drug Metabolism Letters 10 ( 3 ) 200 - 205 2016年9月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Drug Metabolism Letters
© 2016 Bentham Science Publishers.Objective: The aim of the current study was to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating clioquinol and iodoquinol, and to verify the presence of clioquinol/ iodoquinol-sulfating activity in human organ homogenates and cultured cells. Method: An established sulfotransferase assay was employed to analyze clioquinol/iodoquinolsulfating activity of thirteen known human SULTs, as well as cytosols of human kidney, liver, lung, and small intestine. Metabolic labeling with [35S]sulfate in the presence of different concentrations of clioquinol/iodoquinol was performed using cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells. Results: A systematic analysis revealed that six of the thirteen known human SULTs, SULT1A1 SULT1A2, SULTA3, SULT1B1, SULT1C4, and SULT1E1 showed considerable clioquinol/ iodoquinol-sulfating activity. Kinetic parameters of the sulfation of clioquinol and iodoquinol by three SULTs, SULT1A1, SULT1A3, and SULT1C4, that showed the strongest clioquinol/iodoquinolsulfating activity were determined. Moreover, clioquinol/iodoquinol-sulfating activity was detected in the cytosol fractions of human liver, lung, kidney, and small intestine. Cultured HepG2 and Caco-2 cells were shown to be capable of sulfating clioquinol/iodoquinol under metabolic conditions. Conclusion: Collectively, these results provided a molecular basis underling the metabolism of clioquinol and iodoquinol through sulfation.
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Human Cytosolic Sulfotransferase SULT1A3 Mediates the Sulfation of Dextrorphan. 査読あり
Yamamoto, A., Kurogi, K., Schiefer, I.T., Liu, M.-Y., Sakakibara, Y., Suiko, M., Liu, M.-C.
Biol. Pharm. Bull. 39 ( 9 ) 1432 - 1436 2016年9月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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A novel procedure for the assessment of the antioxidant capacity of food components 査読あり
Yoshimura T., Harashima M., Kurogi K., Kurogi K., Suiko M., Suiko M., Liu M., Sakakibara Y., Sakakibara Y.
Analytical Biochemistry 507 7 - 12 2016年8月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Analytical Biochemistry
© 2016 Elsevier Inc.Carbonylation, an oxidative modification of the amino group of arginine and lysine residues caused by reactive oxygen species, has emerged as a new type of oxidative damage. Protein carbonylation has been shown to exert adverse effects on various protein functions. Recently, the role of food components in the attenuation of oxidative stress has been the focus of many studies. Most of these studies focused on the chemical properties of food components. However, it is also important to determine their effects on protein functions via post-translational modifications. In this study, we developed a novel procedure for evaluating the antioxidant capacity of food components. Hydrogen peroxide (H2O2)-induced protein carbonylation in HL-60 cells was quantitatively analyzed by using fluorescent dyes (Cy5-hydrazide dye and IC3-OSu dye), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence determination. Among a panel of food components tested, quinic acid, kaempferol, saponin, squalene, trigonelline, and mangiferin were shown to be capable of suppressing protein carbonylation in HL-60 cells. Our results demonstrated that this fluorescence labeling/SDS-PAGE procedure allows for the detection of oxidative stress-induced protein carbonylation with high sensitivity and quantitative accuracy. This method should be useful for the screening of new antioxidant food components as well as the analysis of their suppression mechanism.
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Sulfate conjugation of daphnetin by the human cytosolic sulfotransferases 査読あり
Han Z., Xi Y., Luo L., Luo L., Zhou C., Zhou C., Kurogi K., Kurogi K., Sakakibara Y., Suiko M., Liu M.
Journal of Ethnopharmacology 189 250 - 252 2016年8月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Ethnopharmacology
© 2016 Elsevier Ireland Ltd.Ethnopharmacological relevance In Turkey, daphnetin-containing Daphne oleoides is used as a folk medicine for treating rheumatic pain and lumbago. A daphnetin-containing traditional Chinese medicine tablet, named Zushima-Pian, is available in China for treating rheumatoid arthritis. The present study aimed to investigate the metabolism of daphnetin through sulfation in cultured human cells and to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of mediating the sulfation of daphnetin. Materials and methods Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [35S]sulfate in the presence of different concentrations of daphnetin. Thirteen known human SULTs, previously expressed and purified, as well as cytosols of human kidney, liver, lung, and small intestine, were examined for daphnetin-sulfating activity using an established sulfotransferase assay. Results [35S]sulfated daphnetin was found to be generated and released by HepG2 cells and Caco-2 cells labeled with [35S] sulfate in the presence of daphnetin. Among the 13 known human SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1B1, and SULT1C4 displayed significant sulfating activity toward daphnetin. Of the four human organ samples later tested, small intestine and liver cytosols displayed considerably higher daphnetin-sulfating activity than those of lung and kidney. Conclusion The results derived from the present study showed unequivocally that daphnetin could be sulfated in cultured human cells and by purified human SULT enzymes as well as human organ cytosols. The information obtained provided a basis for further studies on the metabolism of daphnetin through sulfation in vivo.
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Luo L., Luo L., Zhou C., Zhou C., Kurogi K., Sakakibara Y., Suiko M., Liu M.
Xenobiotica 46 ( 7 ) 612 - 619 2016年7月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Xenobiotica
© 2015 Taylor & Francis.1. This study aimed to investigate the involvement of sulfation in the metabolism of 6-hydroxymelatonin (6-OH-Mel), N-acetylserotonin (NAS) and 4-hydroxyramelteon (4-OH-Ram), and to identify and characterize the human cytosolic sulfotransferases (SULTs) capable of sulfating these drug compounds.2. A systematic analysis using 13 known human SULTs revealed that SULT1A1 displayed the strongest activity in catalyzing the sulfation of 6-OH-Mel and 4-OH-Ram, whereas SULT1C4 exhibited the strongest sulfating-activity towards NAS. pH-dependence and kinetic parameters of these SULT enzymes in mediating the sulfation of respective drug compounds were determined. A metabolic labeling study showed the generation and release of [35S]sulfated 6-OH-Mel, NAS and 4-OH-Ram by HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells labeled with [35S]sulfate in the presence of these drug compounds. Cytosols of human lung, liver, kidney and small intestine were examined to verify the presence of 6-OH-Mel-, NAS- and 4-OH-Ram-sulfating activity in vivo. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-OH-Mel-, NAS- and 4-OH-Ram-sulfating activities than those of lung and kidney.3. Collectively, these results provided a molecular basis for the metabolism of 6-OH-Mel, NAS and 4-OH-Ram through sulfation.
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Sulfate Conjugation of Daphnetin by the Human Cytosolic Sulfotransferases. 査読あり
Han, Z., Xi, Y., Luo, L., Zhou, C., Kurogi, K., Sakakibara, Y., Suiko, M., Liu, M.-C.
J。 Ethnopharmacol. S0378-8741 ( 16 ) 30311 - 30317 2016年5月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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シイタケおよびその加工品による抗酸化ストレス作用 査読あり
近藤知巳,中島有紀子,渡辺朋子,吉山佳世,内田飛香, 黒木勝久,福井敬一,水光正仁,榊原陽一
日本食品科学工学会誌 63 ( 5 ) 199 - 208 2016年5月
記述言語：日本語 掲載種別：研究論文（学術雑誌）
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Human cytosolic sulfotransferase SULT1C4 mediates the sulfation of doxorubicin and epirubicin 査読あり
Luo L., Luo L., Zhou C., Zhou C., Hui Y., Hui Y., Kurogi K., Kurogi K., Sakakibara Y., Suiko M., Liu M.
Drug Metabolism and Pharmacokinetics 31 ( 2 ) 163 - 166 2016年4月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Drug Metabolism and Pharmacokinetics
Copyright © 2016, The Japanese Society for the Study of Xenobiotics.Doxorubicin, an anthracycline, has been reported to be excreted in sulfate conjugated form. The current study aimed to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of sulfating doxorubicin and its analog epirubicin, and to verify whether sulfation of doxorubicin and epirubicin may occur under metabolic conditions. A systematic analysis of thirteen known human SULTs, previously cloned, expressed, and purified, revealed SULT1C4 as the only human SULT capable of sulfating doxorubicin and epirubicin. Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [35S]sulfate in the presence of different concentrations of doxorubicin or epirubicin. Analysis of spent labeling media showed the generation and release of [35S]sulfated doxorubicin and epirubicin by HepG2 cells and Caco-2 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of SULT1C4 in both HepG2 cells and Caco-2 cells. These results provided a molecular basis underlying the previous finding that sulfate-conjugated doxorubicin was excreted in the urine of patients treated with doxorubicin.
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Sulfation of benzyl alcohol by the human cytosolic sulfotransferases (SULTs): a systematic analysis. 査読あり
Zhang, L., Kurogi, K., Liu, M.-Y., Schnapp, A.M., Williams, F.E., Sakakibara, Y., Suiko, M., Liu, M.-C.
J. Appl. Toxicol. 2015年12月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Sulphation of acetaminophen by the human cytosolic sulfotransferases: A systematic analysis 査読あり
Yamamoto A., Yamamoto A., Liu M., Kurogi K., Kurogi K., Sakakibara Y., Saeki Y., Suiko M., Liu M.
Journal of Biochemistry 158 ( 6 ) 497 - 504 2015年12月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biochemistry
© 2015 The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo. This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported.
DOI： 10.1093/jb/mvv062
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Sulfation of 6-Gingerol by the Human Cytosolic Sulfotransferases: A Systematic Analysis 査読あり
Luo L., Luo L., Mei X., Mei X., Xi Y., Zhou C., Zhou C., Hui Y., Hui Y., Kurogi K., Sakakibara Y., Suiko M., Liu M.
Planta Medica 82 ( 3 ) 238 - 243 2015年11月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Planta Medica
© Georg Thieme Verlag KG Stuttgart · New York .Previous studies have demonstrated the presence of the sulfated form of 6-gingerol, a major pharmacologically active component of ginger, in plasma samples of normal human subjects who were administered 6-gingerol. The current study was designed to systematically identify the major human cytosolic sulfotransferase enzyme(s) capable of mediating the sulfation of 6-gingerol. Of the 13 known human cytosolic sulfotransferases examined, six (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1) displayed significant sulfating activity toward 6-gingerol. Kinetic parameters of SULT1A1, SULT1A3, SULT1C4, and SULT1E1 that showed stronger 6-gingerol-sulfating activity were determined. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-gingerol-sulfating activity than those of the lung and kidney. Moreover, sulfation of 6-gingerol was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under the metabolic setting. Collectively, these results provided useful information relevant to the metabolism of 6-gingerol through sulfation both in vitro and in vivo.
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Sulfation of 6-hydroxymelatonin, N-acetylserotonin and 4-hydroxyramelteon by the human cytosolic sulfotransferases (SULTs). 査読あり
Luo, L., Zhou, C., Kurogi, K., Sakakibara, Y., Suiko, M., Liu, M.-C.
Xenobiotica 46 ( 7 ) 612 - 619 2015年11月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Hui Y., Hui Y., Luo L., Luo L., Zhang L., Kurogi K., Zhou C., Zhou C., Sakakibara Y., Suiko M., Liu M.
Journal of Pharmacological Sciences 128 ( 3 ) 144 - 149 2015年8月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Pharmacological Sciences
© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license.Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.
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Abe A., Uchida A., Hoshino Y., Sakakibara Y., Suiko M., Kunitake H.
Acta Horticulturae 1065 1267 - 1274 2015年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Acta Horticulturae
To analyze the protein expression of pollen grains and tubes having self-incompatible reactions in Hyuganatsu (Citrus tamurana Hort. ex Tanaka), we cultured mature pollen and treated it with crude extracts from the styles using a mass or single liquid culture system, and we collected pollen grains and tubes for proteome analysis. The relative expression of each identified protein was quantified by an imager and revealed as a value relative to the pollen grain. Eleven identified proteins were remarkably up-regulated (above 1.2-fold) or down-regulated (under 0.8-fold) in treatment with self-incompatible reactions. Eight of these proteins were predicted to be self-incompatible-related proteins with the reported functions. In this study, F-box protein was identified as showing minimum expression in the treatment with crude extracts from styles of Hyuganatsu (self-incompatible reactions). The S-locus gene product expressed in pollen has been identified as an F-box protein in Solanaceae and Rosaceae. It is not clear whether a mechanism similar to that of Solanaceae and Rosaceae exists in the self-incompatible reaction in Hyuganatsu; however, the reduced expression of F-box protein may induce a self-incompatible reaction.
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タンパク質蛍光標識技術を用いた<i>S-</i>ニトロシル化タンパク質の解析法開発 査読あり
芳村 俊広, 黒木 勝久, 水光 正仁, 榊原 陽一
電気泳動 59 ( 2 ) 123 - 125 2015年
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Japanese Electrophoresis Society
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Nagahama K., Eto N., Eto N., Shimojo T., Kondoh T., Kondoh T., Nakahara K., Sakakibara Y., Sakakibara Y., Fukui K., Suiko M., Suiko M.
Bioscience, Biotechnology and Biochemistry 79 ( 8 ) 1327 - 1336 2015年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Bioscience, Biotechnology and Biochemistry
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry.Natural killer (NK) cells play a key role in innate immune defense against infectious disease and cancer. A reduction of NK activity is likely to be associated with increased risk of these types of disease. In this study, we investigate the activation potential of kumquat pericarp acetone fraction (KPAF) on NK cells. It is shown to significantly increase IFN-γ production and NK cytotoxic activity in human KHYG-1 NK cells. Moreover, oral administration of KP-AF significantly improves both suppressed plasma IFN-γ levels and NK cytotoxic activity per splenocyte in restraint-stressed mice. These results indicate that raw kumquat pericarp activates NK cells in vitro and in vivo. To identify the active constituents, we also examined IFN-γ production on KHYG-1 cells by the predicted active components. Only β-cryptoxanthin increased IFN-γ production, suggesting that NK cell activation effects of KP-AF may be caused by carotenoids such as β-cryptoxanthin.
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Yamasaki M., Nishimura M., Sakakibara Y., Suiko M., Morishita K., Nishiyama K.
Food and Function 5 ( 11 ) 2842 - 2849 2014年11月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Food and Function
© 2014 the Partner Organisations.Here, we examined the effect of tocotrienols (T3) on the growth of adult T-cell leukemia (ATL) cells. All three forms (β-, γ-, and δ-T3) inhibited cell proliferation in a dose-dependent manner; δ-T3 showed the strongest growth-inhibitory effect. δ-T3 increased the G1, G2/M, and subG1 populations and induced internucleosomal DNA fragmentation. δ-T3 treatment also increased the levels of cleaved caspase-3, -6, -7, -9, and poly-ADP ribose polymerase (PARP), and this was accompanied by downregulation of Bcl-2, Bcl-xL, and XIAP. Moreover, δ-T3 decreased nuclear p65 NF-κB levels, indicating downregulation of NF-κB activity. This cytotoxic effect of δ-T3 was abrogated by squalene (SQL) but not mevalonate (MVL), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), or cholesterol (CL). δ-T3 decreased intracellular SQL levels, and inhibition of de novo cholesterol synthesis did not affect the action of SQL. Furthermore, δ-T3 significantly decreased farnesyl-diphosphate farnesyltransferase 1 (FDFT1) expression. Taken together, it is evident that δ-T3, due to its ability to potently induce apoptosis via the depletion of intracellular SQL, shows the potential to be considered a therapeutic agent in patients with ATL.
DOI： 10.1039/c4fo00635f
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Kurogi K., Chepak A., Hanrahan M., Liu M., Sakakibara Y., Suiko M., Liu M.
European Journal of Pharmaceutical Sciences 62 40 - 48 2014年10月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：European Journal of Pharmaceutical Sciences
The current study was designed to examine the sulfation of eight opioid drugs, morphine, hydromorphone, oxymorphone, butorphanol, nalbuphine, levorphanol, nalorphine, and naltrexone, in HepG2 human hepatoma cells and human organ samples (lung, liver, kidney, and small intestine) and to identify the human SULT(s) responsible for their sulfation. Analysis of the spent media of HepG2 cells, metabolically labeled with [35S]sulfate in the presence of each of the eight opioid drugs, showed the generation and release of corresponding [35S]sulfated derivatives. Five of the eight opioid drugs, hydromorphone, oxymorphone, butorphanol, nalorphine, and naltrexone, appeared to be more strongly sulfated in HepG2 cells than were the other three, morphine, nalbuphine, and levorphanol. Differential sulfating activities toward the opioid drugs were detected in cytosol or S9 fractions of human lung, liver, small intestine, and kidney, with the highest activities being found for the liver sample. A systematic analysis using eleven known human SULTs and kinetic experiment revealed SULT1A1 as the major responsible SULTs for the sulfation of oxymorphone, nalbuphine, nalorphine, and naltrexone, SULT1A3 for the sulfation of morphine and hydromorphone, and SULT2A1 for the sulfation of butorphanol and levorphanol. Collectively, the results obtained imply that sulfation may play a significant role in the metabolism of the tested opioid drugs in vivo. © 2014 Elsevier Inc. All rights reserved.